The Science Behind SQUALENE The Human Antioxidant

Dr. BIKUL DAS Foreword By Dr. Sylvain Baruchel This Book discusses the function of Squalene in the human body and has been written for the reader interested in the changing perspective of research on bioactive nutrients, including Squalene. To substantiate the explanations in this book we have included research findings concerning the role of Squalene in the prevention of cancer and heart disease and in the enhancement of the immune function. This book does not suggest that people take Squalene as a dietary supplement, nor that they should not. It is the authors sincere advice that before considering Squalene dietary supplementation, readers consult their physicians as this book is not meant as a substitute for competent medical care. Despite rapid progress in medical research, fundamental biological mechanisms governing our body are still incompletely understood. However, a discussion of these mechanisms is needed to link diverse research findings on nutrients. We encourage readers to take a science- oriented approach on nutrition rather than one based on passive belief. Bear in mind that this book discusses biological hypotheses that should not be taken as established fact. The author has checked the scientific literature referred to throughout this book in an effort to provide credible information on Squalene. However the possibility of human error cannot be discounted and the author, publisher and editor cannot and do not warrant the accuracy of the information contained herein. The author hopes that this book will help to generate a further awareness of the need for environmental protection. Without a clean environment, even the best drug or dietary supplement is fighting a tough battle against both existent and newly-emerging diseases. The author, editor and publisher expressly disclaim responsibility from the use or application of information described in this book. I dedicate this book to my mother Mahindri and my wife Britta whose support is unending and who have graciously accepted my repeated absence from them in my pursuit of knowledge. Foreword Preface Editors note Introduction 11 PART 1 What is Squalene? 14 1 The Primordial balance 14 The first antioxidants 15 Free Radicals 16 Types of antioxidants 16 Oxidant-antioxidant balance 17 Oxidative damage 17 6 8 10

Oxidative stress and antioxidant metabolism 19 Antioxidant metabolism and Squalene 19 Conclusion 20 2 A History of Squalene 21 Squalenes importance in the origins of life 21 Early Squalene research 22 The Isoprenoid synthesis pathway 22 Squalenes importance recognized 23 Clinical applications of Squalene 24 Two forms of Squalene 24 Conclusion 26 3 Squalene the antioxidant 27 Isoprene 27 Biomembrane structure 29 Vitamin E and Squalene compared 29 Squalene in the skin 31 Squalenes role in the immune system 31 Squalene metabolism and cell growth 31 Squalene and aging 32 Conclusion 33 4 Balance and stress in bodily systems 34 Immune imbalance 34 Effective immune balance 35 Oxidative Stress and immune imbalance 36 Apoptosis 37 Metabolic stress 39 An example of metabolic stress 39 Metabolic stress and immune suppression 39 Conclusion 40 Carcinogenesis 41 The evolution of Cancer 41 Cancers secret to success 42 The ras oncogene 42 Squalenes inhibition of cancer proliferation 43 Carcinogenic agents 44 Squalenes preventive/therapeutic potential 44 A growing body of research 45 Potential clinical applications 46 Cytoprotection an undervalued modality 46 Criteria for a cytoprotective agent 47 Squalenes cytoprotective roles 47 A multi-target approach to cancer therapy 49 Conclusion 49 6 Cholesterol and heart disease 50 Coronary heart disease 50 Atherosclerosis 50 Good Cholesterol 51 Bad Cholesterol 51

Cholesterol transport 51 The worst cholesterol of all 53 Cholesterol lowering effects of Squalene 53 Conclusion 55 Part 11 Squalene and Disease Squalene and Disease Contents 5 Squalenes role in cancer & cancer therapy 41 Part 111 Squalene and environmental pollution 56 7 Human skin: first casualty of ozone depletion The epithelium a protective coat 56 Ultraviolet radiation 57 The skin 58 Absorption of UV rays by skin 58 The skins natural antioxidant 59 Evolutionary Adaptation to Loss of Hair 59 Ozone depletion 60 Skin Cancer 60 Immune depression 60 Protecting against UV radiation 61 Are we safe? 62 Conclusion 62 8 Background radiation 63 Sources of radiation 63 Ionizing radiation 63 Medical sources 63 Internal sources 63 Depleted uranium 63 Geological radiation 64 Cosmic radiation 64 Non-ionizing radiation 64 Consequences of background radiation 64 The radio protective role of Squalene 65 Squalene depletion due to radiation 65 Depleted uranium 63 Geological radiation 64 Cosmic radiation 64 Non-ionizing radiation 64 Consequences of background radiation 64 The radio protective role of Squalene 65 Squalene depletion due to radiation 65 Conclusion 66 9 Air pollution, allergies and lung disease 67 Lipid peroxidation and allergies 67 Squalene synthesis a potential adaptive mechanism Conclusion 68 10 The fat cell A Storehouse of Toxins 69

Adipose Tissue Depot or Organ? 69 Toxin Accumulation 70 Detoxification by the Liver 71 Detoxification by Adipose Tissue 71 The Xenobiotic Squalene Link 72 Conclusion 72 11 Metabolic stress and chaos 73 Metabolic response to stress 73 Squalene metabolism 74 Squalene in the skin 74 Squalene in the fat tissue 74 Cyclic and Acyclic Squalene 75 Two metabolic pools 75 State of disequilibrium 76 The mechanism of metabolic stress 76 Chaos 77 Metabolic stress & chaos 77 Conclusion 78 Epilogue 79 Glossary 80 References Index

Foreword
We talk daily about dietary nutrients, yet give little thought to how they work at the cellular level. Many people are amazed to learn that individual cells actually synthesize some of the nutrients they need to maintain inner health. We pay even less attention to how research is progressing and which directions will reveal therapeutic solutions based on dietary nutrients. It is customary in our society to leave such questions in the hands of doctors and researchers. Few people are interested, perhaps in part because eating a salad is much more enjoyable than pondering its nutritional value. However, nutritional research is providing us with information crucial to our long-term health. Soon, more people will want to understand how nutrients work at a cellular level so they can adjust their diets to life style, specific disease risk and perhaps even according to genetic makeup. The emerging awareness of cell signaling in nutrient research suggests that such an era of nutritional self-awareness is approaching. Our under-standing of cell signaling is revolutionizing nutrient research, but lay people remain largely unaware of the fact. Cell signaling describes how cells communicate with each other right into their interior. This information exchange employs molecules like a telephone network, one molecule relaying information to another in a domino sequence that eventually carries it to specific genes. Several such pathways are active in specific cells, just as one home may have several telephones. Cells rely on signaling pathways for growth, proliferation and various functional activities. Hormones and other biomolecules influence cells by acting on these signaling pathways.

In the last fifteen years, great progress has been made in unlocking the code of these signaling pathways, suggesting that many diseases are either caused or exacerbated by faulty cell signaling. This list includes cancer, diabetes, immune disorders and disorders of the nervous system. Signaling research shows that certain nutrients influence the signaling pathways and prevent or inhibit the growth of cancer. This research provides hope for the effective prevention and even the adjuvant treatment of some cancers. The most promising approach seems to be the use of a nutrient combination to influence several signaling pathways at once in the hope that this will be more effective than using a single nutrient. In this multi-target strategy cocktails of various nutrients would be given to people either at risk of a certain cancer or already suffering from it. Such nutrient cocktails may also sensitize tumor cells to chemotherapeutic agents, making conventional anti-cancer therapy both more effective and less toxic. Only vigorous research can determine which signaling pathways to target and how. This explains why research into various bioactive nutrient groups is becoming increasingly important. Isoprenoids are one such group of nutrients and are particularly significant to cellular growth and proliferation. There are hundreds, and some are found to influence various signaling pathways. Squalene and its cyclic derivatives are non toxic Isoprenoids with great promise. The molecular activities of Squalene and its related Isoprenoids ursolic acid, oleanolic acid , geranyl and geraniol can play a role in the anticancer cocktail and may contribute to other nutrient cocktails formulated to prevent such other disease states as elevated cholesterol. This books appearance is very timely and reveals a new approach to nutrient research. It discusses Squalene and its metabolism in the light of the latest understanding of molecular biology, giving an overview of Squalene metabolism and how it seems to be much more than a mere cholesterol metabolite. Squalene is a powerful antioxidant with a potentially crucial role in the cellular antioxidant defense system. However, Doctor Bikul Das points out that this research is still at an early stage and he discusses hypotheses about areas of fundamental biology that are not yet fully understood. This book is also an example of how ideas develop and scientific knowledge grows. Readers should therefore maintain caution and apply a critical eye in their interpretation of this book. Dr. Das also hopes to provoke new directions in the study of pollution-induced disease. The idea of metabolic stress and its relation to oxidative stress outlined in this book, though not yet completely understood, shines a new light on the biology of such pollution-related health problems as UV radiation induced immune suppression. We appreciate innovative thinking and hard work of the author and encourage him to continue his research on nutrients and their metabolism. Dr. Sylvain Baruchel. M.D. Associate Professor of Pediatrics, University of Toronto. Staff Oncologist, Division of Hematology & Oncology. Senior Scientist & Director of New Agent and Innovative Therapy Program The Hospital for Sick Children, Toronto. PREFACE

This book calls attention to Squalene a remarkable nutrient produced in our body and also found in nature. Just a few decades ago, nutrients were thought to have minimal research value. Today, nutrient research is a hot topic among medical circles, the research community and the general public. Several factors account for this changing trend. Firstly, there are today many calls for nontoxic, natural therapies to prevent or treat various disease processes. The hope is that understanding the cellular mechanism of healing nutrients may lead to combination therapies in which several nutrients each target different mechanisms of a disease to achieve a common goal. Rapid advances in cell signaling research provide hope for such natural combinations, which may become more effective than artificial drugs. Squalene and its various cyclic derivatives such as ursolic acid are showing great promise in preventive therapy, particularly in the chemoprevention of cancer. Secondly, there is also a great interest in the innate healing processes of the body. By understanding them we will learn to help and enhance our natural defenses. Individual cells have sophisticated abilities to heal themselves. The cellular DNA repair process is an impressive example. During cell division, DNA divides into two identical strands of daughter DNA. When- as happens on occasion- this process leads to damage, a repairing process corrects it. Another example of cellular healing is when the cell systematically checks the cell membrane for defects or damage and employs several molecules to fix it. There are other examples of self-healing, for example the healing of skin lesions and broken bones. In these processes body tissues synthesize and distribute additional nutrients to facilitate repair and regeneration. For example, the concentration of glutathione a cellular antioxidant increases around a wound and facilitates the healing process. Vitamins and other nutrients are already known to contribute to the healing processes of the body and there are probably many others, either synthesized by our body or obtained from diet. An understanding of their function will shed new insight into innate healing processes. Dietary Squalene has been found to enhance the elimination of toxins and minimize the toxic effect of UV radiation. With this in mind, the presence of very high levels of Squalene in human skin and adipose tissue is attracting significant interest among researchers who are exploring the cellular mechanism of Squalene. The third reason for renewed interest in nutrient research is an appreciation and understanding of the evolutionary dynamics of health and sickness. Evolution focuses on the survival of a species. It helps organisms adapt to changing environments. During our species turbulent evolution, we have learned to protect our health. There is every reason to believe that the tissue distribution of certain nutrients has an evolutionary history. The presence of Squalene in our skin could be an example of such an evolutionary protective mechanism. I have taken Theodosius Dobzhanskys famous quotation, Nothing in biology makes sense except tin the light of evolution, as encouragement to examine the evolutionary aspect of Squalenes presence in our skin. The growing interest in nutrient research has three general themes: medical practitioners are interested in using nutrients in preventative therapy, other individuals are interested in strengthening the innate healing processes through the use of scientifically proven nutrients, and medical researchers and biologists want to understand the evolutionary aspect of health and sickness.

ling processes through the use of scientifically proven nutrients, and medical researchers and biologists want to understand the evolutionary aspect of health and sickness. The evolutionary aspect of nutrient research particularly interests me and I believe it may provide clues as to how pollution is affecting us. Health, evolution, and pollution are very much related to each other. Research into such nutrients as Squalene may help clarify this relationship. In this industrial era, pollution is affecting all biological systems. The question is, how can we best understand the long term impact of pollution: This new theme is discussed in part three. Beyond this emerging frontier of nutrient research in the skin, Squalenes rich history is primitive life, the ancient legends surrounding its healing power and the modern insights into its unique presence in our skin is a fascinating human story that can bridge ancient legends and modern medical research. The cover of the book illustrates a human cell against a background of the Earth. This image came to me as I was writing chapter 1, and thinking of human vulnerability and our interdependence with the entire biosphere. The more we understand about our cells and organs and about nutrients like Squalene, the better we will appreciate the need to maintain our own health and that out our environment. It is my sincere hope that this book will bring a fresh perspective to nutrient research. Dr. Bikul Das Research Fellow Hospital For Sick Children Toronto, Canada, August 2000

Editors Note
Squalene is an Isoprenoid an ancient biochemical that made it possible for archaic bacteria to flourish when our planet was an inhospitable, unrecognizable place. Life began here on surfaces hot enough to boil water, with a virtual absence of atmospheric oxygen and no ozone layer to filter out the fierce ultraviolet radiation of the sun. In fact, Earths surface looked more like Mars than the green orb we now cling to so precariously. Under the protection of this lowly molecule, ancient bacteria proliferated, transforming the atmosphere so that green plants could emerge and harness the suns energy. They too depended on Isoprenoids to avoid being burned into a crisp in the process. Plant life in turn transformed the atmosphere into an oxygen-rich soup that made oxygen respiration possible yet another reaction in which Squalene protects living tissue from oxyradicals. Then something extraordinary happened and has been happening ever since the linear Squalene curled up and provided the basis for the sterol nucleus of cholesterol, without which the modern animal cell would be unthinkable. This biochemical reaction, still not fully explained, remains the most complex single step reaction in the biological world. Among other things, it enabled our forefathers to shed their furry outer coat and expose their naked skin to the suns rays without being destroyed by UV radiation.

Today, Squalene protects the biomembrane of cells and the myriad organelles within from oxidative stress. It helps the cholesterol metabolism maintain order and keep levels of harmful LDL cholesterol to a minimum. It contributes to the cell-renewal cycle and keeps cancer at bay. But now this ubiquitous life-enabling Isoprenoid is under stress. Our environment is undergoing unprecedented oxidative and metabolic attack at the hands of p9ollutants, carcinogens and increased ultraviolet radiation. Has the molecule that has protected life for billions of years finally met its match? Or does this simple substance, for so long considered virtually insignificant hole the clues to our evolutionary survival? I share the authors hope that this book will help to generate further awareness of the need to protect ourselves and our fragile planet. We must acknowledge that there is no guarantee of ultimate victory unless we act together to ensure that our environment remains amenable to life. Stephen Schettini Montreal May 2000 Stephen Schettini is a writer, illustrator and designer of medical books for professionals and the general public. He has developed extensive training guides and learning programs on the cardiovascular system, arthritis, hormone replacement therapy, allergies & antihistamines, dermatology and prostate cancer. He has also coauthored Glutathione [GSH]: Your Bodys Most Powerful Healing Agent [with Dr. Jimmy Gutman] and the Osteoporosis Remedy [with Dr. I. William Lane]

Introduction It has been clear for some years that Mediterranean peoples tend to suffer considerably less than others from heart disease and certain cancers. Scientists have linked this to relatively high levels of olive oil consumption, but they arent first to make this connection. Many ancient Mediterranean cultures believed that olive oil increases strength and longevity, and indeed the olive tree is a rich source of Squalene! The olive tree has always been more than just another food source. Since the earliest days of Mediterranean civilization it has been surrounded by a wealth of cultural symbolism. Ancient kings ruled with scepters of olive wood, priests still anoint the worthy with olive oil, and the olive branch is a recognized symbol of peace in many cultures. Early Mediterranean peoples considered it a gift from the gods, worthy of be adored and defended. Old Greek and Roman writers referred to it as the Eternal Tree of Peach and the Jewish bible includes dozens of references to the tree. Ancient Indian physicians were well aware of the great healing power of the olive tree and its oil. The Koran too describes it as a Precious Condiment. The city of Athens was mythically named after Pallas Athena, goddess of peace and wisdom. She was said to have created a tree able to light up the night [olive oil lamps produce a warm, gentle light], to soothe wounds and to produce a food rich in flavor and energy. According to Roman legend, Hercules strode along the shores of the Mediterranean, his olive staff sending out roots every time it struck the ground and

sprouting new trees. The Phoenicians helped spread the tree westward from Asia Minor and around the shores of the Mediterranean. Olive trees live for centuries, even millennia. When a main trunk dies, new shoots sprout from its base and grow. Add to this the many uses which it is put and the distinctive cuisine that has emerged from it, it is not surprising that it has become so rich in myth. And now we discover there may be some factual basis to its reputation for health and healing. Another rich source of herbal Squalene is the amaranth plant, an extremely robust type of grain that can survive both scorching heat and extremely dry soil. It produces six-foot stalks with brilliant feathery red or magenta plumes. The Greek word amarantus means never withering. In India the amaranth herb has been widely used for thousands of years. It is as rich in Squalene and as common in that region as the olive tree in the Mediterranean basin. In the great epics of the ancient Indian cultures the herb is believed to be empowered with immortal strength and fertility. The Sanskrit word for the plant amaranth [not amaranth] means King of Immortality. Once a North American staple and the preferred grain of Aztec royalty, the broad leafed grain was believed to have sacred healing power. Aztec soldiers are a very thick soup of this herb before going to war, and the amaranth plant was outlawed by Spanish missionaries who were disturbed by its association with human sacrifice. In fact, they believed the key to suppression of the Aztec culture was the annihilation of the plant. The Swedish Order of the Amaranth dates back to the 1653 reign of Queen Christina, who brought European culture to her country and negotiated the landmark Peace Treaty of Westphalia in 1648. The amaranth has frequently represented distinction and honour, and was formed into the Amaranthine Wreath symbol of the Swedish Order and of the Bond of Fraternal Friendship representing the strength and power of the plant. In Japan, a rich source of Squalene is shark liver oil [sharks belong to the species squali]. In ancient times, this rich oil was thought to increase strength and longevity. This miraculous healing power of shark liver oil is even mentioned in some old Japanese folk stories. The shark was considered a legendary creature living in the ocean depths. Many Japanese people believed the liver of the shark to contain powerful healing agents. Like the Aztecs who drank a soup of amaranth, ancient warriors of Japan and China and even the Maoris of New Zealand- were known to drink shark liver oil before leaving for war. We can therefore trace cultural recognition of Squalene-rich products with unique survival qualities to the Mediterranean region, Scandinavian, the Indian subcontinent, the Far East and Central America. The claims made by the ancient peoples of these lands may not be based on science, but they lend color and warmth to our research and prompt us to place the full glare of the scientific spotlight on this intriguing substance. Chinese healers were the first to conduct pre-scientific research into a rich natural source of Squalene. In 1596 Lee Ji Chin, a Chinese healer of the Ming Dynasty [1369 1644] composed a 52 volume compendium of some two thousand herbs, including the liver oil of the deep sea shark. Chinese traders subsequently brought the book to Japan, where it was known as Honzokomoku. Samurai warriors used this oil to increase their strength. Villagers of Suruga Bay on the Izu Peninsula of Japan were accustomed to drinking the same oil. The local name of this special extract was Samedawa or cureall.:

In 1906, Dr. Mitsumaru Tsujimoto, a Japanese industrial engineer, discovered that Samedawa contains extremely large quantities of an unsaturated hydrocarbon. He named the hydrocarbon Squalene, from the Latin root squalus [shark]. Dr. Tsujimoto was presented the Imperial Award of the Japan Academy in honor of his achievement. Squalene was first found in the human body in the 1950s, when the cholesterol metabolism was first identified. This is a complex pathway by which glucose is converted to the allimportant cholesterol molecule. The pathway entails dozens of transformations of one biochemical to another. Squalene is just one of those intermediate steps. At the time the significance of the transitional biochemicals was lost in the excitement of the greater picture. They were considered just a means to an end, and not significant in their own right. However, three of the biochemicals in that pathway molecules that perform vital functions including the regulation of cell growth and proliferation through a process named after them isoprenylation. Without this process, cell membranes could not anchor proteins vital to cell growth. Some Isoprenoids notably Squalene- are also strong antioxidants. They have lately been discovered to play protective roles not only in the antioxidant system but also in the immune function. Isoprenoids also play a part in the regulation of apoptosis [programmed cell death]. The history of Isoprenoids goes back billions of years. There are thousands of varieties, some of which played a crucial role in the protection of early life on this planet. This Isoprenoid nature of vitamin A, vitamin E, beta-carotene and other well known nutrients is discussed in chapters 2 and 3. Indeed there has been a flurry of experimental activity around Isoprenoids, and things are now known about these humble molecules that were never imagined by the researchers who first identified Squalene. This book is a synopsis of that research and weaves the threads of these various experimental findings into a overall picture. and things are now known about these humble molecules that were never imagined by the researchers who first identified Squalene. This book is a synopsis of that research and weaves the threads of these various experimental findings into a overall picture. In fact this bigger picture is still growing. Squalene has been found abundantly in the skin, the membranous lining of the gastrointestinal and respiratory tracts and in adipose tissue [fat]. There is serves independent functions of great importance to hour health. This skin of primates has virtually no Squalene but the sebum secreted by human skin contains about 12% -a huge proportion. This appears to be an evolutionary requirement of our unique nakedness, protecting us from our unparalleled exposure to the suns ultraviolet radiation. As environmental pollution leading to ozone depletion exposes us to rising levels of these harmful rays, the burden on Squalene in our inner and outer protective coatings may exceed its ability to cope. This could stress Squalene metabolism and contribute to immune suppression. This book examines our bodys natural use of Squalene as well as research suggesting its potential dietary usefulness. Emphasis has been placed on the impact of pollution upon antioxidant balance, using Squalene metabolism as a model. It has three parts: PART ONE WHAT IS SQUALENE? Squalene is a strong antioxidant Isoprenoid. It helps our cells avoid oxidative stress and prevents lipid peroxidation. It also contributes to a balanced immune

response. Part One describes Squalenes role: As an antioxidant As a regulator of the Isoprenoid metabolism In an effective immune response. PART TWO SQUALENE AND DISEASE Squalenes influence on the vital pathway that transforms glucose into cholesterol may be pivotal. It regulates or controls the rate of synthesis of the enzyme HMG Co-A reductase, and may contribute to effective treatments for cancer and heart disease. Several research studies have already demonstrated Squalenes anticancer and cholesterol lowering activities. Part Two describes the potential use of Squalene in disease management, especially against; Cancer, and Heart disease. PART THREE SQUALENE AND ENVIRONMENTAL POLLUTION Squalene is present in high concentrations in human skin and in fat cells. This may be an evolutionary requirement. Most likely the pressure of evolution in the human protective coat and the requirements of its macrophage system have led to the storage of large amounts of Squalene in the cellular protective system of the skin and underlying fat tissue. Rapid changes in the environment may be placing oxidative pressure on the protective coat of our body with potentially disastrous effects. Part Three describes: Environmental pollution, and Pollution-induced oxidative stress leading to stress in Squalene metabolism Part 1 - What is Squalene? This part of the book describes Squalene and how it works in the healthy individual, suggesting the possibility that stress in Squalene metabolism contributes to immune suppression. It also raises questions about the possible therapeutic and preventive role of Squalene modulation. 1 - The Primordial Balance During most of the twentieth century medical researchers were preoccupied with identifying various disease states and treating them as they arose. Extensive studies of the immune system contributed to the development of effective pharmaceutical drubs that today successfully deal with a wide variety of bacterial and viral illnesses. However, many diseases remain only partly understood and relatively intractable, including Alzheimers disease, Parkinsons disease, diabetes, mellitus, rheumatoid diseases and most common and most feared of all, heart disease and cancer. New research and technology is at last enabling doctors and scientists to better explain the mechanisms of these and many other diseases. Some of the most striking advances in recent years are emerging from research into free radical biology. It is now clear that specific disease agents are not the only source of ill-health. Cells and tissue can also be destroyed by a breakdown of the

very molecules of which they are composed. Polluting free radicals such as oxyradicals noxious by-products of the energy production process in each cell constantly threaten the inner environment of our body. Free radical molecules are unbalanced by having too few or too many electrons. They are dangerous because in their attempt to regain balance they upset the stability of surrounding molecules, Sometimes initiating chain reactions that can spiral out of control. Still, free radicals are necessary for our survival as well. The are used in intracellular communication, and immune cells produce huge amounts of free radicals to attack bacteria and other harmful agents. Sometimes they produce to many, yet the free radicals do not harm the cell. Each cell possesses its own defense mechanism the antioxidant defense system that maintains a dynamic internal balance between free radicals and antioxidant nutrients. Without this balance the cell would not survive, tissue would degenerate and we would be unable to maintain our health. This oxidantantioxidant balance is presumably a primordial equilibrium without which life would never have been possible. Increased generation of free radicals can lead to oxidative stress, producing imbalance and resulting in oxidative damage, cell death, tissue damage and disease. Fortunately, cells and tissues implement a compensatory mechanism, tending to minimize oxidative stress by synthesizing more antioxidants as they need them. This dependence on endogenous rather than dietary antioxidants suggests that a study of the metabolism of endogenous antioxidants will enlighten our understanding of the relationship between oxidant- antioxidant balance and oxidative stress. The purpose of this chapter is to discuss the role of endogenous anti-oxidants in oxidant-antioxidant balance. Since Squalene is an endogenous antioxidant that is where we will begin. The First Antioxidants. Primitive living cells first emerged on this planet about four billion years ago. They survived by converting light energy into chemical food, just as plants harvest the suns energy. Plants initiate photosynthesis with chlorophyll, which is activated by sunlight and helps carbon dioxide interact with water to produce glucose and release oxygen. This activation is sometimes transferred to oxygen molecules in the form of extra electrons, and they become oxyradicals. In addition, the early biosphere lacked any protective ozone layer and was subject to fierce ultraviolet radiation. This too generated free radicals. For years, biologists have wondered how these primitive organisms protected themselves from such strong radiation and so many free radicals. It was presumed that the skin enclosing their single-celled bodies must have contained a protective agent. Recent breakthroughs by NASA scientists may provide a piece of the puzzle. They have speculated that the powerful antioxidant quinine was deposited on Earth by incoming meteorites. It seems that quinone was the first Isoprenoid. Quinone is especially able to neutralize the free radicals generated by ultraviolet rays and may have enabled a few lucky cells to become evolutionary survivors. Without quinone and other antioxidant molecules primordial organisms would not have survived the hazards of unshielded ultraviolet radiation. Today, quinone is one among many Isoprenoid antioxidants found abundantly in the plant and animal kingdoms. Light harvesting complexes [LHCs] are another type of Isoprenoid that

protects plants. These are a group of carotenoids antioxidants that surround the chlorophyll molecules and use an electron transfer reaction to protect them from ultraviolet radiation damage, principally by preventing the oxidation of molecular oxygen. They also help absorb extra electrons before they can damage other molecules. The outer coating of many seeds and fruits also contains Isoprenoids. For example, tomatoes contain the antioxidant Isoprenoid lycopene. Unlike plant cells, animal cells absorb oxygen and derive energy from its reaction with glucose. In this process, protons flow through special molecules in the mitochondria [energy producers] of each cell. Electrical energy is produced and moves through the mitochondria as electrons are shunted from one molecule to another in a process known as the reduction-oxidation [redox] reaction. Molecules in the mitochondria are alternatively reduced [receive electrons] and oxidized [donate electrons]. This generates electricity, which is converted into chemical energy. During redox reactions electrons sometimes escape, damaging either the redox molecule itself or the cell. However, redox molecules such as ubiquinone have an Isoprenoid tail capable of neutralizing numerous free radicals and keeping the mother molecule extremely stable. Once again, Isoprenoids helps protect the organism. The Isoprenoid ubiquinone [also known as coenzyme Q10] is synthesized inside the cell and is involved in the energy releasing process of the mitochondria. Many antioxidants are either isoprenoids or have an Isoprenoid tail. Vitamin E, vitamin A and flavonoid are all isoprenoids. Free Radicals The damage caused by free radicals occurs at a subatomic level. In any stable atom, electrons orbit the nucleus in pairs and any breakup of this pairing makes the atom unstable. It or the molecule of which it is part is said to be in a state of imbalance and is called a free radical. One of the most common sources of free radicals is the process burns glucose with oxygen to produce energy through oxidation reduction [redox] reactions in the mitochondria. The type of free radical it releases an oxyradicals is a routine by-product. As you might expect, the production of oxyradicals increases when our energy requirements increase for example when exercising, fighting sickness or eating. Not all free radicals are manufactured within the cells of our body. They are everywhere. Ultraviolet radiation can create them in the skin and even turn ground level oxygen molecules into free radicals. We are surrounded by pollutants, some of which are themselves free radicals and some of which interact with metabolic processes, causing further subatomic damage. Types of Antioxidants Antioxidants are antidotes to free radicals, including oxyradicals. Many substances act as antioxidants, but they all have one thing in common the capacity to stabilize the imbalance of unpaired electrons and neutralize the harmful potential of free radicals without themselves becoming unstable. If it is true that the first isoprenoids arrived from outer space, we living organisms can literally thank the stars

for our evolutionary survival. However, even though we still benefit from environmental antioxidants chiefly from food sources most forms of life have learned to produce antioxidants endogenously [within the cells where they are needed]. In the human body they include glutathione sulfhydride [GSH], superoxide dismutase [SOD] catalase, Squalene and coenzyme Q10 [ubiquinone]. These last two are both isoprenoids. The protective coat of many organisms from the biomembranes of cellular organelles to human skin are protected from free radical damage by antioxidant isoprenoids. Other antioxidant isoprenoids are obtained from nutrients and are said to be exogenous. The include substances like vitamins E and A, lycopene and beta-carotene. These are usually found in various foods but they are also frequently taken as concentrates in pills, food supplements or nutriceuticals. They are used by healthconscious individuals in many ways, usually in the hope that they will maintain good health, prevent all sorts of disease and hopefully slow the aging process. Their usefulness is not in question, but there is wide disagreement over what constitutes an appropriate dosage. It is well known, for example, that taking too much Vitamin A or E can have harmful toxic consequences. There is also growing evidence that having too many antioxidants is just as harmful as not having enough. In fact our body as a whole must maintain a proper balance between oxidants antioxidants. Oxidant-Antioxidant Balanc-Antioxidant Balance The idea of oxidant-antioxidant balance emerged from research showing that for any given level of free radicals, tissue damage is prevented most effectively by just the right concentration of antioxidants. We have long known that during periods of oxidative stress the antioxidant defense system increases the synthesis of antioxidants. Now we are learning that at other times our body actually decreases production because too many antioxidants can harm the body as well. Such adjustments suggest the existence of any antioxidant defense system an overseeing mechanism used by the body to monitor and maintain an appropriate balance, just as the immune defense system controls the synthesis and activity of immune cells. However, in the same way that the immune defense system can lose its balance, this system too can be disrupted. Our focus should not be to take as many antioxidants as possible but to help the system maintain its oxidant-antioxidant balance. It alone knows its precise needs, and therefore endogenous antioxidants-those synthesized in the cell will play a greater role in oxidant-antioxidant balance than exogenous [dietary] ones. To do this of course, the body must have the necessary raw materials [precursors] at hand. Aging makes it increasingly difficult for us to maintain this balance, because synthesis of Squalene, GHS and coenzyme Q10 all decline as we get older. The direct result of a disrupted oxidant-antioxidant balance is cellular damage, and one of the worst types of damage caused by free radicals is to the cell wall [biomembrane]. Oxidative Damage Cell and tissue damage caused by oxidant-antioxidant imbalance is referred to as oxidative damage. The first step of this damage process is the lipid peroxidation chain reaction, which breaks down cell membranes.

Every living cell is enclosed by a membrane a double layer of lipids [fats]. A cell also has organelles [functional parts] that help it grow, replicate and do its work. These organelles include the nucleus, mitochondria and ribosomes. Like the cell itself, each organelle has its own membranous wall, known as the biomembrane. The biomembranes surface is a sort of complex electric fence, with biological devices such as receptors [entry points for specific molecules] and electrical channels through which energy is exchanged. All these devices generate free radicals. In fact, free radicals are produced constantly within and outside the cell, some as by-products of energyreleasing oxidation in the mitochondria, others quite usefully, as in the case of immune cells that use them as weapons against invading pathogens. The biomembranes themselves are highly vulnerable to free radical damage, especially in the hydrophobic [waterless] band between the two lipid layers. The fatty acids that constitute the two layers of the cell membrane are lipid molecules and like gasoline, are flammable hydrocarbons. They are packed very close to each other and are oxidized [catch fire] quickly. When one molecule is ignited it quickly triggers the same reaction in its neighbors a chain reaction called lipid peroxidation. Lipid peroxidation [the burning of liquids] quickly damages the entire biomembrane and threatens adjoining cells. Lipid peroxidation is the most common and pernicious source of damage to biomembranes. It impairs the cells function and leads to its eventual death. Diseased cells spill their contents into surrounding tissue and cause inflammation. As more cells are damaged or destroyed tissue damage follows and disease sets in. The effects of lipid peroxidation are initially microscopic and not immediately apparent, but they accumulate over time and are important factors in the progression of chronic diseases such as atherosclerosis and the rheumatoid group of disorders. In fact such diseases are turning out to be more numerous than was ever imagined. A large portion of the biological molecules that form the basic building blocks of life are glucose, fatty acids and amino acids and the constituent atoms of these molecules can be destabilized by many types of biochemical reactions leaving them with unpaired electrons. Free radicals spontaneously seek to correct their imbalance by stealing electrons from any available molecule. Hopefully they encounter antioxidant molecules, which render them harmless. Not infrequently however they encounter defenseless neighboring molecules that in turn lose electrons and become free radicals themselves. This can promote an ever-widening chain reaction leading to a disruption and destruction of living tissue leading to oxidative damage. Even when these chain reactions are eventually stopped by our bodys antioxidant defenses, the damage already caused to cells, tissues and organs accumulates over time and provides a foothold for incoming pathogens [disease causing substances]. The damage also contributes to the progression of such diseases as cancer, heart disease, arthritis and AIDS. An unconventional but significant segment of the scientific community even considers aging itself to be largely preventable disease, caused mostly by free radical damage. Nowadays there is enormous scientific and public interest in reinforcing the antioxidant defense system as a way to break the cycle of free radical damage, slow the aging process, extend life-span and improve overall health. Diseases associated with oxidative damage 1. Cancer

2. Diabetes Mellitus [peripheral neuropathy of this disease is due to free radical damage to the nerve heath 3. Heart failure and ischemia-reperfusion injury 4. Autoimmune disorders e.g. the rheumatoid group 5. Kidney disorders e.g. glomerulo-nephritis, tubulointerstitial diseases 6. Infective disorders e.g. AIDS and AIDS related dementia, pulmonary fibrosis due to tuberculosis, secondary anemia due to malaria 7. Neurodegenerative disorders such as Alzheimers disease 8. Dermatological disorders such as photosensitivity disorder, psoriasis, pemphigus vulgaris 9. Atherosclerosis Free radicals are thought to cause many pathological conditions including the transformation of a normal cell into a cancer cell. Also, redox molecules which are normally immune to free radicals can sometimes be overcome by them, with disastrous consequences. At the root of these many specific occurrences of oxidative damage lies the bigger and even more disturbing picture of oxidative stress. the bigger and even more disturbing picture of oxidative stress. Oxidative Stress & Antioxidant Metabolism Oxidative stress results when antioxidant balance fails. Although oxidative damage is implicated in the progression of many diseases, it develops only when oxidative stress rises above a certain threshold. As soon as cells and tissues experience oxidative stress, the compensatory mechanism of the oxidant-antioxidant balance immediately tries to preempt oxidative damage by synthesizing antioxidants. Only if and when the stress crosses a certain threshold does the mechanism fail and oxidative damage occur. The mechanism of oxidative damage is not unlike that of heart failure. In response to failure, the heart accommodates the increased load by increasing in size, but beyond a certain limit its pumping ability declines sharply and eventually fails. Similarly, although the antioxidant defense system can increase its synthesis of antioxidants, oxidative stress may cross the threshold at which the compensatory mechanism fails. Endogenous antioxidants [those synthesized in the cell] probably play a greater role in this compensatory mechanism because their rate of production is within the control of the body. Exogenous antioxidants like Vitamin E may help reduce oxidative stress but cannot directly affect the compensatory mechanism. This is supported by the evidence that as age-related tissue levels of endogenous antioxidants decrease, the body is more prone to oxidative stress. Therefore oxidative stress results from the failure of endogenous antioxidants. Such a reciprocal relationship between oxidative stress and endogenous antioxidants suggests that endogenous antioxidants and their metabolisms require further study. If we can understand when and why the compensatory mechanism fails, we may find ways to prevent its failure. We hypothesize that metabolic stress determines the failure of the compensatory mechanism. Examining this hypothesis will provide valuable insight into the relationship between oxidant- antioxidant balance and oxidative stress. Antioxidant Metabolism and Squalene The above discussion suggests the great importance of clearly defining metabolic stress, why it occurs and how it leads to oxidative stress. These questions may lead to new insights into balance and stress in the immune system. They may also

provide more insight into oxidant-antioxidant balance, which we can justifiably consider primordial balance. Since Squalene is synthesized in our cells, it is an endogenous antioxidant. It may therefore serve as an appropriate model for the study of antioxidant metabolic stress its influence on oxidative stress. We examine this possibility in chapters 4 & 11. We must also consider Squalenes history as a antioxidant. The cell membranes of archae and bacteria that lived about 3.5 billion years ago were rich in Squalene. Along with coenzyme Q, lycopene and some other isoprenoids, Squalene was among the first antioxidant group to take part in the primordial balance of life. Indeed, research reveals that Squalenes antioxidant properties resemble those of vitamin E and other Isoprenoid antioxidants. The natural presence of this strong antioxidant in human cells and its particularly high concentration in human skin do not seem coincidental. Our job is to identify the exact antioxidant role it plays in the body. Conclusion All life forms must deal with the everyday threat of free radicals. While early life first benefited by exogenous [environmental] antioxidants, those organisms that were able to manufacture endogenous antioxidants in their own metabolism gained the evolutionary upper hand. The new science of free radical biology suggests that oxidative damage is responsible for some of the most intractable diseases of the twentieth century and that maintaining an oxidant-antioxidant balance is a crucial first step to overcoming some of them. Since levels of endogenous antioxidants decline with the aging process, it becomes increasingly difficult to maintain this balance and the question naturally arises of whether the process of disease and aging can be slowed by therapies that might arrest or reverse this antioxidant decline. A History of Squalene Squalenes Importance in the Origin of Life Squalene has served as an antioxidant for terrestrial life for over three billion years, a fact due in great part to its nature as a pure Isoprenoid. Without isoprenoids, the history of life on Earth would be unrecognizable, if indeed there were any life at all. About 3.5 billion years ago during the Precambrian era, the surface of Earth was very hot and the atmosphere was filled with methane, ammonia and other toxic gases. Nevertheless, huge colonies of archae and cyanobacteria [organisms resembling bacteria seem to have thrived. They left a vivid fossil record. Archae which date from that time survived in temperatures of 100 deg Celsius and on very little oxygen. The membrane of their cells was rich acyclic isoprenoids mainly Squalene. These Isoprenoid-rich life forms dominated the Earth right up to the Cambrian era a period of more than three billion years. Fungi, plants and animal life forms appeared only about 500 million years ago and primitive humans barely two million. The fossils of these three billion year old isoprenoids are found well preserved in the deep sediments under the ocean floor. Marine biologists routinely use Squalene, lycopene and other isoprenoids as biological markers of these sediments. Isoprenoids may also one day help astrobiologists trace the evidence of life beyond this planet. Just as several billion year old isopreniods fossils are found in all sorts of terrestrial rock, they hope that similar fossils on meteorites will indicate the existence of life in outer space. A 1999 scientific paper published in the journal Science traces the probably

origin of quinone an early Isoprenoid molecule right back to the cosmos. Author Max P. Bernstein writes that isoprenoid molecules were probably carried to Earth by meteorites and that their presence enabled primitive cells and organisms to protect themselves from the harsh living conditions of early life, especially exposure to extreme ultraviolet radiation. At that time, the ozone layer of Earth was not fully formed and the surface of the planet was relatively unshielded from the full effects of the suns rays. The earliest evidence of life on Earth is a several billion year old isoprenoid fossil pentamethyleicosane [PME], the molecular structure of which is similar to Squalene. PME is an acyclic isoprenoid, the product of primitive methane-producing bacteria. This is why the PME fossil is so significant, especially given its striking molecular resemblance to Squalene. A present day study of Squalene will help us understand the probable function of PME and other acyclic isopreniods in ancient life forms. Early Squalene Research In 1936, Nobel laureate Paul Karrer described the biochemical structure of Squalene for the first time. This medical researcher was already famous for his account of the chemical structures of Vitamins E and A. He was surprised to find that Squalene had a similar structure to these two antioxidant vitamins. This may have hinted at Squalenes antioxidant characteristics, but far more interesting things were yet to be discovered. In the 1950s Squalene was found to occur naturally in the human body, although it was not considered particularly significant at the time. Researchers were trying to describe how cholesterol is synthesized in the cell when it was discovered that Squalene happens to be one of the steps in the transformation of glucose into this vital substance. High cholesterol levels are so feared these days that many people are unaware that normal levels are absolutely essential to health. Cholesterol is manufactured in individual cells in a complex series of biochemical steps known as the mevalonate pathway [see below]. Glucose is first converted into mevalonic acid, and this in turn produces isoprenoids geranyl, farnesyl and squalene. Some two dozen steps later, the cell has a supply of cholesterol, essential for the manufacture of hormones and bile salts. The Greater mevalonate pathway Glucose Mevalonic acid Geranyl Farnesyl Co-enzyme Q10 Squalene Dolichol Cyclic squalene About 20 steps Cholesterol The Isoprenoid Synthesis Pathway Within the greater mevalonate pathway, the four steps from mevalonate to squalene are known as the isoprenoid synthesis pathway. We now know that they are of critical importance in a range of functions including cell growth and proliferation as well as cholesterol synthesis. Many of the discussions in this book are concerned with this pathway as well as the step immediately preceding it the synthesis of HMG Coenzyme-A reductase [HMG Co-A]. It turns out that these are mot merely intermediates, but also rate-controlling steps reactions that slow down or speed up

according to the bodys requirements. They have profound implications for the function, growth, replication and life span of individual cells. The presence of squalene in cells affects the rate of synthesis of HMG Co-A reductase, which in turn affects the entire synthesis of cellular isoprenoids and cholesterol, offering tantalizing possibilities for the prevention and treatment of high blood levels of bad cholesterol one of the most important risk factors for heart disease. But we are getting ahead of the scientific detective story. When its natural occurrence was discovered in the body in the 1950s Squalenes antioxidant function was still unknown. function, growth, replication and life span of individual cells. The presence of squalene in cells affects the rate of synthesis of HMG Co-A reductase, which in turn affects the entire synthesis of cellular isoprenoids and cholesterol, offering tantalizing possibilities for the prevention and treatment of high blood levels of bad cholesterol one of the most important risk factors for heart disease. But we are getting ahead of the scientific detective story. When its natural occurrence was discovered in the body in the 1950s Squalenes antioxidant function was still unknown. Squalenes Importance Recognized There was in face a delay of over a decade before the spotlight was finally placed upon this antioxidant agent found in olives, amaranth and shark liver oil. The abundant folk tales and anecdotal stories created a negative bias in the scientific community squalene was considered a mere folk cure and its potential was ignored. In any case, limited research funding and the relative immaturity of biochemical technology hindered further understanding. Research avenues reopened in 1963, when an article in the scientific journal Nature demonstrated that squalene stimulates macrophages the principal immune cells in the inner and outer protective coat of our body. This empirical observation was exciting news, but an explanation of how and why it was so remained elusive. In 1950, researchers led by McKenna found that human skin secretes very high levels of squalene. Subsequently, C.K. George and others at Rockefeller University, New York demonstrated the widespread occurrence of squalene in subcutaneous and submucosal human tissue. This finding of significant squalene levels in the protective coat of the body raised the immediate question of whether it plays a protective role. This second research group also found a separate source [pool] of human squalene in the body, quite independent of the cellular cholesterol metabolism. Its biological function there remained unknown but its existence could not be ignored. There was no longer any doubt squalene was much more than a simple by product of cholesterol metabolism. In 1982, R. Tilvis and his group found another pool of squalene synthesis this time in fat cells. And in 189 C. De Luca and colleagues compared the squalene content of human skin to that of other primates and found it to be much greater. The sebum of gorillas, for example, contains about 0.1% squalene, whereas human sebum contains about 12% - 120 times more! This led to an entirely new notion that the presence of squalene in human skin may be an evolutionary requirement. In an earlier study conducted in 1977, M. Gloor and A. Karenfield had found that the bodys consumption of squalene increases when skin is exposed to ultraviolet [UV] radiation. Then in 1993, O. Salamoto and his research group conducted more specific tests and demonstrated that the first molecule targeted by UV rays as they enter the human skin is squalene. Since ultraviolet radiation is known to harm unprotected

tissue, the implication is that they protect the skin from UV damage at least until they are depleted Several research groups got to work on this question and finally in 1995 a Japanese team clearly demonstrated that squalene can prevent UV induced oxidation of lipids in skin a key finding that finally placed squalene in the scientific spotlight. 23 In 1982 squalenes detoxifying function was demonstrated in several research experiments and in 1993 its radioprotective effects were revealed. These discoveries set the stage for the medicinal use of squalene. Clinical Applications of Squalene During the early part of the twentieth century, it was believed in Japan that squalene could reduce the risk of cancer, and combat tuberculosis and other microbial diseases. Dr. Keijiro Kogami, a medical research from the Tokyo Imperial University, conducted extensive research and developed a squalene treatment for tuberculosis. His work apparently showed significant success but the hospital where he worked and his records were destroyed in 1945 by World War two bombings and his work was taken up later by the Yokota Research Institute in Tokyo. Two prominent Japanese physicians and researchers Dr. Ryosuke Yokota and his son Dr. Takashi Yokota pioneered research on clinical applications. They established the Yokota Health Institute and worked together from 1968 to 1989. There they undertook the longest running clinical research trials on the health benefits of highly purified and carefully preserved squalene obtained from the deep sea shark. Several laboratory research studies showed that dietary squalene can strengthen the immune response, especially against radioactive poisoning. In 1996 a human clinical trial of squalene was performed to examine its effectiveness in lowering blood cholesterol. As a result of these and consequent research studies, dietary squalene has been found to: 1. Lower blood cholesterol 2. Enhance the anti-tumor action of chemotherapeutic agents 3. Inhibit cancer growth 4. Increase the efficiency of the immune system In order to understand these developments, we will examine the isoprenoid synthesis pathway in which squalenes role has been expanded from that of a mere precursor to an important metabolic player in its own right. This is discussed in chapter 3. First we must describe the squalene molecule. Two Forms of Squalene There are two molecular forms of squalene - linear and cyclic. Both have the same constituents but each one has its own distinct molecular shape and biological properties. Linear squalene is a long wavy strand an extremely stable and powerful antioxidant that protects the biomembrane [envelope] of living cells from lipid peroxidation [the most dangerous form of free radical damage]. Under certain conditions acyclic squalene loses this stability, curls in on itself and becomes cyclic, losing much of its antioxidant potential but becoming the sterol nucleus. The transformation of acyclic to cyclic squalene has defied synthesis in the

laboratory. It is still a mystery how the molecules form is reconfigured without any change in its composition. All we know is that the acyclic molecule enters the deep pocket of a cone shaped protein complex where it is broken down into its six component isoprenes and then reassembled in a new configuration. Only then can the molecule become the nucleus of cholesterol and many other sterols. It has been said that this cyclization reaction is the most complex single step reaction in the biological world. molecule become the nucleus of cholesterol and many other sterols. It has been said that this cyclization reaction is the most complex single step reaction in the biological world. This transformation first took place several billion years ago, during the Precambrian period, opening up whole new possibilities for life on this planet. The wavy shape of linear squalene provoked considerable interest when scientist noticed that it resembled the molecular structures found abundantly in very primitive cells. It turns out that the cell membranes of archaea one of the oldest forms of life are composed of acyclic isoprenoids like lycopene and squalene. In addition, marine geologists discovered large amounts of the isoprenoids squalene and lycopene in deep sea sediments dating back to primordial geological ages [the Precambrian era]. Squalene is a principal constituent of the cell membrane of the archaea that live in such inhospitable environments as sulfur springs and deep sea volcanic vents. It is believed that these archaea survived these environments because of the protective acyclic squalene and other antioxidants in the biomembrane. As we move up the evolutionary ladder we find that the eubacteria and cyanobacteria are liberally composed of hopanoids cyclic squalene molecules that undergo further modifications to form triterpene compounds. This transformation of acyclic to cyclic squalene was an evolutionary step of enormous significance. Cyanobacteria are a product of a relatively temperate oxygen environment and do not need to maintain such robust protective mechanisms as archaea. Instead, they developed genes able to provoke the enzymatic transformation of acyclic squalene into hopanoids, resulting in a stronger cell membrane able to engage in more sophisticated evolutionary activity. In modern mammals cyclic squalene forms the vital sterol nucleus the building block of such essential substances as steroids, other hormones, vitamin D and of course cellular cholesterol. Although the emergence of cyclic squalene is considered an evolutionary development, living organisms continued to depend on linear squalene. A very significant portion of this protective isoprenoid does not become cyclic and the organism benefits from its antioxidant properties. Today acyclic isoprenoids like squalene and lycopene are found throughout the plant world. In the animal kingdom, acyclic isoprenoids remain an invaluable constituent of cellular membranes. Linear squalene is found abundantly in human skin, where it is also reduced to squalane, a reverse step that cannot be taken by cyclic squalene. [Squalane is formed when hydrogen ions saturate squalenes double bond neutralizing its antioxidant abilities. Squalane is nevertheless an effective moisturizer used in cosmetic preparations]. Humans are the only primates with such concentrations of Squalene in their skin, apparently because of our susceptibility to ultraviolet B radiation. Medical research has shown that squalene is the first target molecule of UV radiation it apparently takes the brunt of UVs damaging ionizing potential. This radiation may nevertheless induce stress in squalene metabolism an idea explored in chapters 4 & 11.

Conclusion The first hints of squalenes importance in our body were uncovered when it was found to be an integral part of the cholesterol synthesis pathway in individual cells. Later discoveries that it stimulates the activity of macro-phage immune cells and also protects the skin from UV radiation damage have left no doubt as to the bodys day to day dependence upon squalene. Following centuries of traditional use in Japan, researchers using natural squalene in therapeutic applications were rewarded with success, but their findings were not clearly understood. Recent understanding of squalenes isoprenoid origins and its metabolism began to unravel the puzzle, but the big breakthrough was the realization that it performs a regulatory role in the cholesterol synthesis pathway. There is good reason to believe that continued research into this regulatory role may help explain and refine the successes of the applications developed in Japan. The presence of very high levels of squalene in human skin may have to do with its ability to protect against UV radiation and other sources of free radicals. Squalene the Antioxidant Squalene is an isoprenoid antioxidant a type of organic molecule vital for the very existence of life on Earth. Hundreds of thousands of isoprenoids are found in nature, of which squalene and some others are potent antioxidants notably Vitamin E, beta-carotene, lycopene, phytol and coenzyme Q10. They all contain isoprene units. This chapter outlines the antioxidant mechanism of squalene and its significance in cellular protection. Isoprenes Isoprene units are very small molecules containing five carbon atoms and are found in all isoprenoids. There are many isoprenoids on Earth perhaps several hundred thousand. Each contains isoprenes attached in different configurations or to different molecules. Molecules containing only isoprenes like Squalene are said to be pure isoprenoids. Those with one or more isoprene units attached to the mother molecule like Coenzyme Q10 and Vitamin E are mixed isoprenoids. The isoprene unit contributes to the antioxidant properties of the whole molecule but does not by itself account for them. Dolichol, for example is a pure isoprenoid of twelve isoprene units with no significant antioxidant abilities, whereas coenzyme Q10, a mixed isoprenoid with ten units, is a good antioxidant. Squalenes six units give the molecule an effective and stable antioxidant configuration. All these isoprenoids, it should be noted, are intermediate metabolites of the mevalonate pathway. The usefulness of an antioxidant is largely determined by its ability to stop a lipid peroxidation chain reaction. Lipids [fatty acids] in the biomembrane [outer envelope] of a cell are arranged like long chains of molecules, aligning themselves in parallel. When a free radical approaches one of these fatty acid chains, it liberates a lipid peroxide radical a fatty acid chain with an oxyradicals at the end. This radical then goes on to attack another fatty acid chain, liberating a new peroxide radical. However, when it encounters an antioxidant molecule, it is quenched and the chain reaction is stopped. Antioxidant isoprenoids are ideal for stopping lipid peroxidation chain reactions in the biomembrane. They are water insoluble and can be easily incorporated into the lipid bilayer. Squalene and coenzyme Q10 two endogenous antioxidant

isoprenoids are indeed found there and laboratory research has shown them to be effective. Not every isoprenoid has antioxidant properties, and even an antioxidant can be overwhelmed by free radicals. Its effectiveness all depends upon the stability of the molecule. Like any other molecule encountering a free radical, antioxidants are obliged to either donate or receive an electron. Unlike other molecules, the loss of the electron does not destabilize them. Squalene is an excellent antioxidant because of its great capacity to receive or donate electrons without suffering molecular disruption. There are two ways in which it can neutralize [or quench] a free radical, either physically or chemically. Physical quenching involves the donation of an electron by squalene, stabilizing the radical without itself becoming unstable. In a chemical quenching reaction the radical is chemically incorporated with the squalene molecule, producing squalene hydroperoxide a new molecule. Squalene hydroperoxide is not an antioxidant but is an excellent emollient that in the skin serves as a natural sunscreen. In the case of physical quenching, the squalene molecule contributes an electron without changing its own nature. This is a necessary prerequisite for any antioxidant, but squalene does so in a particularly effective way. The key to its stability is the configuration of atoms in its constituent quaternary carbon and methyl group, known as its pi electron system. It is a quaternary carbon group because the central carbon atom is directly connected only to other carbon atoms, making it particularly stable. In additions, the pi electron systems of six isoprene units enable each unit to interconnect forty-four hydrogen atoms, providing it with extraordinary stability. Because of all this, squalene is said to have a low ionization threshold its atoms are held in place by a strong natural bond that is maintained with little expenditure of energy. Therefore, even when it donates electrons its energy field remains stable. Squalenes very low ionization threshold accounts for its very large capacity to donate electrons, like vitamin E. This unique stability is the key to squalenes ability to terminate a lipid peroxidation chain reaction. Once a lipid peroxidation chain reaction is underway it will damage one lipid molecule after another unless it is stopped by the intervention of a terminator an appropriate antioxidant molecule standing in its path. Most terminator molecules contain isoprene units. Isoprenes have excellent shock absorbing capabilities and are the right molecule to terminate the chain reaction. This is why antioxidant isoprenoids are vital to such a vast range of living tissue. The antioxidant property of squalene has been known for a long time, and laboratory research has shown that squalene specifically terminates lipid peroxidation chain reactions in the skins surface. Y. Kohno and his colleagues have shown that squalene is stable [i.e. cannot be easily oxidized] and is capable of terminating chain reactions. It is reasonable to assume that it performs a similar function wherever it is found, for example within individual cells and in the biomembrane. M.K. Rao first wrote of squalenes antioxidant properties in 1968 in an article published in the Journal of the American Oil Chemical Society. However the origin of these properties was still unknown. Only recently in the light of free radical biology have scientists realized that they derive from squalenes isoprenoid nature. In both plant and animal life isoprenes play a significant role in protecting the cell or redox molecule from free radical induced damage. In the cytoplasm [ the whole cell except the nucleus] where most of the cells work takes place, electrons are

produced by many of its metabolic reactions. Especially, large numbers are released as by-products of the energy generating activity of the mitochondria. Electrons reacting with water inside a cell will generate hydroxyl ions {OH} and hydrogen ions [H] both free radicals and highly toxic to lipid and protein molecules within the cell. Leaked electrons may also collide with molecular oxygen inside the cell, producing superoxide ions [O]. Most importantly, free radicals may convert molecular oxygen in the cytoplasm into free radicals. Plant chloroplasts contain LHC [light harvesting complex] isoprenoid molecules to prevent molecular oxygen from being rendered into oxyradicals. However, we still do not know whether similar defense mechanisms exist in the mammalian cell. Does cytoplasm contain isoprenoid molecules to recognize and neutralize these free radicals? It is highly possible that squalene may act in animal cells Biomembrane Structure The part of the body where the biological properties of isoprenes are in greatest demand is the biomembrane. This two layered lipid skin envelops each cell and each of the various organelles within. In particular, the biomembranes of the mitochondria the site of energy releasing electrical activity are particularly prone to a lipid peroxidation chain reaction. There, nutrients are oxidized, constantly releasing free radicals in the midst of this intense concentration of lipids. Each of the biomembranes two layers is a collection of longitudinally arranged lipid molecule chains, including cholesterol, vitamin E and fatty acids. The molecule chains have one hydrophobic [water avoiding] end and one hydrophilic [water seeking] end. Like a magnet, they are said to be polarized, and naturally arrange themselves in a n orderly pattern. By avoiding the watery environments both outside and inside the cell, the hydrophobic ends enclose a waterless band between the two layers. Acyclic squalene is entirely hydrophobic and is not anchored in the biomembrane. It is therefore attracted to this waterless band and accumulates there where it performs its crucial antioxidant function. Squalenes ability to protect the biomembrane from free radical damage is discussed below. Vitamin E & Squalene Compared Of all isoprenoids that can act as antioxidants, we so far know most about vitamin E. Its structure of three isoprenoid side chains and two phenol rings makes it a very effective antioxidant. It is commonly believed that Vitamin E is the principal antioxidant of lipid peroxidation chain reactions in the biomembrane, but this may not be the case after all. An ideal antioxidant should fulfill three criteria. It must: 1. stop the chain reaction 2. be synthesized and regulated on demand [so the body does not need to depend upon an outside supply]: 3. incorporate itself into the biomembrane without altering or damaging the membrane We can compare vitamin E and squalene in the light of these requirements. Vitamin Es ability to completely protect the biomembrane is limited by its uneven distribution throughout the biomembrane. Because it is an exogenous antioxidant and must be obtained from dietary sources, the body has no control over where and when it is available. Squalene on the other hand, is synthesized within the cell from glucose

a readily available nutrient under normal circumstances. Also, the large benzene structure in vitamin E limits its integration into the biomembrane, which can incorporate no more than two molecules per 2,000 lipid molecules. Too many vitamin E molecules disturb the biomembranes physiological properties. It has been shown that large quantities of squalene do not alter the physiological property of the biomembrane. Squalene has additional advantages over vitamin E. It does not require recycling, whereas Vitamin E must be continuously recycled by endogenous 29 antioxidants such as glutathione and squalene. And, during periods of oxidative stress, the availability of antioxidants decreases, limiting the potential of recycling Vitamin E. For all these reasons, the usefulness of vitamin E as the primary antioxidant in the biomembrane may be exaggerated and the role played by squalene may be more significant. Also, squalene is not fixed in the biomembrane and scavenges free radicals in the hydrophobic intermembrane band. It is also highly resistant to free radical attacks on itself. Squalene has an additional advantage it can recycle Vitamin E. Antioxidant molecules such as Vitamin E neutralize free radicals by donating electrons but then become radical themselves. However they are recycled by the intracellular antioxidants glutathione and squalene and sent back to work. This recycling is a reduction process involving a biochemical ENE reaction which stabilizes a free radical by donating a hydrogen atom. Comparisons of vitamin E and squalene Vitamin E. A mixed isoprenoid of three isoprene units and very good antioxidant capacity Exogenous [dependent upon dietary sources] and not necessarily available when needed Cannot be synthesized in the body available only in certain foods Limited integration into the biomembrane, where it becomes embedded in the lipid bilayers Is fixed in the lipid layer and cannot move freely Too many vitamin E molecules alter the biomembranes physiological properties and structural configuration Vitamin E requires recycling by endogenous antioxidants such as glutathione and squalene Is itself susceptible to free radical attacks The usefulness of vitamin E as the sole terminator in the biomembrane is limited

Squalene: A pure isoprenoid of six isoprene units and very good antioxidant capacity Endogenous [manufactured on demand] readily available under normal circumstances Manufactured within the cell from readily available glucose Strongly attracted to the hydrophobic band between the two lipid layers of the biomembrane, where risk of lipid peroxidation is greatest Can move freely thoroughly the biomembrane Large quantities do not alter the physiological properties of the biomembrane Does not require recycling Relatively resistant to free radical attacks on itself The role played by squalene as a terminator in the biomembrane is significant Usually a molecules methyl group supplies the hydrogen atom for the ENE reaction. Squalene has six methyl groups capable of participating in the ENE reaction. This may explain squalenes ability to recycle oxidized vitamin E back in to recycled vitamin E. 30

Squalene in the Skin The human cell synthesizes two isoprenoid antioxidants: ubiquinone [coenzyme Q10] and squalene. The former is mostly distributed within the mitochondria [energy production centres] of our cells. Squalene is found mainly in the biomembrane, but also in the fatty layer just beneath the skin. In addition, the outer surface of our skin is covered by a coating of squalene rich fat. Laboratory research has shown that squalene is capable of protecting the skin surface from free radical induced lipid peroxidation confirming its antioxidant properties. Squalenes Role in the Immune System Squalenes antioxidant properties also contribute to protective functions in the immune system. As primitive organisms learned to survive the threat of free radicals and proliferate in number and variety, they needed increasingly to protect themselves from viruses and bacteria, which were also proliferating. Only those able to do so avoided extinction. The first step was the emergence of the macrophage an immune cell that kills, eats and digests bacteria and viruses. Today the macrophage remains the principal immune defense of fish and many invertebrates. Humans have developed a more sophisticated army of immune mechanisms, including lymphocytes that can attack different antigens in specific ways and can even recognize particular invaders and know how to overcome them. But we still depend largely on the macrophage for protection. In fact it remains the frontline defense in the present day human body especially in the protective coat those parts of the body in direct contact with the outside environment. Laboratory tests have shown that squalene enhances the function of macrophages. It seems that the concentration and distribution of squalene in skin and adipose tissue is an evolutionary requirement to strengthen the defense function of the protective coat of our body. Squalene Metabolism & Cell Growth Researchers developed ways to arrest the mevalonate pathway by blocking it at the squalene stage, preventing the progress of glucose into cholesterol. But there was an unexpected consequence cell growth came to a halt, obviously because something crucial was missing. However, adding cholesterol to the cell culture medium afterwards did not correct the problem. The researchers deduced that one or more intermediaries of the pathway were essential for cell functions. These intermediate metabolites obviously play a more significant role than was previously thought. The squalene synthesis segment of the mevalonate pathway produces some isoprenoids essential for cell growth and proliferation. Of these four products, the two isopreniods geranyl and farnesyl are derived from mevalonic acid and are direct precursors of squalene. In order to perform its work and particularly when it undergoes division the cell depends upon certain proteins and growth factors. The isoprenoids geranyl 31

and farnesyl help the cell by providing an anchorage in the biomembrane for these proteins. This is called protein isoprenylation. Deficient squalene precursors at this stage can prevent protein isoprenylation and inhibit cell growth. For cells to function efficiently, squalene and its precursors are essential. These precursors also function as signaling molecules for cell growth and proliferation. Close to the beginning of the mevalonate pathway the synthesis of HMG Co-A immediately precedes the four steps of the squalene synthesis segment mevalonic acid to geranyl to farnesyl to squalene [see below]. This step is of particular interest because it determines the mevalonate pathways rate of production as a whole and is the key to many cellular functions. The production of HMG Co-A is dependent upon the availability of HMG Co-A reductase, which determines several important cellular functions including: Control of the rate of production of cellular cholesterol The entire mevalonate pathway in the cell, including production of geranyl and farnesyl two isoprenoids crucial to cellular growth and proliferation Squalene has been found to regulate HMG Co-A activity The regulatory role of squalene on HMG Co-A reductase and the roles of squalenes molecular cousins and precursors geranyl and farnesyl are the key to understanding squalenes full metabolic role in the cell. The Squalene synthesis segment of the mevalonate pathway controls the isoprenoid metabolism by regulating enzyme HMG Co-A reductase levels Glucose HMG Co-A Reductase Mevalonic acid Squalene synthesis segment Geranyl Farnesyl SQUALENE Cyclic Squalene About 20 steps CHOLESTEROL HMG Co-A reductase is the rate-limiting enzyme of the mevalonate pathway and therefore the controller of the mevalonate production factory Squalene & Aging For unknown reasons the distribution and concentration of squalene and other isoprenoids including coenzyme Q10 changes as a person ages. The activity of HMG Co-A reductase too is age specific. Generally speaking isoprenoid

concentrations decrease with aging, particularly in the brain. It is not clearly known why such changes occur but this undoubtedly affects our health adversely. More research is required to uncover the implication of the age specific change in the isoprenoid metabolism and to investigate the possibilities of modulating it. 32

Conclusion Squalene is an ancient and potent isoprenoid antioxidant. Its isoprene constituents and its special molecular structure provide its antioxidant properties. It is stable enough to resist oxidation itself when quenching free radicals and also recycles vitamin E. The biochemical structure of squalene not only makes it a stable antioxidant but also a regulator of cellular growth. It has recently been discovered that squalene plays a key role in the regulation of the cholesterol synthesis pathway through its influence on the enzyme HMG Co-A reductase an enzyme crucial for growth and cellular proliferation. There is no doubt that squalenes protective role in the body is profound and the question arises of whether squalene levels in the body can be modulated to provide antioxidant protection in ways similar to dietary vitamin E. This is especially significant in elderly people whose isoprenoid concentrations are particularly depleted. 33 4 - Balance & Stress In Bodily Systems Our body is an extraordinary network of interconnected operating systems. In good health, the balanced interaction of these systems keeps our cells healthy and happy. However as we know from the second law of thermodynamics entropy [the tendency to fall into disorder] universally increases in all systems. For example, the homeostatic conditions of water flow when the flow rises or falls beyond a certain threshold, entropy disrupts the phenomenon and it disappears. Systems in our bodies are no exception when balance is disturbed, disease results. However, biological systems are sophisticated masters of entropy that work hard to maintain a state of balance [low entropy] and some biologists consider life to be the antithesis of entropy. Life has its own control mechanisms that maintain constant, dynamic balance. Scientists call this homeostasis a sort of inherent wisdom of the biological system. It is also called health. Invading viruses or bacteria interfere with our health by targeting bodily control systems and creating imbalance. More complex disease processes such as diabetes and rheumatoid arthritis also break down control systems. Those most commonly targeted are the immune control system, the oxidant-antioxidant control system and the metabolic control system. Each has evolutionary niches that can be targeted by pathogens, resulting in increased entropy for all. A good example is the way the human immuno-deficiency virus [HIV] targets the immune system and leads to AIDS. There are some experimental evidence to suggest that it upsets key processes of the oxidant-antioxidant control system by creating a deficiency of glutathione. This results in oxidative stress and immune imbalance that undermine the oxidantantioxidant control system eventually contributing to its collapse. This finding suggests that a proper balance in the antioxidant metabolism is a prerequisite for an effective immune system and indeed, squalenes immune enhancing

properties have been long known, Since squalene metabolism is an antioxidant metabolism, a study of its role in the immune balance may provide more insight into the crucial relation between antioxidant metabolism and immune function. Such insight will contribute to the development of useful therapies to reinforce the immune system. This chapter describes the fundamentals of immune balance and the role of squalene and its metabolism in effective immune response. Immune Imbalance The balanced immune response defines the meaning of immunodeficiency. The function of the immune system is to keep the internal environment of our body free of pathogens and xenobiotics. To do this it relies on a network of special cells and molecules, principally macrophages, lymphocytes and cytokines. It was long thought that a large number of immune cells resulted in a stronger and more balanced internal environment. In parallel to the concept of oxidant-antioxidant balance however, it has become increasingly apparent that too active or too many immune cells can lead to immune deficiency s readily as too few. Our bodys ability to resist disease does not depend on the brute strength or numbers of immune cells but on their state of balance. 34

Consider the case history of a thirty-eight year old woman suffering from chronic pharyngitis [sore throat] for two years. When she eventually developed lesions in her mouth, her doctor diagnosed bacterial pharyngitis. His examination led him to suspect her immune status. A battery of tests revealed large numbers of lymphocytes [immune cells] and high levels of immunoglobulin [antibodies] in the blood and yet infectious bacteria were found in a throat swab. The doctor wondered If she has so many antibodies and immune cells, why arent they killing the viruses and bacteria? He was forced to conclude that something was wrong with the womans immune response. -eight year old woman suffering from chronic pharyngitis [sore throat] for two years. When she eventually developed lesions in her mouth, her doctor diagnosed bacterial pharyngitis. His examination led him to suspect her immune status. A battery of tests revealed large numbers of lymphocytes [immune cells] and high levels of immunoglobulin [antibodies] in the blood and yet infectious bacteria were found in a throat swab. The doctor wondered If she has so many antibodies and immune cells, why arent they killing the viruses and bacteria? He was forced to conclude that something was wrong with the womans immune response. The imbalance in this womans immune system is probably due to some underlying disorder that prevents it from effectively responding to outside attacks. Such diseases are characterized by an excessive immune response to a real or imagined threat, resulting in damage to the host. Thus immunodeficiency does not always imply a reduced number of immune cells or fewer antibodies. Rather, it refers to the lack of a balanced immune response in which the internal functioning of immune cells plays the main role. Effective Immune Balance Individual immune cells are the functional units of effective immune response. Among them, macrophages are the frontline protective cells. They are large white blood cells that simply engulf invaders and metabolic debris old red blood cells, dead tissue and even cells that have become malignant. Once it is safely contained within the macrophage, the material is digested and conveyed safely out of our body. Other immune cells such as lymphocytes use extremely sophisticated means to fight off equally sophisticated viruses, but macrophages operate by brute force. They create within themselves vesicles [sacs] filled with the free radical hydrogen peroxide, which are transported to the outer cell membrane and squirted into the path of oncoming enemies. Once released, the enormous quantities of these free radicals are as potentially dangerous to the immune cells own membrane as to the invaders. In a battle situation, immune cells are often called upon to grow and proliferate rapidly. Their ability to do so depends upon effective biomembrane protection which is a function of the antioxidant defense system. Antioxidants such as glutathione and vitamin E prevent and stop lipid peroxidation chain reactions. Squalene has been found to optimize the macrophages function. Evidence suggests that the immune cells biomembrane s protected by squalene during phagocytosis, and the synthesis and consumption of squalene probably increases at this time, as do other antioxidants such as glutathione.

Quite apart from the oxidant-antioxidant balance within individual immune cells, the immune response as a whole must maintain its own balance. Many types of immune cells must work together as a team. This is maintained by a system of communication that not only distinguishes friendly from enemy cells, but also identifies the type of threat and encourages the growth and activity of the appropriate immune response. Like a ball game in which players cooperate for the sake of the whole, each one recognizes different needs in different parts of the field and communicates with the others through words and signals. 35 The communication mechanism that ensures a balanced response and lets immune cells know what to expect from each other is called negative feedback and is a universal law of bodily control systems. According to our bodys needs, over excited cells receive signals to slow down and follow an overall strategy, while sluggish cells are encouraged to become more active. In short, macrophages, lymphocytes and other immune cells work as a team, exciting or inhibiting each other with chemical signals to maintain over balance. The messengers of the immune system are cytokines chemicals released by immune cells that travel quickly through the blood, lymph and other body fluids to and from the battle site. They carry excitatory or inhibitory messages, encouraging or restraining other immune cells. Simply put, cytokines regulate the immune response. Keeping the immune system working as a unit requires a balanced expression of cytokines. Each immune cell synthesizes both excitatory and inhibitory cytokines and releases them on demand. This cytokine balance depends upon a balanced internal environment in the immune cell and here the squalene metabolic process plays an important role. Two precursors of squalene geranyl and farnesyl have been found to influence cytokine synthesis and secretion. Experimental research shows that deficiency of these two precursors inhibits the macrophages ability to synthesize cytokines causing the cytokine system to lose its balanced expression. Individual cells use squalene to protect their biomembrane, and two squalene precursors are used for cytokine synthesis and secretion. In addition, research on oxidative stress induced immune imbalance shows that endogenous antioxidant metabolisms are factors in an effective immune balance. It therefore appears that squalene and its metabolism may contribute to effective immune balance. This is hardly surprising when we consider that squalene is an endogenous antioxidant. The metabolism of glutathione another endogenous antioxidant also contributes to effective immune balance. Oxidative Stress & Immune Imbalance A system falls into stress when it is overburdened by the demands placed on it. Disease agents such as HIV encourage the release of large numbers of free radicals. These destructive agents either burn up the cell or trigger its suicide [apoptosis]. These combined assaults on the cells of our body are referred to as cellular oxidative stress. Oxidative stress occurs when our body cannot access enough antioxidants and free radicals gain the upper hand. Once balance is lost, the deficiency of

antioxidants becomes increasingly acute and the cytokine expression becomes overexcited. Our bodys defense systems spiral out of control. Instead of slowing down, they frantically secrete excitatory cytokines, worsening the situation. The immune system becomes more and more aggressive, desperately trying to maintain our health but in fact pushing the body into greater and greater levels of imbalance. This is like a ball game in which the players see themselves facing defeat, become desperate, lose their emotional balance and enter into uncoordinated attack leaving themselves increasingly vulnerable. Excessive levels of excitatory cytokines cause untold damage to healthy tissue. 36

Over excitation of cytokines can initiate positive feedback, further increasing oxidative stress. This soon depletes endogenous antioxidants such as glutathione and squalene leaving the cell membrane open to lipid peroxidation. Each state of imbalance causes further imbalances, amplifying the damage in an ever-widening spiral of destruction. The greater part of acute damage leading to disease occurs through this vicious circle of amplification. Positive feedback poses two dangers in particular the appearance of autoantibodies and further unrestrained amplification of cytokines induced tissue damage. oxidative stress. This soon depletes endogenous antioxidants such as glutathione and squalene leaving the cell membrane open to lipid peroxidation. Each state of imbalance causes further imbalances, amplifying the damage in an ever-widening spiral of destruction. The greater part of acute damage leading to disease occurs through this vicious circle of amplification. Positive feedback poses two dangers in particular the appearance of autoantibodies and further unrestrained amplification of cytokines induced tissue damage. 1. While antibodies are normally produced by the immune response to pathogens, autoantibodies turn on our bodys own proteins. Normally they are controlled when negative feedback causes antibodies to inhibit their production. When oxidative stress disrupts negative feedback however, positive feedback takes over and autoantibodies can cause considerable damage. The appearance of autoantibodies in AIDS is an example of positive feedback initiated by stress. 2. The other danger of positive feedback is an unbalanced cytokine expression leading to lipid peroxidation and subsequent tissue damage. The damage caused by too many excitatory cytokines leads to progressive and wideranging destruction of all sorts of tissue, as demonstrated by the activity of the human immunodeficiency virus {HIV] in the human brain. Although HIV cannot replicate in the brain as it does in other parts of the body, AIDS patients frequently develop marked symptoms of dementia, such as memory loss and shortened attention span. The damage is triggered by the few HI viruses that do pass into the brain. Although they cannot themselves act, their presence stimulates immune cells in the brain to secrete an aggressive cytokine {TNF-alpha]. This cytokine in turn amplifies cytokine secretion by activating more immune cells, resulting in oxidative stress that further increases the secretion of excitatory cytokines. The production of TNF-alpha is caught in the vicious circle of positive feedback. By disrupting the balance expression of cytokines, this tiny population of HI viruses unable even to replicate can initiate a snowball leading to extensive brain tissue damage. Such effects are seen in many ailments, including allergies, tuberculosis and kidney failure. Increasing oxidative stress in brain leads to extensive damage at the hands of apoptosis. In other words, oxidative stress pushes normal cells towards programmed cell death [apoptosis]. And apoptosis is a major contributing factor to oxidative stress induced immune suppression. Apoptosis Every second 25 million cells die [and 25 million are born] inside our body, and the body needs to maintain order. This order is maintained by apoptosis a natural process of cell death which is normally harmless. Healthy cells have several

inherited genetic mechanisms that, when triggered, force them to commit suicide. If apoptosis were not a controlled mechanism and cell deaths were random, toxic debris would be strewn about surrounding tissue. Many cells contain free radicals and other toxic substances. For example, pancreatic cells 37 have powerful digestive enzymes. Macrophages and neutrophils contain hydrogen peroxide and digestive enzymes within special sacs in their cells. If the cell membranes break for example due to a lipid peroxidation chain reaction the release of these substances into surrounding tissue causes extensive inflammation. Apoptosis is a way for our body to contain these toxins. Dead cells are engulfed by the trash-collecting macrophages before their membranes break open. Apoptosis is an essential part of overall health and development of the body. It is for example, responsible for the resorption of a tadpoles tail, the removal of webbing between the fingers and toes of a human fetus, the formation of adequate gaps, [synapses] between neurons in the brain and the sloughing off of the inner lining of the uterus [the endometrium] at the start of menstruation. Apoptosis is not the random death of worn-out cells but highly organized behavior, which is why it is defined as programmed cell death. Without apoptosis, these processes would not proceed in a systematic fashion and would result in severe inflammation. Some body cells only undergo apoptosis at the end of their useful life, when they begin to malfunction. But other cells are discarded as part of a particular bodily function for example, endometrial cells die not according to their age or condition but according to the rhythms of the menstrual cycle. The complex cellular mechanisms that decide a cells fate are known as apoptosis regulators. An example is the tumor suppressor gene [a killer gene] that identifies cells about to become cancerous and forces them to undergo apoptosis. A link between apoptosis and the squalene synthesis pathway has been established experimentally, although the precise mechanism has not yet been explained. In these research experiments, cells underwent apoptosis when their squalene synthesis pathway was inhibited. It has been found that apoptosis can also be induced by free radicals. During periods of oxidative stress, for example, the incidence of apoptotic cell death increases. This has been blamed for the development of Alzheimers disease, Parkinsons disease, diabetes mellitus and other illnesses. In cases of myocardial infarction too, many cardiac cells die of apoptosis. In AIDS a large number of healthy immune cells die due to forced apoptosis triggered by oxidative stress. Disease agents and disease processes generate additional free radicals, resulting in increased production of excitatory cytokines. This in turn promotes uncontrollable excitement in the immune response, increasing the generation of free radicals and thus oxidative stress levels. The response of the endogenous antioxidant metabolism is to increase production of antioxidants, in order to meet increased demand. For reasons which are still unclear however, the metabolism fails, resulting in diminished antioxidant synthesis. The cause of this failure must be clarified. In chapter one, we hypothesized that stress in the antioxidant metabolism may cause such failure. What is this metabolic stress and why does it occur?

38

Metabolic Stress The cell must produce various biochemicals on demand and its ability to respond depends on the competence of metabolic pathways. These pathways are sequences of events that in the healthy body lead to the manufacture of the right molecule in the right place at the right time. When our body is in a state of metabolic stress, the competence of these metabolic pathways is easily disturbed. Once consequence of this disturbance is metabolic stress. We have already explained that a system falls into stress when it is overwhelmed by the demands placed upon it. Just as oxidative stress can lead to an oxidant-antioxidant imbalance; this imbalance in turn can lead to the greater problem of metabolic stress. An Example of Metabolic Stress A well known cause of metabolic stress is hypoxia reperfusion injury a common consequence of blocked coronary arteries. The blockage cuts off the blood supply to the heart tissues, resulting in hypoxia inadequate levels of oxygen. If the tissue is reperfused replenished with oxygenated blood it consumes the incoming oxygen as desperately as we gasp for air after being under water for too long. The first part of the problem is that much of the tissues antioxidant supply has been exhausted during the period of hypoxia and it is unable to cope with even normal levels of oxyradicals, resulting in oxidative stress. The second part is that the production of cellular antioxidants suddenly increases to counteract their increased consumption. However, this production depends upon the metabolic process of antioxidant synthesis, which is overwhelmed by the demands placed on it, leading to metabolic stress. Tissue levels of antioxidants then plummet, leading to hypoxia-reperfusion injury. Thus, oxidative stress increases demand for a particular nutrient, leading to stress in its metabolism and in turn compounding oxidative stress. Metabolic Stress & Immune Suppression This idea that metabolic stress may lead to an ineffective immune response or even immune collapse is central to this chapter. This immune system is normally able to operate effectively even in the midst of free radicals because the endogenous antioxidant metabolisms synthesize enough antioxidants to counter the oxidative threat. However, this stress reaches a certain threshold when the bodys ability to synthesize endogenous antioxidants has reached its limit, at which point the antioxidant metabolism falls into a state of stress. Cytokine synthesis, membrane protection and other normal cellular functions are then impaired, leading to immune imbalance. Since metabolic stress therefore limits the ability of the immune system to operate in normally acceptable situations of oxidative stress, prevention of metabolic stress should help to maintain the immune function in spite of oxidative stress.

Conclusion Immune suppression is a result of an imbalance in the immune system rather than a simple lack of immune cells. Oxidative stress may increase the consumption of antioxidants, create stress in the antioxidant metabolism and lead to immune suppression. Experimental studies have shown that squalene-supplemented diets lead to increased performance of the immune system. This can be explained by squalenes essential roles in the protection of the biomembrane of immune cells against oxidative stress. Most importantly, the squalene metabolic process is an antioxidant metabolism. It is there for vulnerable to oxidative stress induced metabolic stress. Such stress may contribute to free radical induced immune suppression particularly in the skin. This is discussed in detail in the last chapter of the book: Metabolic Stress and Chaos. 39 40

Part 11 Squalene & Disease This part of the book describes how squalene has the potential to be used as a cytoprotective agent against cancer and as a cholesterol lowering agent. 5 -Squalenes Role in Cancer & Cancer Therapy: Carcinogenesis Cancers biological mechanism has been so mysterious for so long, that of all diseases in the modern world it is one of the most difficult to treat. During the last decade however, many of its secrets have been revealed, including its genetic evolution. It is commonly believed that healthy cells suddenly and inexplicably become cancerous and develop quickly into tumors. Nothing could be further from the truth. Cancer develops in a single cell through a process of evolution, and in most cases excepting acute childhood cancers cancer cells must struggle for years to divide and proliferate into a tiny mass of one-thousand cells. The initial development of cancer is extremely slow but once a certain limit is reached tumors progress rapidly and aggressively. This chapter describes how a healthy cell becomes cancerous and how it develops into a large mass. It also discusses the role of squalene metabolism in the evolution of cancer cells and how dietary squalene shows significant potential for cancer chemoprevention and Cytoprotection. The Evolution of Cancer Cancer is like a sleepy settlement that hardly changes for centuries, and then in a few decades suddenly grows into a town, a city and finally a metropolis that consumes the surrounding countryside in concrete, smoke, dust and noise. Cancer begins when a cell suddenly breaks away from the control systems governing surrounding tissue and looks after itself, irrespective of the needs of the body. It grows into a multicellular mass and develops its own autonomous biomechanisms that can survive attacks by the immune system and by even the most brutal radiation and chemical therapy. This development is known as the clonal evolution of cancer. As it divides and evolves, a cancer cell transfers its hereditary blueprint to the child cell. This child cell acquires all the strengths of its parent cell, most notably its ability to grow independently and to survive attacks from immune cells. This child cell itself acquires new techniques to grow more rapidly and aggressively. When it multiplies, its own child cells inherit and benefit from the parents hard earned techniques. Thus, with each cell division, the cancer becomes stronger and more resilient. Like any other living thing, only those cells that can acquire strong survival properties will make it. Even cancer obeys the Darwinian law of natural selection. Through evolutionary processes the cancer becomes autonomous,

resistant to immune attack and increasingly dangerous. The cloned child cells eventually grow into a small colony. At some point they differentiate [take on different roles] and acquire new properties. Some become invaders, some metastasize [mobilize and seek new sites to colonize] and some become specialists in the art of survival. Their environment [the body] is extremely hostile. After all, the immune system recognizes the threat, uses every weapon at its disposal and is often victorious. Cancers that survive this hostility are inherently tenacious. All but a few types of cancer develop very slowly. Breast cancer cells divide every 100 days and a one centimeter growth in diameter [about a billion cells] takes an average of nine year to evolve. Lung cancer growths reach this size in ten to twelve years. However the next step is considerably more rapid. Both breast and lung cancers progress from a one gram mass to a one kilogram mass within three years. Cancers Secret to Success Every cell in the body has the potential to become cancerous. Most of those that tend to do so inadvertently activate a gene called p53, which generally triggers their self destruction through a process called apoptosis. Alternatively, the immune system routinely detects cells with cancerous tendencies and sends macrophages to engulf and destroy them. Only an extremely small number of cells that attempt to become cancerous actually succeed in gaining a foothold. Still, even one is enough to eventually kill the whole organism. Cancers secret to success lies in the early, clonal stage of its growth. During this time it learns to survive as an independent mass by controlling its genetic expression and gaining immortality in the sense that it is no longer subject to apoptosis. Cancer cells bypass the bodys defenses by activating oncogenes [cancer genes]. Oncogenes are usually inactive in normal cells, but are relocated and mutated in cancer cells so that they are switched on. The Ras Oncogene Ras was the first and most common family of oncogenes to be discovered it is found in some 30% of cancers. Although many others have since been identified, ras remains the most dangerous and most triumphant because in combination with other oncogenes an evolved ras oncogene can transform a normal cell into a cancerous one in a single step. It also plays a somewhat central role in the activation of other oncogenes. In normal cells ras acts as a switch to trigger cell growth when conditions are right. In many human cancers, ras is hyperactivated - permanently switched on enabling the cancerous cell to grow autonomously. Oncogenes are a potent factor in cancer evolution in fact they are the backbone of cancer evolution. Without them few cancers if any would survive 41 42

the coordinated opposition of the immune system and the apoptosis control system. Cancers perverse ability to invade and consume increasingly large areas of healthy tissue distinguishes it as a powerful evolutionary force in the biological system. system. Cancers perverse ability to invade and consume increasingly large areas of healthy tissue distinguishes it as a powerful evolutionary force in the biological system. Cancer cells activate the ras gene, which in turn synthesizes ras proteins and performs ras functions. The activation of these proteins depends upon the isoprenoid metabolism in the cancer cells. Through protein isoprenylation, farnesyl anchors the ras protein to the cell membrane. Without this vital step, the ras oncogene cannot get to work. This is extremely interesting because of farnesyls place in the isoprenoid synthesis pathway and its dependence upon HMG Co-A reductase, which is regulated by squalene. A dietary supply of exogenous squalene can inhibit isoprenoid production in cancer cells and hamper their growth and development. It has been suggested that this may explain olive oils anticancer property. Several experiments have demonstrated that the anticancer property of dietary squalene. Before we recount them we will see how chemical carcinogens induce oncogene activity and the role of squalene in the detoxification of such carcinogens. Squalenes Inhibition of Cancer Proliferation When dividing and multiplying, cells go through four phases. Cancer cells are no exception. These phases are G1 [pre DNA synthetic phase], S [DNA synthesis phase], G2 [Post DNA synthesis phase], and M [Mitosis or cell division phase]. A G1 phase cell moving towards the S phase requires two products of the mevalonate pathway for protein isoprenylation geranyl pyrophosphate and farnesyl pyrophosphate. Protein isoprenylation attaches some important proteins to the cell membrane or nuclear envelope. However, the presence of exogenous squalene sets up negative feedback inhibition by down-regulating the enzyme HMG Co-A reductase, decreasing farnesyl synthesis, disrupting the mevalonate synthesis pathway and inhibiting protein isoprenylation. Protein isoprenylation is even more important in tumor cells than in normal cells, especially when the ras oncogene is hyperactivated [permanently turned on] so its disruption is calamitous to the cancer cell. By locking the cell in the G1 phase, squalene prevents cancer cell growth and proliferation. Both caution and further research are necessary. The degree of squalenes inhibitory effects varies from one cancer to another. Also, squalene has other mechanisms that may prevent cancer, apart from its role in cell growth. Carcinogenic Agents 43

Certain carcinogens [cancer causing chemicals] can activate oncogenes or cause mutations, making a normal cell cancerous and initiating the ominous evolution of clonal proliferation. Many carcinogens such as 4-[methylnitrosamino]-1 [pyridyl] 1 butanone [NNK] found in tobacco smoke target the ras oncogene. With increasing frequency, environmental pollutants and some types of radiation promote the transformation of normal cells into cancerous ones. Increased amounts and varieties of industrial carcinogens are blamed for the growing rate of cancer throughout the world. We are only just beginning to identify these substances and learning to avoid them. Squalenes Preventive/Therapeutic Potential Squalenes potential detoxification properties may be useful against chemical carcinogens. T.J. Smith and his colleagues demonstrated that dietary squalene can prevent lung carcinogenesis induced by NNK 4-methylnitrosamino-1-3-pyridyl-1butanone] and also proved squalene able to detoxify NNK in laboratory mice. Squalene has been also found to inhibit the carcinogenesis induced by TPA [12tetradecanoyl phorbol-13-acetate] a [7-12-dimethylbenz[a]anthracene]. C.V. Raos team successfully used squalene to neutralize the potent carcinogen AOM [azoxymethane]. Two properties of squalene may contribute to its anti-carcinogenic activity its separate abilities to prevent ras activation and to detoxify harmful chemicals. Any way to inhibit oncogene activation is an extremely interesting and potentially powerful weapon in the anticancer arsenal. Because it combats cancer at the earliest stages, squalenes preventive and therapeutic possibilities are extremely promising. Several research findings show that squalene and other closely related isoprenoids may play a very important role and deserve in depth investigation. Squalene has been shown to: 1. prevent the occurrence of certain cancers 2. prevent carcinogenic agents from inducing cancer 3. act directly against tumor activity 4. optimize the activity of chemotherapeutic agents. It has been found that several plant-derived isoprenoids share squalenes cancer inhibiting mechanism, and apparently exogenous isoprenoids are the most effective ras inhibitors. Although the precise mechanism has yet to be clarified, the cancer preventing action of squalene is supported by many epidemiological and laboratory findings. 44

A Growing Body of Research Several laboratory experiments have shown squalene to be anticarcinogenic. A particularly interesting article appeared in the April 1998 issue of Carcinogenesis, a peer review journal for physicians specializing in Cancer. It describes an experiment in which three separate groups of female mice were fed a diet containing 5% corn [control group] 19.6% olive oil [second group] and 2% squalene [third group], starting 3 weeks before being given a single dose of the potent carcinogen NNK. Sixteen weeks after the NNK had been given all the mice in the control group had multiple lung tumors averaging 16 tumors per mouse. The mice in the olive oil and squalene groups exhibited significantly decreased lung tumor multiplicity 46% & 58% respectively. The squalene diet also decreased lung hyperplasia by 70%. Hyperplasia is the abnormal proliferation of normal cells a first step towards cancer. A research paper published two months earlier in the same journal reported research carried out by the Nutritional Carcinogenesis division of the American Health Foundation, Valhalla, New York. Male mice on a diet enriched with one-percent squalene were exposed to AOM azoxymethane a chemical which can cause colonic aberrant crypt foci, a pre-cancerous condition of colon cancer. These mice did not develop colonic aberrant crypt foci, while the control group on a normal diet did so. These results are considered a highly significant indication of squalenes cancer preventing properties. Olive oil contains a certain percentage of squalene and several studies have been conducted in Mediterranean countries to examine the oils possible cancer prophylactic properties. Mediterranean people consume relatively large amounts of olive oil and high consumption levels have been associated with lessened risk of breast and prostate cancers among others. For many years researchers have been trying to understand why. It was first thought that oleic acid a monounsaturated fatty acid might account for the cancer protective effects of olive oil. However research by major research institutions around the world has found that oleic acid may not possess such properties. Now there is an increasing tendency to link the cancer preventive role of olive oil to its high concentration of squalene. Several laboratory experiments have found that squalene is beneficial even as an adjunct to conventional cancer treatments. Usually, cancerous cells develop a mechanism that will pump out any anti-tumor drugs. Squalene somehow promotes their accumulation in the cancer cell, making the drug much more effective. More research is underway. There are many similar research experiments. However one report deserves particular attention. Published in the October 1985 issue of the Japan Journal for Cancer Research, it describes an experiment showing squalenes ability to enhance the action of anticancer drugs. This research finding strongly suggests several major therapeutic advantages to using squalene as an adjunct to anticancer chemotherapy and radiotherapy. By inhibiting the development of drug resistance, tumors can be overcome with a decreased dose of anticancer agents. This has the two fold advantage of attacking the cancer more aggressively while causing considerably less damage to

healthy tissue a common undesirable and often dangerous side effect of conventional chemotherapy and radiotherapy. Similar research findings from several such 45 experiments are encouraging more and more physicians to include squalene in the anticancer arsenal. Potential Clinical Applications Squalenes powerful antioxidant and cytoprotective effects are very significant. Chemotherapeutic agents such as cyclophosphamide and cisplatin induce bone marrow and kidney damage by generating free radicals or enhancing the oxidative metabolism. Since it is a strong antioxidant, squalene may be able to minimize such tissue damage. Also, squalene has been found to potentiate the cancer killing abilities of some chemotherapeutic agents. In addition squalenes ability to protect cells from the effects of radiation makes it a suitable protector of healthy cells against cancer radiotherapy. Cytoprotective therapy promises to play a much greater role in future cancer treatments. Squalene in combination with some cytoprotective agents such as amifostine may bring considerable relief to cancer patients. Further clinical and laboratory research is necessary to explore such possibilities. We outline these possibilities below. Cytoprotection an Undervalued Modality The last decades have seen the development of many anticancer drugs, some of which have shown great promise. However, most of them have the short coming of producing severe side effects including the free radical destruction of bone marrow and kidney failure. Another problem is that cancer cells are ferociously adaptable and soon learn to tolerate or resist these drugs. To achieve effective results, it is often necessary to increase the dose and the corresponding side effects. This includes cellular DNA damage and mutation leading to further cancers. One emerging avenue of research focuses on substances that help our bodies tolerate anticancer drugs and radiation and/or make cancer cells more susceptible to these treatments. To be truly useful, these substances must have a differentiating action and must maximize the benefits of the anticancer agent at a minimum dose. This is called cytoprotective therapy. A good cytoprotective agent must: 1. discriminate a normal cell from a cancer cell 2. protect the former but not the latter So far a mere handful of cytoprotective agents has been developed and of these only one is considered in any way satisfactory amifostine. It has broad range of cytoprotective action against several anticancer drugs, but is not without its drawbacks it promotes hypotension and allergic reaction. It is also very poorly tolerated by children. It must be given intravenously and since it rapidly loses its effectiveness, it cannot protect against the long term accumulation of drugs in the bodily tissue. The development of a powerful and effective cytoprotective agent would be a significant advance in anticancer therapy. However the list of necessary requirements is 46

long and every potential candidate must undergo extensive laboratory and clinical testing. This chapter examines these requirements and considers squalenes qualifications as a cytoprotective agent. clinical testing. This chapter examines these requirements and considers squalenes qualifications as a cytoprotective agent. Criteria for a Cytoprotective Agent Although it primary function is not to actually attack the cancer, an ideal cytoprotective agent will promote some sort of direct anticancer action however slight as a reassurance that it does not protect cancer cells in any way. This is asking a great deal. Most early candidates for this sophist aced job turn out to either have no anticancer activity or to indiscriminately protect both cancerous and normal cells. Another factor that must be countered is the direct activity of cancer cells against pharmaceutical threats an efflux mechanism enables them to pump anticancer drugs out of the cell, while healthy cells have no such protection. An ideal cytoprotective agent should do precisely the opposite protect healthy cells from the toxicity of anticancer drugs while disarming the cancer cells self protecting mechanism. A cytoprotective agent should therefore provide selective protection of normal tissue against chemotherapeutic agents in two ways: 1. by entering more readily into normal tissue than cancerous tissue a higher accumulation in normal tissue and lesser in cancer cells is the key to selective protection 2. by decreasing the cancer cells efflux [ability to pump out anticancer drugs] the unique efflux mechanism of cancer cells may differentiate them from normal cells and enhance selective action After successful laboratory experiments a potential agent should be evaluated in human clinical trials. Experimental therapy in cancer patients may reveal a reduction of side effects to the anticancer therapy, but this is not enough. Researchers should not lose sight of the most important criterion of all without measurable tumor shrinkage; no candidate can be considered a true cytoprotective agent. Squalenes Cytoprotective Roles Six properties of dietary squalene lead us to suggest its great potential as a cytoprotective agent: 1. ANTI CANCER ACTION: Squalenes ability to inhibit protein isoprenylation prevents the unrestrained growth characteristic of cancer cells. Like many molecules with isoprenoid side chains squalene may also act as a differentiating agent, making cancer cells less dangerous by prompting them to divide normally. 2. ANTI EFFLUX ACTION: Squalene has already been found to increase the accumulation of chemotherapeutic drugs like adriamycin and bleomycin in

cancer cells by decreasing the cells ability to pump them out. This biochemical resistance is effective against a variety of drugs. The pump is built from the p-glycoprotein complex and usually expels large amounts of 47 hydrophobic compounds. Squalene is a hydrophobic compound and could potentially monopolize the cells pumping functions, enabling the anti-cancer drugs to destroy the cell before they are expelled. In this way, Nakagawa and colleagues found that dietary squalene supplementation caused cisplatin, adriamycin and bleomycin to accumulate in cancer cells. This is an important property of squalene. Research is needed to further explore these mechanisms and to determine the effective doses that produce such a result. 3. ANTIOXIDANT ACTIVITY: Many anti cancer drugs damage body tissues by generating highly toxic free radicals. Adriamycin a widely used drug generates a superoxide anion that damages heart tissues. Cyclophosphamide, another very potent and important anti-cancer drug is metabolized in the kidney into a highly toxic free radical, chloroacetaldehyde, which may generate oxidative stress leading to kidney damage. Anti cancer drugs that employ platinum cause bone marrow damage by generating free radicals. Squalene protects against oxidative stress and free radical damage. Squalenes proven antioxidant properties may neutralize these free radicals and protect normal tissue. 4. EFFECTIVE TISSUE DISTRIBUTION: The tissue distribution of dietary squalene has been studied in laboratory animals. Dr. H. M. Storm and his colleagues at the Kansas Medical Center fed mice a squalene rich diet. After two weeks the squalene concentration in their intestinal mucosa increased fifteen fold. There is every reason to expect a similar distribution in humans. This finding is extremely significant since both anti cancer drugs and radiotherapy can damage the intestinal mucosa and disrupt the cytokine network, threatening the integrity of the epithelium. The researchers concluded that squalene protects intestinal cells from high doses of radiation by increasing the cellular metabolism and thus minimizing tissue damage. 5. SAFETY PROFILE: Complementary health practitioners usually promote a dietary supplement of one to four grams of squalene per day as an anti cancer therapy. So far, no serious side effects have been reported. Generally speaking, squalene can be consumed safely as a dietary supplement in food or capsules [but it should not be drunk since this may result in accidental inhalation, leading to lipoid pneumonia]. This does not , however, mean that all squalene dietary supplements on the market are safe. Some have been found to contain PCBs and other carcinogens. In other words, squalene is safe as long as it is carefully extracted and a purity of 99.9% is maintained at every stage of production. 6. IMMUNE RESPONSE BOOSTER: Cancer induces a nonspecific immune response, increasing opportunistic infections and diminishing quality of life. Squalenes ability to protect and enhance the immune response is therefore one more advantage. 48

A Multi-Target Approach To Cancer Therapy -Target Approach To Cancer Therapy The progress of potential anti cancer treatments was for some time frustrated by our poor understanding of the generation and progression of cancer cells. Recent advances in cancer biology are helping us see cancer cells in a whole new light. Step by step cancer biologists are learning the survival secrets of cancer. Much effort is made to understand how cancer cells feed on the same glucose and amino acid nutrients as normal cells, and yet achieve such enormously successful proliferation and growth. So far we know that the hallmark of cancers success is its ability to survive even in the midst of genetic and metabolic instability. Although our knowledge of what goes on within these cells is still incomplete, we do know that they explore many avenues of survival. A new approach to cancer treatment is to target as many of these avenues as possible by natural agents such as flavonoids and isoprenoids. This multi target strategy aims to challenge cancer from as many directions as possible by targeting its metabolism, its cell signaling, its angiogenesis, etc. Cancer researchers no longer seriously expect to find a single miracle drug to defeat cancer as definitively as penicillin was able to defeat bacterial infections. Squalene contributes to at least two of these approaches. Most promising is squalenes cytoprotective activity and research is ongoing in Canada to discover its full potential. Also, it can control the isoprenoid metabolism by inhibiting HMG Co-A reductase and activating the differential pathway that pushes cancer cells to slow down and grow like normal cells. Conclusion Various epidemiological and laboratory data suggest that squalene may help prevent cancer at its outset and can also fight established tumors. These findings are consistent with the known role of squalene in the regulation of the isoprenoid metabolism. The isoprenoid metabolism in the cancer cell is highly active, and protein isoprenylation is an essential first step in the activities of oncogenes such as ras. However, exogenous isoprenoids rapidly enter cancer cells, which are very susceptible to their action. Their production of isoprenoids is inhibited as the enzyme HMG Co-A reductase is down regulated and their growth is curbed as they become more susceptible to the bodys natural defense system. Squalene may also exert its anti cancer effect through other mechanisms, notably by detoxifying carcinogens and augmenting the natural defense systems of the body. Extensive research into the link between carcinogenesis and the anti-cancer properties of squalene is imperative. Squalene may have a valuable role to play as a cytoprotective agent in cancer chemotherapy. At present, ongoing laboratory and clinical research is exploring the cytoprotective role of squalene in cancer chemotherapy 49 6 Cholesterol & Heart Disease.

Coronary Heart Disease Coronary heart disease [CHD] is a widespread disease often blamed on modern lifestyle. In the United States, the number of victims is 1.5 million per year, of whom about one quarter do not survive. An additional 150,000 people die within one year of the attack. In 1995, 40% of all deaths in the USA were due to CHD. In Britain 300,000 people suffer from a heart attach each year, of whom more than half approx 170,000 die. CHD is the leading cause of death, disease and health care spending in the modern world. In India, Brazil, China and other developing nations the death rate due to CHD is rising alarmingly and the cost of hospitalization for survivors is extremely high. The World Health Organization [WHO] warns that unless this trend is reversed the increasing costs of treating heart diseases and the rapid growth of a sedentary middle class in developing nations will rapidly erode their advances in healthcare. Governments have generously funded scientific research and scientists have made enormous efforts to identify the risk factors of CHD. Four major risk factors are known to increase the chances of coronary heart disease: 1. high blood pressure 2. cigarette smoking 3. lack of physical exercise 4. high levels of bad cholesterol in the blood During the last two decades of the twentieth century, the world of healthcare has had some success in controlling blood pressure levels. Social groups are always hard at work trying to lower smoking rates and more people than ever before though still only a fraction are excising regularly. The fight against cholesterol however remains an uphill battle. More than nine out of ten heart attacks are precipitated by the narrowing of the inner passage of coronary arteries as they are blocked by plaque that includes cholesterol deposits. Any artery can be damaged by atherosclerosis. The most common sites of damage are the blood vessels of the heart, abdomen, lower extremities and brain. Atherosclerosis The heart is a muscular pump that forces oxygenated nutrient rich blood through the arteries to all the cells of the body. These nutrients include sugar, amino acids and fats. Like any other part of the body, the heart itself requires fresh blood, which is delivered through the coronary arteries. If these arteries become blocked and worse still, the tissue may die due to lack of oxygen and the patient suffers cardiac arrest. There are two major coronary arteries, right and left. Each divides and spreads over the surface of the heart rather as the roots of a tree spread over a rock. 50

Hundreds of branches form small arteries, creating a network over the surface of the heart. Each artery is a conduit. Cholesterol is sometimes deposited in the lumen [interior] of these arteries just as silt accumulates on a river bed. The deposits of cholesterol harden, constricting the artery and reducing its flexibility. These are the mechanics of atherosclerosis. Its exact cause has still not been clearly explained, although many risk factors have been identified. Cholesterol deposition on blood vessel walls is a principal one. heart. Each artery is a conduit. Cholesterol is sometimes deposited in the lumen [interior] of these arteries just as silt accumulates on a river bed. The deposits of cholesterol harden, constricting the artery and reducing its flexibility. These are the mechanics of atherosclerosis. Its exact cause has still not been clearly explained, although many risk factors have been identified. Cholesterol deposition on blood vessel walls is a principal one. Artery walls have two layers inner [intima] and outer [media]. Atherosclerosis begins when the smooth muscle cells of the media migrate to the intima. If bad cholesterol levels rise in the blood, build up of cholesterol plaque in the intima gradually hardens and narrows the arteries. Good Cholesterol Cholesterol is widely believed by the general public to be an unmitigated evil. It is in fact vital to life an important lipid in the cell membrane structure and in nerve fiber sheaths. It is also the basic molecule for the production of certain important hormones, including corticosteroids and sex hormones. It is only harmful when having built up high concentrations in the arteries it becomes a major constituent of atherosclerotic plaque. Bad Cholesterol Medical researcher Dr. Joseph L. Goldstein awarded the 1985 Nobel Prize for Medicine following his research into the cholesterol regulatory mechanism of the liver once remarked that cholesterol is . A Dr. Jekyll & Mr. Hyde thing, because it is both necessary and a harmful agent. Dr. Goldstein discovered how the liver filters excess cholesterol from blood. He and his colleague M.S. Brown published a research article in the Journal Science, prompting the major pharmaceutical companies to develop drugs to increase the livers cholesterol filtering function. For good or bad, the liver is the bodys main manufacturer of cholesterol which is manufactured along the mevalonate pathway as in any other cell. Quite apart from this, cholesterol also enters the body from dietary source. The blood circulation transports it to the liver where it is normally filtered. However some of it leaves the liver and circulates in the blood supply. This is the cholesterol that contributes to plaque build up in the arteries and is known as bad cholesterol. It is not chemically different from good cholesterol but is associated with fats that transport it through the body differently. Cholesterol Transport Cholesterol cannot swim. Or to put it more scientifically, it cannot travel alone through the body because it is not water soluble. It is carried through the

bloodstream on a boat of fat and protein called a lipoprotein. There are three types 51 of lipoprotein high density [heavyweight], low density and very low density [lightweight]. 1. High density lipoproteins [HDLs] transport cholesterol into the liver without harming the body this is known as good cholesterol. 2. Low density lipoproteins [LDLs] and very low density lipoproteins [VLDLs] transport cholesterol out of the liver and around the body, where it is often deposited in arteries. This is known as bad cholesterol. One of the livers functions is to filter excess cholesterol from the blood. LDL receptors constitute the filtering system of the liver. These receptors [attractive doorways] protrude from liver cells and snag the LDLs, which are either put to metabolic use or if not required by the body are consumed by the macrophages that populate the liver. However if the livers macrophages cant keep up with the flow of incoming LDLs they end up back in the blood stream where they contribute to the buildup of atherosclerotic plaque. The filtering mechanism depends greatly on the feedback inhibition of HMG Co-A reductase. Researchers have found that if this enzyme is inhibited the production of LDL receptors is stepped up, thus increasing the livers filtering capacity. As a result of these findings, pharmaceutical drugs were developed to inhibit HMG Co-reductase notably a group of drugs called statins. During the nineteen nineties, three large long term epidemiological studies comparing different population groups examined the efficacy of these drugs with encouraging results. Harvard School of Medicine researchers described the positive results of the Cholesterol and Recurrent Events [CARE] study, which employed pravastatin. The other two studies the West Scotland Coronary Prevention Study {WOSCOPS] and the Scandinavian Simvastatin Survival Study [4S] showed that either pravastatin or Simvastatin can significantly reduce heart attack deaths. The success of these trials provides empirical support for the theory of cholesterol and LDL receptor synthesis. These large scale research results have encouraged doctors to prescribe these cholesterol lowering drugs in order to reduce the chances of heart attacks and to prolong the lives of heart attack survivors. However, they are expensive and their side effects are very much at issue since their effectiveness depends on long term use. A report from the United Kingdom National Health Service Center for Reviews and Dissemination criticized the routine use of cholesterol lowering drugs such as pravastatin, commenting that the intervention represents poor value for money. The report estimated that the cost of one year of life gained from taking Simvastatin is 7,240 pounds [about U.S. $11,656]. The report reflects the wider concerns of hearth service economists around the world. A huge number of people are at high risk for CHD and the costs of pharmacological prevention of coronary heart disease are simply becoming unmanageable. Patients must take statins for at least four years to derive positive results. Since the drug can damage the liver its use must be monitored during this period along

with certain blood parameters. Prolonged use can cause myopathy [muscle damage leading to inflammation], especially if dosage is raised. Newer variations like pravastatin can also damage the liver, pancreas, muscles and skin. Accounts of such negative effects have been published in various clinical journals. Statins interrupt the cholesterol synthesis pathway before the synthesis of squalene and ubiquinone [coenzyme Q10]. Perhaps at the same time that they preempt 52

the buildup of harmful cholesterol levels in endothelial cells, they also deprive them of these two vitally important endogenous antioxidants and give rise to some of the long term side effects of the drug. Indeed, it has recently been suggested that statins may actually help turn bad cholesterol into the worst cholesterol. these two vitally important endogenous antioxidants and give rise to some of the long term side effects of the drug. Indeed, it has recently been suggested that statins may actually help turn bad cholesterol into the worst cholesterol. The Worst Cholesterol of All It appears that an imbalance in the cholesterol metabolism underlies CHD and atherosclerosis. However, bad cholesterol alone is not the only culprit in CHD. When bad cholesterol is attacked by free radicals it becomes an even more harmful substance oxidized cholesterol [oxLDL] increasingly known as the worst cholesterol. This may be the greatest risk factor in atherosclerosis. And oxidized LDL may increase the proliferation of vascular smooth muscle cells [SMC] a crucial event in atherosclerosis. Lipid peroxidation of LDL leads to the MDA-modification [malondialdehyde] of oxidized LDL, which has been found in atherosclerotic lesions. The body responds by producing MDA specific antibodies in these sites and the presence of these antibodies is used to predict the progression of carotid atherosclerosis and CHD. Oxidized LDL cholesterol accumulates as plaque on the inner walls of the blood vessels in the heart and sets up oxidative stress in the affected areas. This irritates the smooth muscle cells just beneath the intima and they migrate inwards towards the lumen where they are subjected to oxidative stress by oxLDL free radicals. The result is lipid peroxidation and inflammation. In an attempt to correct the situation, macrophages accumulate, engulf the cholesterol and soon become fatty themselves [foam cells]. Instead of clearing up the mess, they become part of it. The result is a thick plaque and significantly narrowed blood vessels. Cholesterol-Lowering Effects of Squalene Dietary squalene has been found to lower cholesterol levels in blood, apparently increasing the livers filtering capacity. This mechanism seemingly derives from Squalenes ability to down regulate the HMG Co-A reductase, which in turn enhances the livers capacity to filter bad cholesterol. These laboratory findings are supported by epidemiological correlations of squalene rich olive oil consumption with a low incidence of CHD. The laboratory evidence of the cholesterol lowering ability of squalene has prompted pharmacologists to combine statin drugs with squalene. This may lead to reduced statin doses, thus reducing its side effects. At the same time, squalene may prevent the lipid peroxidation of bad cholesterol and prevent the formation of worst cholesterol. A large clinical trial conducted in Taiwan and published in the Journal of Clinical Pharmacology documented the effectiveness and safety of squalene in combination with pravastatin the statin drug used in the Harvard CARE study. 53

A double blind, placebo controlled, 20 week trial was conducted on a randomized selection of 102 elderly people, all suffering from high cholesterol levels. They received 10 mg pravastatin and /or 860 mg squalene daily, either separately or in combination. The results showed that both pravastatin and squalene effectively reduced levels of total cholesterol and LDL cholesterol, while increasing levels of HDL cholesterol. Interestingly, combination therapy reduced all bad cholesterol parameters and increased HDL cholesterol to a greater extent than either agent alone. The research paper concluded, Co-administration of pravastatin and squalene combines the specific effects of the two drugs on lipoprotein in elderly patients with hypercholesterolemia, who might have a higher incidence of side effects when using larger doses of pravastatin alone. The protective role of a diet rich in squalene [such as olive oil] suggested by some epidemiological studies may be due not only to the squalene0induced reduction in LDL cholesterol levels, but also to its role in preventing oxidation of LDL. Squalenes powerful antioxidant properties are well known but further research is expected to clarify its ability to prevent conversion of bad cholesterol to the worst type. Historical research into squalenes cholesterol-diminishing potential In 1953 D. Kritchevsky and his fellow researchers explored squalenes HDL cholesterol diminishing role in a laboratory study on four groups of rabbits on various diets for seven weeks. Their aortae were subsequently examined for signs of atherosclerotic plaques and it was concluded that, unlike cholesterol, dietary squalene did not induce atherosclerosis. In 1974, another experiment conducted by I. Prance and his colleagues found that squalene protects laboratory mice against gallstone formation. They hypothesized that squalene reduced cholesterol synthesis in the liver. In 1985, T.A. Miettinen presented findings to the seventh International Atherosclerosis Symposium in Melbourne, Australia suggesting that a squalene rich olive oil diet could effectively reduce serum cholesterol levels. In 1989, T.E. Strandberg and his fellow researchers reported that rats given 1% dietary squalene for 5 days experienced strongly suppressed [-80%] HMG Co-A reductase activity in the liver cells. In 1990, Miettinen and his colleagues published the results of another study in the Journal of Lipid Research. They fed human subjects 900mg of squalene per day for seven to ten days and produced a seventeen fold increase in serum squalene, with no significant increase in serum cholesterol level. In 1994, Miettinen and his colleagues proposed that the cholesterol reducing mechanism of dietary squalene may result from the down regulation of HMG Co-A reductase activity, leading to decreased cholesterol synthesis in the liver. The relationship between cholesterol synthesis and LDL receptor synthesis in liver cells was first proposed by Dr. Joseph L. Goldstein [1985 Nobel Laureate] who

proposed that increased cholesterol levels in the liver decreased LDL receptor synthesis. 54

Conclusion The livers filtering mechanism removes bad cholesterol from blood. Central to this mechanism is the livers LDL receptor. Synthesis of this receptor is linked to the activity of the enzyme HMG Co-A reductase, which is involved in cholesterol manufacture in the liver. By inhibiting this enzymes activity, squalene increased the bad cholesterol filtering capacity of liver cells. Squalene may also play an important role in preventing the oxidation of bad cholesterol into worst cholesterol. Further research is required to explore this potential therapeutic application in combination with existing cholesterol lowering drugs. Such use may reduce the cost and toxicity of the statin groups of drugs while simultaneously enhancing their effectiveness. 55 Part 111 Squalene & Environmental Pollution In this part of the book, we seek a model to explain the impact of pollution upon the evolutionary mechanism of our body. Readers may be surprised to encounter the term evolution in this book, but without it we cannot properly explore the impact of pollution, which in evolutionary terms has come upon us suddenly and with great force. Theodosius Dobzhansky, one of the greatest biologists of the twentieth century remarked that Nothing in biology makes sense except in the light of evolution. We ourselves are biological systems and in order to understand the impact of pollution in our bodies we must understand the full implications of this new evolutionary pressure. The arguments in Part Three depend upon an understanding of the martial presented in Part One. Human Skin: First Casualty of Ozone Depletion In this chapter, we try to identify the bodys natural protective system against UV radiation and to examine how it is affected by ozone depletion. We have already discussed oxidative stress in the immune system and its impact on squalene metabolism. Now we will apply this knowledge to the effects of oxidative stress in the protective coat of our body, comprising the skin [external surfaces]. Ultraviolet-B rays from the sun generate free radicals in exposed skin. They also convert ground-level oxygen to ozone, a powerful oxidant that attacks the internal protective coat. As the ozone layer is depleted, more UV rays reach the Earths surface, increasing these threats. Both the external and internal surfaces of the protective coat therefore suffer oxidative attack due to the diminishing ozone layer and increasing levels of UV radiation. The Epithelium A Protective Coat

The epithelium coat is a fine layer covering the skin and the lining of the mouth, throat, lungs and digestive tract and constitutes a protective coat with a surface area greater than a tennis court. These surfaces of the body are all in direct contact with environmental substances air, food and drink, plus of course, bacteria, viruses and toxins. The layer covering the outermost surface of the skin is also in direct contact with sunlight and is covered by a combination of lipids called sebum of which almost one part in eight is squalene. The inner parts of the protective coat are covered by a mucosal layer. The adipose [fat] layers beneath both skin and mucosal cells also contain a large proportion of squalene. 56

Skin contains a large number of macrophages that defend the body against outside invaders. They may be part of the original immune system developed in mammals. We have already seen that their main function is to destroy and consume incoming bacteria and viruses. They also bring a part of the dead intruder to the internal immune system a process called antigen presentation. This enables the body to recognize the antigen in the future so that it can respond more efficiently. t outside invaders. They may be part of the original immune system developed in mammals. We have already seen that their main function is to destroy and consume incoming bacteria and viruses. They also bring a part of the dead intruder to the internal immune system a process called antigen presentation. This enables the body to recognize the antigen in the future so that it can respond more efficiently. Unlike the internal mucosae, the skin is subjected to direct sunlight, which includes UV-B radiation, a source of free radicals and potential skin damage. This may help explain why sebum contains such a high proportion [12%] of squalene. If the performance of macrophages is increased by squalene, it is reasonable to assume that the substantial presence of squalene in the skin is helpful to their role, providing a protection unique in the animal world. This high concentration may be an evolutionary requirement for the defense of the protective coat against UV radiation and other outside threats. Because the skin and mucosae are constantly exposed to the outside environment, the chances of oxidative stress here are high. We know that the threat of UV-B rays is very serious, and that many environmental toxins can also initiate oxidative stress in the skin. Therefore we must ask: Does skin have a normal defense against oxidative stress and if so, Could ongoing ozone depletion overwhelm this defense? It is known that intense UV-B radiation can suppress the immune function in the skin, perhaps because of increased oxidative stress and subsequent stress in squalene metabolism. Ultraviolet Radiation Sunlight includes a wide spectrum of radiation; of which visible light is just one tiny slice. All energy from the sun moves at the speed of light and comes in waves of varying frequency. Waves of shorter length are more energetic [vibrate more frequently] than those with a longer wavelength. The wavelengths of visible light rays range from 400 to 700 nm [nanometers] and produce the different colors we see. Ultraviolet light has a shorter wavelength than visible light, ranging from 400nm to 10nm in length. It is generally divided into categories, UV-A [from 400nm to 32nm], UV-B [from 32nm to 290nm] and UV-C [from 290nm to 10nm]. UV-B and UV-C are harmful to the body. The upper atmospheres ozone layer usually absorbs all rays shorter than 310nm, blocking them from the Earths surface. Normally, only UV-A and a small portion of UV-B rays reach us. A paradox of nature is that although UV-B rays can profoundly damage skin DNA and lead to

skin cancer, small portions are actually used in the skin to synthesize vitamin D, a nutrient of great importance to normal bone maintenance. Even slight exposure of the skins surface to UV-B rays can turn molecular oxygen into singlet oxygen, which attacks skin lipids, creates lipid radicals and starts a chain reaction. Skin lipids are particularly vulnerable to such oxygen derived free radicals and these chain reactions can lead to inflammation and oxidative stress in the skin. 57 It is believed that increased UV-B radiation is responsible for worldwide increase in skin cancer. Other possible consequences may include immune suppression, allergies, memory loss, blood cancer and cataracts. On the one hand UV-B in direct sunlight damages our skin directly. On the other hand it converts normal oxygen molecules into ground level ozone free radicals that threaten our protective coat around the clock. Ozone [O3] may accumulate near the ground in polluted cities. Ozones volatile union of the three oxygen atoms make it a powerful free radical. Ozone is an irritant to the skin and can be a major source of oxidative damage to the protective coat. It may also be responsible for the rising incidence of asthma and other respiratory diseases within urban populations. The Skin The skin is the largest organ in the body and among other functions serves as a protective covering. It has an outer [epithelial] and an inner layer. The cells of the outer layer produce three important substances: 1. sebum a mainly lipid secretion of the sebaceous glands 2. keratin a fibrous protein that acts as a waterproof barrier [letting us swim in fresh water without swelling, or in salt water without shrinking] 3. melanin secreted by melanocytes, acts as an ultraviolet filter, controlling the passage of ultraviolet rays into the skin. Our discussion focuses on sebum since it coats the outermost skin surface. The total surface area of the skin is between 16 and 20 square feet. The outer layer is only 1 millimeter thick and its surface layer of fatty sebum measures about one quarter of a millimeter. Sebum keeps the skins surface smooth and moist and also serves an antibacterial and antifungal function. Absorption of UV Rays by Skin Ultraviolet rays penetrate the skin and product vitamin D without harming surrounding tissue at least for a while. However, UV-B radiation also reacts with atmospheric oxygen to produce singlet oxygen [oxygen with an extra electron] and can set up severe oxidative stress throughout the entire skin surface, Sebum is the first victim of this stress and lipid peroxidation would normally set in immediately. Our ancestors spent most of their waking hours out under the sun. Without some sort of protection, even very low doses of UV-B rays would produce harmful

consequences, and they must have developed a skin defense against UV-B rays. Human skin should have sufficient antioxidants to neutralize these free radicals while still allowing UV-B radiation to penetrate for the purpose of vitamin D synthesis. Therefore we can expect to find some natural protection in the skin against UV-B radiation. 58

The Skins Natural Antioxidant The skins natural antioxidant would have to fulfill four roles to protect the skin from UV radiation and its consequent oxidative stress: 1. It should be able to prevent or limit UV radiation induced lipid peroxidation. 2. It should not become toxic or harmful after absorbing UV rays. 3. It should not transform into a pro-oxidant [an agent that at first behaves like an antioxidant, then becomes a free radical] 4. The skin should be able to synthesize and accumulate it without any harmful effects Three isoprenoids in the skin fulfill the first criteria vitamin E, vitamin A and squalene. The two vitamins are relatively scarce in the skin, which cannot synthesize them. Squalene on the other hand is abundant [ 12 % of the sebum] and is also synthesized in the skin. In fact, squalene is one of the skins major surface lipids and is also present in underlying fat [subcutaneous tissue]. Given that it is an excellent antioxidant, this makes squalene a likely candidate to be the skins best natural protector and has led researchers to test its antioxidant properties in the skin. In a unique research project sebum was tested for its ability to prevent lipid peroxidation or to neutralize singlet oxygen. Y. Kohno and colleagues compared the antioxidant capacity of squalene with that of sixteen other fats found in skin. They showed that squalene more than any other could protect the skins surface from lipid peroxidation. They also discovered that squalenes stable molecular structure resists outside attack by peroxide radicals meaning that it does not act as a pro-oxidant. Finally, they found that squalene more efficiently recycles oxidized vitamin E than such strong antioxidant skin fats as methol oleate. Other research reports substantiate squalenes role in protecting skin from UV radiation. Unlike vitamin E, squalene is readily available and is synthesized locally. So under normal circumstances squalene is instrumental in protecting the lipid content of sebum from UV induced lipid peroxidation. In fact, the action of squalene in skin and subcutaneous fat resembles that of the light harvesting complex [LHC] in plant leaves. Both seem to prevent molecular oxygen from undergoing oxidation. Evolutionary Adaptation to Loss of Hair Compared to human beings, apes and other primates have insignificant amounts of squalene in their skin. C. Luca and his colleagues studies the skin surface lipids of nine different species of monkeys and found only trace amounts. The sebum of gorillas for example is only 0.1% squalene [one thousandth] compared to a humans 12% [almost one eighth]. Indeed, human skin secretes 125-420 mg of squalene daily. What accounts for such a discrepancy: The obvious answer is that in the process of losing our fur, squalene protected our increasingly naked skin from oxidative stress. Apart from its fur covering, the skin of nonhuman primates is much thicker, adding to

its protection against UV-B radiation. Evolution may have covered human skin with squalene for good reason. 59 However, the world we live in is changing more quickly than our evolutionary ability to adapt. Squalene and its synthetic pathway are likely suffering from increasing stress. This danger is exacerbated by the fact that the squalene synthesis pathway in skin is not only involved in vitamin D synthesis but also in effective immune response. Ozone Depletion The ozone layer in the uppermost layer of the Earths atmosphere is continuously formed by the interaction of very strong, short-wavelength ultraviolet rays [ below 100nm] with oxygen and acts as a shield to prevent strong UV rays [below 290nm] from entering the earths atmosphere. Chlorofluorocarbons [CFCs] deplete the ozone layer, thinning our protective shield and making pollution a constantly growing threat. Industrial societies have long been releasing two atmospheric pollutants CFCs and methyl bromide into the upper atmosphere. These have already thinned the ozone layer to the extent that holes are appearing in it, so more UV-B radiation is getting through to our skin. The planets surface at the end of the twentieth century was getting four times as much ultraviolet radiation as it had done a half century earlier. Scientists have found that UV rays can now penetrate non turbulent ocean water to an unprecedented depth of nine feet. This has very tangible health implication. Skin Cancer Industrialization has changed the world, and this change is mirrored in our protective coat skin and respiratory and intestinal mucosae. During the last two decades of the twentieth century the incidence of skin cancer has risen significantly around the world. Medical research has confirmed that strong UV-B rays break the molecular bonds of skin DNA, leading to mutation and significantly increasing the risk of skin cancer. The United Nations Environmental Program estimates that each 1% decrease in upper atmospheric ozone will result in a 2% increase in UV-B radiation and a 6% increase in squamous cell carcinoma one of the most common forms of skin cancer. The incidence of melanoma another common and lethal skin cancer has doubled in the United States in 20 years. This is the result of both direct ultraviolet radiation on the skin and indirect oxidative stress caused by UV induced free radicals in the air. Immune Depression Doctors worldwide are also seeing an increased incidence of allergy related disorders. Not only are viral infections more common, they are appearing on a global scale and their behavior is changing even the common cold now takes much longer to overcome than it used to. HIV and its attack on the immune system [AIDS] continues to create havoc, despite the developments of potent drugs. All of these threats have a common theme a weakened immune system. One factor known to contribute to weakened immunity is excessive UV-B radiation, which alters the 60

function of skin macrophages and promotes the secretion of inhibitory cytokines, leading to depression of the entire immune system. secretion of inhibitory cytokines, leading to depression of the entire immune system. Another source is ground level ozone, which takes up where UV radiation leaves off. Cities with highly polluted air are partially shielded from the suns rays by dust and smog, but are breeding grounds for ozone. This toxin can damage the protective coat, particularly in the bronchi and bronchioles [air passages of the lungs], causing chronic coughs, lung infections, asthma, and even lung cancer. It is also capable of generating oxidative stress which in turn leads to immune suppression. Protecting Against UV Radiation There is no realistic hope that ozone depletion will cease in the foreseeable future. The most optimistic outlook is a possible stabilization or slight reduction in atmospheric pollution within one or two generations. The burden therefore falls on medical science to protect us from its harmful effects. The first way to avoid UV triggered disorders is to minimize our exposure to sunlight something easier said than done. Almost everybody enjoys the sensation of sun on skin from time to time, and it is claimed that a sunscreen with a Sun Protection Factor of at least 15 blocks harmful UV radiation. However the effectiveness of such products is more theoretical than real. Even under ideal conditions it must be reapplied every two hours. Sweat and water not to mention towels easily displace even so called waterproof sunscreens, and exposed patches quickly appear. Researchers have therefore been searching for other protective agents. Their first thought was that topical antioxidants would help, but any product layered on the surface of the skin suffers the same disadvantages as sunscreen. In any case, UV rays rapidly consume these antioxidants and they must be reapplied even more frequently then sun blocking lotions. Nevertheless, the idea of an enhanced antioxidant defense has not been abandoned. Vitamin A and its related compounds carotenoids are known to protect skin against oxidative damage and lipid peroxidation. OF all carotenoids, lycopene provides the best UV protection but also causes abnormal skin pigmentation and may lead to a growth known as lycoma Researchers have also turned to vitamin E a natural antioxidant in the skin. However it has recently been discovered that upon exposure to UV rays, vitamin E acts as a pro-oxidant it turns into a free radical itself. So far no truly effective method exists to protect the skin from UV-B radiation. The only effective escape is to stay indoors during periods of bright sunlight and to cover the skin when outside. Similarly the best way to escape ground level ozone is to stay away from polluted cities or to wear a protective mask. However for most people these solutions are both inconvenient and socially extreme, and widespread adherence is unlikely. The development of an ointment with a combination of such natural antioxidants as squalene, vitamin E and glutathione might prove very effective but cannot guarantee protection from ozone depletion. The only

sure solution is a reduction in ozone depleting pollutants. 61 Are We Safe? The slightest exposure of any part of the protective coat to UV rays or polluted air may promote oxidative stress. The protective coat is highly vulnerable after all, its total surface area exceeds that of a tennis court. How significant might oxidative stress be over this huge surface? It depends on the extent to which oxidative stress exacerbates metabolic stress. Oxidative stress generated by even a brief period of exposure to UV radiation or polluted air might induce stress in squalene metabolism. O. Sakamoto and colleagues found that the first target of UV-B radiation in skin is squalene and that exposure of skin to UV-B radiation increases its synthesis and consumption. Considering what we know of the relation between oxidative stress and squalene metabolism in the immune system, a larger picture emerges. A UV-B induced increase of squalene synthesis and consumption in the skin may not remain localized and could spread into the general squalene metabolism throughout the body and this effect may remain even after the initiating pollutant or UV-B radiation is removed. Indeed, metabolic stress may be a nonlinear phenomenon in which biological events can product effects out of proportion toi their stimuli. Such a possibility is the subject of chapter 11. Therefore although we tend to focus on the localized effects of UB-B radiation such as DNA damage or cytokine synthesis by immune cells the internal environment of our body may be undergoing very slow, systemic, long term damage. Such gradual changes could make us a victim of Darwinian natural selection. Conclusion The presence of high concentrations of squalene in the skin presumably results from an evolutionary survival mechanism. Ozone depletion and the consequent exposure of the body to significantly stronger UV-B radiation is increasing this requirement beyond the bodys ability to cope, placing squalene metabolism in stress, with unknown consequences. New, more effective types of skin protection from ultraviolet radiation may evolve from the use of internal rather than topical substances, and their impact may go beyond mere protection of skin and help diminish the impact of general metabolic stress, which is discussed in chapter 11. The only sure solution however, is a reduction in the use of ozone depleting pollutants. 62

8 - Background Radiation - Background Radiation The Universe and the environment in which we live have always been permeated with radiation. It rises out of the ground and pours down from the cosmos. Light itself is a form of radiation. The sun and every star in the cosmos emit incalculable amounts of energy in the form of radiation. It is found in rocks and air. Even our bodies generate radiation, though negligible amounts. Some types of radiation pose much more of a threat than others and there are many each with its own wavelength and frequency. Sources of Radiation Our bodies long ago developed mechanisms to protect ourselves from the normal threat of background radiation. However our exposure to it has increased considerably in the last century since the discovery of radioactivity and mankinds use of uranium and other radioactive materials. Dozens of nuclear bombs have been exploded in the atmospheres and underground, and their radioactive residue though highly diluted, takes centuries or millennia to lose its toxicity. We are exposed repeatedly to X-rays and other forms of medical radiation. Nuclear power reactors occasionally leak small amounts of radiation. Some nuclear weapons lie disintegrating on the sea floor along with the reactors of sunken and irretrievable nuclear submarines. The storage or transport of growing piles of nuclear waste sometimes leads to leakage too. In general background radiation threatens and destroys fewer lives than the toxic by-products of burned fossil fuels, but it is one more form of pollution and it is not insignificant. Overall background radiation can only be detected with the help of sensitive equipment but it is on the rise and is harmful to living cells. Pressure placed by background radiation on the internal environment of the body may consume squalene in new ways, adding to our overall need for this protective substance. The consequences of such new requirements and stress in squalene metabolism are discussed in chapter 11. Background radiation is either non-ionizing [longer wavelengths, to the left] or ionizing [shorter wavelengths, to the right]. IONIZING RADIATION Various frequencies of ionizing radiation are so called because they ionize body tissue tear electrons away from their constituent atoms and molecules. There are several natural and man made sources of ionizing radiation. MEDICAL SOURCES Diagnostic X-ray equipment and other machines such as those used in nuclear medicine, including radiation therapy, all emit ionizing radiation. Exposure to these sources has doubled in the past 20 years. INTERNAL SOURCES Our bodies are composed of radioactive potassium and carbon, accounting for about 8% of total background radiation absorbed per year. DEPLETED URANIUM

Mankinds use of refined radioactive materials has made increased exposure to radiation a problem of our times. There are many sources, some quite unexpected. 63 For the 1991 Gulf War, for example, a new, exceptionally hard, armor casing was manufactured from depleted uranium [DU] alloy. DU is a form of nuclear waste mainly from uranium-238. The casing itself carries minimal radioactivity, but upon impact it contaminates the atmosphere with a highly radioactive and easy to inhale uranium oxide dust in particles as fine as 0.5 microns. These highly toxic armaments, designed and financed by the Pentagon, are exported around the world. The subject of depleted uranium and its radioactive nature was initially understated or hidden for obvious reasons. Since it has become public knowledge, however, it has become the subject of an emotionally charged political debate that has unfortunately made objective information very hard to come by. GEOLOGICAL RADIATION Geological radiation results from the radioactive decay of thorium and uranium radio nuclides in the Earths crust. Billions of years ago, gravitation caused the Earth to collapse into its present size, forming elements like uranium-238. Its various stages of radioactive decay have led to the present day emission of alpha, beta and gamma rays much stronger than UV radiation and able to promote cancer and immune suppression. Electrically charged subatomic particles emitted by such radiation can penetrate several centimeters into the body. Colorless, odorless radon [also known as uranium gas] originates from both uranium deposits and nuclear waste and is believed to account for about 55% of our background radiation. COSMIC RADIATION Cosmic radiation is composed of protons, electrons, neutrons and heavy nuclei from galactic sources, contributing about 9% of the total background radiation. The amount doubles with every 1500m increase in altitude. NON_IONIZING RADIATION Longer wavelength rays from UV radiation, radio waves, microwaves and infrared sources do not penetrate out body that is to say, they do not product free radicals inside the body. For example, ultraviolet [UV] rays are classified an nonionizing because they do not penetrate the body deeply like X-rays or gamma rays. However, they can ionize molecules in the skin, leading to lipid peroxidation. Infrared waves, microwaves and radio waves on the other hand, do not ionize even the outermost layers of skin. Very low frequency radio and other electromagnetic waves have been found to modulate ion flow and interfere with cellular RNA transcription and DNA synthesis, but their overall effect in humans remains unknown. Consequences of Background Radiation Our increased exposure to background radiation has both immediate and long term consequences. In the short-term we can expect an increase in all kinds of cancers, and a generally weakened immune response. The long range prognosis includes

accelerated aging and changes in psychological behavior. Some scientists have warned of decreased fertility human sperm count may be declining because of increased overall radiation exposure. 64

Those exposed to chronic radiation injury commonly experience fatigue, non restorative sleep, joint pain, weight gain, low self esteem, memory loss, rashes, headaches, allergic tendencies, asthma, urticaria, sore throats and fever. igue, non restorative sleep, joint pain, weight gain, low self esteem, memory loss, rashes, headaches, allergic tendencies, asthma, urticaria, sore throats and fever. The high energy of background radiation generates free radicals in our bodies and initiates oxidative stress. It can increase synthesis and secretion of excitatory cytokines, unbalancing the immune response. Exposure to very strong ionizing radiation, such as whole body irradiation with X-rays beyond levels of normal medical use, leads to a massive release of excitatory cytokines, resulting in severe inflammation, cell death and the ultimate death of the organism. The Radioprotective Role of Squalene Ionizing radiation can inflict either acute or chronic oxidative stress on the body, depending on its source. The antioxidant defense system, including glutathione, vitamin E and SOD [superoxide dismutase] can reduce this oxidative stress. Laboratory experiments show that squalene can protect the body against acute ionizing radiation, although its exact mechanism remains unknown. Squalenes antioxidant nature, its immune stimulant action and its ability to protect cellular structures and improve cellular repair response may all play a role. A laboratory study conducted by the Walter Reed Army Institute of Research entitled The Ability of Squalene to Protect Against Radiation Injury was submitted to the Cosmetics, Fragrance and Toiletry Association [CFTA] in February 1960. In the experiment, 20 mice were fed 2000 mg/kg of undiluted squalene 15 minutes prior to receiving 575 roentgens of X-rays. Sixty percent of the animals survived for 30 days. In a control group [mice not given squalene] only 25% survived. More recent research at the University of Kansas Medical Center, USA, confirmed the radioprotective action of squalene. Dr. H. M. Storm and his colleagues at the Kansas Medical Center published their report in the June 1993 issue of the medical research journal Lipids. Healthy mice were fed a diet rich in pure squalene and 14 days later exposed to gamma rays. Seven days later total while cell counts and total lymphocyte counts revealed that the squalene-fed groups blood count was consistently 18-19% higher than a control groups. The survival of the squalene fed mice was much higher than control fed mice [P=0.0054]. So it seems that squalene provides a type of cellular and systemic radioprotection. Squalene Depletion Due to Radiation Radiation induced oxidative stress may affect squalene metabolism with adverse results. In the human body, squalene is distributed in the fatty tissues of various organs including lungs, kidneys, spleen and brain. Strong ionizing radiation may consume the squalene content of these sites when the body suffers chronic radiation injury. In fact, during each of the experiments described above, the animals squalene levels were high before irradiation and low afterwards, confirming that squalene was consumed. People suffering from chronic radiation injury usually experience fatigue and loss of energy, perhaps due to impaired energy distribution and

65 consumption in our body. Given the link between squalene synthesis and coenzyme Q10 synthesis which is intricately linked to the energy metabolism there may reasonably be a connection between increased squalene consumption and fatigue. Radiation exposure also leads to immune suppression which may have to do with increased consumption of squalene and other antioxidants. Thus, the increased background radiation may place new pressure on squalene metabolism, the consequences of which are discussed in chapter 11. Conclusion The health hazards of chronic exposure to ionizing radiation are many. However the ways in which chronic low doze ionizing radiation are a health hazard are still not clearly known. It is likely that squalene is rapidly consumed when exposed to high levels of ionizing radiation, leading to the increased synthesis and subsequent redistribution of squalene and its immediate isoprenoids in the vital organs of the body. Moreover, squalene metabolism coincides with the metabolism of coenzyme Q10. The latter is part of the energy production process inside the cell. Therefore, by affecting squalene metabolism, chronic radiation may affect the energy metabolism in the body. There is every reason to believe that research into squalene replenishment during or following radiation exposure would be justified. 66

9 - Air Pollution, Allergies & Lung Disease - Air Pollution, Allergies & Lung Disease Each breath we take of the polluted air in todays towns and cities fills our lungs with two nasty toxins carbon monoxide and the total suspended particulate matter [TSP]. Carbon monoxide combines rapidly with blood, reducing its capacity to transport oxygen. TSP from vehicle exhaust reduces lung function over a period of time. The All-India Institute of Medical Sciences in New Delhi a city particularly afflicted by vehicle emission and other pollutants recently conducted a two year large scale study of over 100,00 people that showed a clear correlation between air pollution and emergency room admissions for asthma, bronchitis and heart complaints. The study found a 41% increase in asthma cases, 39% in chronic bronchitis, and 30% in heart attack cases over a ten year period. Many such studies have been conducted in recent decades and the link between air pollutants and allergies especially asthma is no longer in doubt. Lipid Peroxidation & Allergies Carbon monoxide vehicle emissions and ground level ozone are a deadly combination. They each act as powerful free radicals, causing lipid peroxidation in the mucosae of mouth, throat, nose and lungs. When combined this attack can lead to increased oxidative stress and an imbalance in the cytokine expression of the mucosaes macrophage cells. Without balanced immune protection, the mucosal cells then become prone to amplified allergic processes. An example of this amplification is seen when a simple normally tolerable cold requires hospital admission. The hospital admissions for asthma and other pollution exacerbated conditions have grown at an alarming rate in cities where pollution levels are particularly high. Squalene Synthesis A Potential Adaptive Mechanism Squalene concentrations in the mouth and respiratory mucosae are minimal compared with skin levels, possibly because frequent exposure to toxic air is a relatively recent evolutionary pressure on the body. In earlier centuries, there was no need for such extensive protection of the inner mucosae because air was much cleaner. The increase in air pollution may be exerting pressure on squalene metabolism in the lungs to increase the synthesis and consumption of squalene in the lung. But such changes take time. The adaptability of a biological system to acute and massive environmental pressure is quite unknown. What we do know of evolutionary change in various species has been the product of millions of years of gradual environmental change. 67

It is important to know whether squalene consumption increases in the lung mucosae after exposure to air pollutants. The long term effect of a consistent increase in Squalene consumption in the respiratory coat may be deleterious, leading to stress in squalene metabolism. In particular, it is important to understand the sensitivity of the mast and other immune cells of the respiratory mucosae to squalene metabolic stress. Conclusion The allergic response of the bodys immune system is increasing in urban environments due to the overwhelming effects of air pollutants on the lung mucosa. This may increase the consumption of and demand for squalene, leading to metabolic stress with adverse consequences. Further research is needed to explore the link between pollution induced lipid peroxidation and amplified allergic conditions such as asthma and allergic rhinitis, as well as the connection between the isoprenoid metabolism and the immune response to allergens in the lung. Research is also needed to find ways to augment the immune response throughout the internal protective coat in which squalene clearly plays such a profound role. 68

10 -The Fat Cell: A Storehouse of Toxins -The Fat Cell: A Storehouse of Toxins Fat. The word quickly brings to mind oil and grease, or the ring around your middle which just wont go away! In the medical world fat is called adipose tissue. Surgeons slice through it happily because it is soft, hardly bleeds and has no apparent function other than storage. However, medical attitudes towards adipose tissue are changing considerably. New findings suggest that fat could be regarded as an important organ. Adipose tissue has a highly organized communication network with an connection to the brain. It also synthesizes large amounts of squalene and stores it in droplet form. Notably, adipose tissue is found in the protective coat of the body and is engaged in energy metabolism. Considering what we have learned so far about squalene it seems unlikely that such rich stores of this valuable substance are there by accident. We need to understand the significance of squalene stores in adipose tissue. It is known that many organic solvents including dioxins are deposited in fat cells. These man made chemicals are called xenobiotics and several researchers have discovered that squalene enhances the elimination of xenobiotics. Squalene may play a role in this detoxification. To find out, we much investigate the potential impact of accumulated toxins on squalene synthesis in fat cells. Adipose Tissue: Depot or Organ? A depot is merely a place for storage and distribution. An organ is a coherent element of the body with an organized structure and a specific function. The heart is an organ and so is the skin. Adipose tissue has long been considered a lowly depot, but scientists are beginning to reconsider this simplistic view. It is true that adipose tissue stores and later distributes fat according to energy requirements [it also acts as a heat insulator] but it is slowly gaining recognition as a full fledged organ for some people, the largest in the body. Fat cells can grow very large, increasing their diameter by as much as twenty times and their volume by a thousand. The average person s 10 20 kg of fat stores some 90,000 to 180,000 calories. Fat cells store triglycerides long chain hydrocarbons bound to a glycerol molecule in liquid form. Triglycerides can be broken down into fatty acids by the enzyme lipase and are released when the body requires extra energy during physical exertion for example, or to compensate for starvation. Indeed, fat is by far the richest food source of energy. The new attitudes towards adipose tissue follow remarkable discoveries about the activity of the fat storage cell the adipocyte. This humble cell was thought to be no more than a storage manager until the hormone leptin [a secretion of the adipocyte] was discovered in the blood. In 1995 a research article appeared in the journal Cell. Author J.S. Flier, a researcher at the Beth Israel Research Institute, Bethesda, Maryland, described the hormone leptin in an article entitled, The Adipocyte: Storage Depot or Node on the Energy Information Superhighway. Flier was the first to recognize the high activity levels of adipose tissue and to describe it as an organ.

69 Fat cells synthesize leptin and secrete it into the blood. This hormone is just one component of a highly organized control system that regulates the long term balance of energy intake and expenditure. Obesity is generally believed to result from overeating but this is not quite true. Rather it is is a result of this lost balance when intake chronically exceeds expenditure. Some obese people believe that their bodies are more energy efficient, meaning that their internal metabolism uses less energy. This too is not quite true. Obese people in fact burn more energy than the non-obese. Adipose tissue and the brain regulate leptin secretion via an as yet unidentified control system. However, a hypothetical model suggests that leptin is an afferent loop [a loop that sends information from the periphery to the central regulatory body] of a brain controlled energy regulatory system. The discovery of leptin and the suggestion that adipose tissue is an organ triggered a multi-million dollar race as the pharmaceutical industry searched for ways to control obesity with drugs. Unfortunately, they soon discovered that obese people are leptin resistant. Although their leptin blood levels may be high, their brains become insensitive to it. Nevertheless, the discovery of leptin reinvigorated fat cell research and after the commercial euphoria died down several other surprising molecules were discovered in adipose tissue. TNF-alpha, a cytokine secreted by fat cells, can cause fatigue and increase insulin resistance in diabetic patients. This may help explain why obese people are particularly prone to diabetes. Some diabetics particularly obese ones need more insulin than others. When their requirements exceed 200 units per day, they are said to be insulin resistant. This was previously thought to result from some genetic predisposition or a decrease in insulin receptors. Now it is thought that more fat leads to increased secretion of TNFalpha, and therefore greater insulin resistance. Large amounts of adipose tissue are also thought to contribute to high blood pressure. Sufferers are often prescribed angiotensin converting enzyme [ACE] inhibitors that effectively reduce blood pressure by limiting production of angiotensin 11 a peptide synthesized in the lungs from angiotensionogen and secreted by the kidneys. It is now known that fat cells too secrete angiotensionogen. Previously the kidneys were believed the only source. Since increased adipose tissue has been associated with an increased circulating level of leptin and angiotensionogen, the links connecting obesity, diabetes and hypertension are becoming clearer. Fat cells also maintain large stores of squalene. The average persons 10 20 kilograms of fat contain 5 10 grams of squalene, 90% of which is stored unchanged by the fat cell only 10% being converted to cholesterol. Of the stored portion, fourfifths are maintained as liquid droplets within the cell and the remainder is bound to the cell membrane. However the function of this stored squalene remains unknown. Toxin Accumulation Many man made toxins enter the body and accumulate in adipose tissue without ever being eliminated. The discovery of leptin may have made the fat cell an

object of renewed respect among obesity researchers, but environmental scientists still 70

view it as a reservoir of dioxins, PCBs and other xenobiotics, including natural compounds, drugs, environmental agents, carcinogens and insecticides. luding natural compounds, drugs, environmental agents, carcinogens and insecticides. Some xenobiotics act as pro-carcinogens. Some mimic estrogen receptors. Some are not inherently harmful but become toxic following biotransformation within the body. For example, the liver enzyme system cytochrome P450 turns some xenobiotics into dangerous carcinogens. Increased deposits in adipose tissue of dioxins, dibenzofurans, polychlorinated biphenyls [PCPs] DDT, DDE, hexachlorbenzene, alkylphenols, and several organochlorine pesticides have recently been associated with testicular cancer, lymphoma, leukemia, prostate cancer, malignant melanoma and endometrial cancer. The many ways in which xenobiotics can harm our bodies have been only partially uncovered. Most man-made chemical compounds are lipophilic they are irresistibly drawn to fat. The liver breaks down xenobiotics substances into smaller molecules in an attempt to eliminate the, but some end up more toxic, not less. Detoxification By the Liver The detoxification processes of the liver have two phases. In phase 1, the toxic molecules are modified by oxidation, reduction, hydroxylation, methylation and other biochemical processes. The reactions are carried out with cytochrome P450, glutathione S-acyltransferase, and other molecules synthesized by the liver cells. In these reactions, drugs such as diazepam are inactivated. However, the same chemical reactions may activate some other drugs such as prednisone, which is activated to prednisolone. The phase 1 detoxification process can render some molecules of relatively low toxicity even more dangerous. For example, the tuberculosis drug isoniazid may cause hepatic failure. Inactive carcinogens are sometimes converted to active carcinogens by the same process. The phase 11 reaction attempts to render fat- soluble molecules water-soluble so they can be more easily excreted through bile or urine. Unfortunately, the liver is unable to eliminate all toxins. Xenobiotics that do not submit to this process are carried by the blood to the adipose tissue and stored in fat cells. There they accumulate, reach toxic doses and overflow into surrounding tissue where they may act as carcinogens. It has even been suggested that the increased incidence of breast cancer may result from the accumulation of xenobiotics in breast tissue. Detoxification By Adipose Tissue An organisms ability to detoxify itself is a crucial step in its evolution. Given the significance of adipose tissue where xenobiotics accumulate the question arises, does adipose tissue have its own detoxification system? There is good reason to believe that squalene stored in fat cells may act as a detoxifying agent. Four independent researchers have tested the detoxifying abilities of

squalene by measuring the extent to which squalene helps cleanse the bodies of laboratory animals of xenobiotics. Results have been encouraging. Squalene-rich diets 71 leading to increased squalene blood levels do seem to improve the elimination of organochlorine xenobiotics such as hexachloro-benzene [HCB] and hexachlorobiphenyl [6-CB]. Other experimental studies also suggest that squalene may be a useful antidote to drug overdosing. Oral squalene seems to enhance the elimination of theophylline, Phenobarbital and strychnine in rats. Although its detoxifying mechanism is still not clearly known, it is thought that squalene may possibly increase the mobilization of lipid soluble xenobiotics enabling elimination through the intestine. T.J. Smith and colleagues suggested that squalene may exert its detoxifying effects by stimulating the bodys central detoxification system in the liver [cytochrome P450]. Fat cells also contain cytochrome P450 so the question naturally arises of whether the squalene stored in fat cells also has a detoxifying function. There is more to squalenes detoxifying activity. When xenobiotics accumulate in fat cells, stored squalene may be released into the general circulation, stimulating bile flow and enhancing xenobiotics elimination. Scientific testing of this hypothesis could lead to the development of effective new means of detoxification. The Xenobiotic-Squalene Link Xenobiotics stored in fat cells may alter the fat cell metabolism. One way to explore this possibility would be laboratory studies on the sensitivity of squalene metabolism to various xenobiotics. Since squalene metabolism is found to play an important role in the cytokine secretion of immune cells, it may also take part in the cytokine secretion of fat cells. Indeed, the accumulation of xenobiotics may incline the isoprenoid pathway to either increase or decrease squalene synthesis. This could alter the way fat cells secrete cytokines such as tumor necrosis factor [TNF]-alpha an excitatory cytokine. TNF-alpha is known to sometimes cause fatigue, lethargy, fever, and increased insulin resistance in diabetics. Two disorders chronic fatigue syndrome [CFS] and fibromyalgia are commonly found in populations living a modern lifestyle. CFS is characterized by increased secretion of TNF-alpha, perhaps from fat cells. Studying the relationship between squalene metabolism and various xenobiotics may help clarify the link between environmental pollutants and modern illnesses. Most importantly the accumulated xenobiotics stored in fat cells place additional stress on squalene metabolism and are yet one more factor contributing to overall metabolic stress. Conclusion When considered as an organ, adipose tissue is responsible for maintaining the bodys energy balance by producing and secreting leptin an important hormone in the highly organized energy control system. It is probably that fat cells must remain toxin free for optimal functioning and the considerable squalene store of the fat cells may help keep them so.

72

11 -Metabolic Stress & Chao-Metabolic Stress & Chaos What is the fate of dietary squalene? Where does it go? Is it converted into cholesterol? And why does skin contain such high levels of squalene? Why is it not converted into cholesterol? As they attempt to answer these questions, scientists are slowly discovering another side of squalene research its metabolic response to pollution. The increased synthesis and consumption of squalene in skin following UV exposure is one illustration of this. However metabolic response may eventually fall into a state of stress. When does metabolic stress occur and how does it affect the immune response? [These questions came up in our chapter 4 discussion of the negative influence of oxidative stress on effective immune response]. The existence of a state of disequilibrium makes squalene metabolism an ideal place to seek answers to these questions. We will first elaborate on the concept of metabolic response to stress, then briefly discuss the physiology of squalene metabolism, and finally discuss the mechanism of stress in squalene metabolism. Metabolic Response to Stress We believed that an understanding of stress in antioxidant metabolism is best approached by examining the metabolic response to stress. In many cases, such as a response follows oxidative stress. It is well known, for example, that surgery causes oxidative stress. Recovering patients tend to lose weight for a week or so afterwards, even when fed above normal levels of good food. The body responds to surgical stress by burning more protein and fat in an attempt to accelerate healing. This normal metabolic response to stress is perfectly understandable and is generally helpful to the patient. However, this response may cross a threshold at which point the metabolic response to stress becomes itself a source of stress. Positive feedback causes the metabolic response to continue in an uncontrolled way damaging the body. This is metabolic stress. The consequences of metabolic stress have been well studied in the context of protein energy malnutrition and prolonged fasting. In both cases, doctors observe accelerated tissue damage, wasting and even immune suppression actually induced by the over responsive protein energy metabolism. Hypoxia reperfusion injury is one example of metabolic response to oxidative stress. Increased squalene synthesis during UV exposure to skin is another. Thus, the metabolic response to stress is an event of the early adaptive mechanism. It is not itself a state of stress. Only when the response reaches a certain threshold does the adaptive mechanism fail and give way to stress. Once that threshold is passed, antioxidant levels decline in spite of increased antioxidant synthesis. We will try to understand the threshold concept in antioxidant metabolism by examining the physiology of squalene metabolism.

Squalene Metabolism To identify stress in squalene metabolism, we must consider how squalene metabolism is related to the cholesterol metabolism. As a precursor of cholesterol, squalene was commonly believed by scientists to be entirely converted to cholesterol. The suggestion of an independent squalene metabolism is new, and based on research findings that only a very smell portion of dietary squalene is actually converted. The rest remains either unchanged or converted into some metabolite other than cholesterol. Various human tissues maintain separate squalene concentrations, as follows: 100mcg/dl in plasma, 500mg/kg in adipose tissue, 1g/kg [dry weight] in skin and 50mg/kg in liver. Increased dietary intake of squalene can increase these tissue concentrations by several times their normal values. The question remains, how does tissue maintain its own squalene concentration and why is this squalene not converted into cholesterol? By discussing squalene metabolism in skin and fat we present evidence that squalene metabolism is independent of the cholesterol metabolism and explain why different tissues maintain independent squalene levels. Squalene in the Skin Squalene is found abundantly in skin, where it acts to protect against free radicals. When stimulated, synthesis of squalene in the skin increases independently of cholesterol synthesis, suggesting that an independent squalene metabolism exists in the skin. Both cyclic and acyclic forms of squalene are present in skin. Acyclic squalene serves as a potent antioxidant and cyclic squalene is used in the synthesis of vitamin D, cholesterol and other sterols. Squalene in Fat Tissue Fat cells have an entirely independent squalene metabolism. The acyclic squalene remains linear at all times and only 10% is transformed into its cyclic form. This raises the question of whether there is an acyclic squalene metabolism separate from the cholesterol metabolism and is supported by the phylogenetic [evolutionary] evidence that the linear squalene metabolism in fat cells is an unchanged descendant of this archaic squalene metabolism. Perhaps the skin too retained that metabolism even after is acquired a cholesterol [cyclic squalene] metabolism. Experimental evidence suggests that squalene bound in the biomembrane of the microsome [a cellular organelle] is metabolically active and that approximately 90% of the newly formed squalene is stored in a lipid droplet and only 10% is used in cholesterol synthesis. This suggests that 90% of membrane-bound squalene may remain in its active, linear form. 74

Cyclic & Acyclic Squalene Acyclic squalenes history in metabolic processes began with archaea, the ancient life form that lives on in deep sea volcanoes under extreme pressures [200 atmospheres] and temperatures [85deg celcius]. Archaes biomembrane is a teeming soup of biomolecules that includes isoprenoids and acyclic squalene molecules. We have seen that although vitamin E has been considered one of the most powerful terminators of lipid peroxidation chain reactions, its large size limits its ability to fit into the biomembrane. Acyclic squalenes excellent antioxidant properties make it as effective as vitamin E, but because of its small size and mobility it encounters far more lipid molecules and probably neutralizes more oxyradicals than Vitamin E. This is probably why archaes biomembrane contains squalene and not vitamin E. As we have noted earlier, both skin and adipose tissue contain acyclic squalene. Is this acyclic squalene ever rendered into cholesterol? Perhaps the body somehow determines which portion fo squalene becomes cyclic and which does not. These are very big questions to which the answer at the moment is a resounding Dont know. However, the presence of two distinct forms of squalene in our body tissues strongly suggests that squalene metabolism takes place in two metabolic pools one cyclic and one acyclic. Two Metabolic Pools Our hypothesis that squalene metabolism is independent of the cholesterol metabolism is first supported by evidence turned up at Rockefeller University where in 1974 K. Liu and his colleagues investigated the squalene content of the body. The squalene content of the body was measured back in the days when it is only known role was as a cholesterol precursor. However, the Rockefeller University team found that barely one-tenth of plasma squalene is actually converted into cholesterol. They referred to it a active squalene in contrast to the approximately 2.6grams of so called inactive squalene that mysteriously does not. To identify these two squalene stores in the body we refer to them as separate pools. The smaller squalene pool is metabolically transient because it proceeds on its way down the cholesterol pathway. The other is metabolically stable because it does not. It is tempting to think that the transient pool is cyclic squalene and the stable pool is acyclic. The transient pool can also be considered inactive, as it is rapidly converted into cholesterol. The stable [acyclic] pool can be considered active since it provides the antioxidant function. Most dietary squalene is probably acyclic and therefore active. We have shown that both skin and fat contain very high levels of active [stable] squalene that is not converted into cholesterol. The active squalene concentration in skin is about 1g/kg dry weight of skin. However, skin also contains a very small amount of inactive [transient] squalene which is rapidly converted into cholesterol. We have also shown that there are two squalene pools in fat tissue. This distinction between active and inactive pools suggests the significant possibility of squalene metabolic stress. 75

State of Disequilibrium So far we have found that active squalene pools [ASPs] exist in the skin and in fatty tissue for specific functions, such as protection of skin from UV damage. We hypothesize that ASPs also exist in immune and other cells. The discussion in the first part of the book reveals squalenes independent role in biomembrane protection. It seems natural that an independent squalene metabolism exists in the cell to control the cellular distribution of squalene and requires an ASP. It is thus likely that ASPs exists even at the cellular level. ASPs may also exist in the liver as hepatic cholesterol synthesis is affected by dietary squalene. Of the four active squalene pools, each one has its own level of active squalene. The skin pools contain about 1g/kg, adipose tissue contains about 500 mg/kg, the body as a whole holds about 20mg/kg in individual cells and the liver contains about 50mg/kg. The body contains a total of about three grams of active squalene. Its inactive squalene pool [destined for conversion into cholesterol] adds up to about 300mg. There is thus a state of disequilibrium between the active and inactive squalene pools [3gm to 300mg]. The force maintaining this disequilibrium is crucial to the existence of the active squalene pool. Without it, all active squalene would be converted into cholesterol. K. Liu and colleagues were surprised to discover such a state of disequilibrium. It may be visualized as a force that keeps a liquid in the two arms of a U-tube at different levels. The Mechanism of Metabolic Stress A state of disequilibrium similar to that among the squalene metabolic pools may be common to all endogenous antioxidant metabolisms. It is well known that oxidant and antioxidants are never in a state of equilibrium. An example is the ratio of reduced to oxidized glutathione [GSSH: GSSG] in cells and tissue. The value never settles down to 1:1 This state of disequilibrium suggests the possibility of stress in the antioxidant metabolism. If sufficient liquid is added to one column of the tube, the state of disequilibrium diminishes and difference in levels will fall. Similarly, when antioxidant synthesis reaches a certain threshold, the disequilibrium will be disturbed, leading to rapid consumption of antioxidants. Thus, the very existence of a state of disequilibrium makes the antioxidant metabolism vulnerable to stress. In the U-tube example, the magnitude of the force of disequilibrium determines how much liquid must be added to column A to upset the disequilibrium. Similarly, the performance of the adaptive mechanism determines the threshold at which stress is provoked in the antioxidant metabolism. In the squalene metabolic pool, metabolic stress would occur when the compensatory or adaptive mechanism fails to maintain the state of disequilibrium. What is this compensatory or adaptive mechanism? How does it operate on the squalene metabolic pool? When and why will it fail to maintain the state of disequilibrium? To seek an answer to above questions, we turn to the chaos theory. 76

Chaos Biological systems are organized differently from mechanical systems. They are chaotic, but not disorganized. Chaotic organization can fluctuate, enabling biological systems to adapt to new situations quickly. Chaos theory grew out of a need to explain phenomena that could not be explained by linear dynamics, in which an effect is proportional to its cause. Many bodily processes such as the normal variations of a heartbeat can only be well described by nonlinear dynamics [NLD]. Nonlinear dynamics begins by setting aside the concept of proportionality. The causal components of an nonlinear system form a network with multiple interactions, so a small change in a system can have large, sometimes unanticipated consequences. The complex fluctuations in the heart rate of a healthy individual are a typical example. Contrary to the expectations of linear thinking, the beat of a diseased heart one for example suffering congestive cardiac failure is less chaotic than a healthy heartbeat. It seems that disease states render the biological system more predictable. Metabolic Stress & Chaos It seems most likely that the squalene metabolic pools are organized in a nonlinear fashion. As an example of chaotic organizations, even the small metabolic pool of an individual immune cell mirrors that of a large one. All metabolic pools, no matter how large or small, maintain a state of organized disequilibrium. This organization is not dissimilar to the computer generated leaf, in which even the smallest details always resemble the largest. This non-linear organization of all squalene metabolic pools enables the adaptive mechanism to effectively maintain the state of disequilibrium so that under normal conditions metabolic stress is preempted. However, the generation of free radicals for example by the action of UV radiation on skin stimulates squalene synthesis. When this synthesis passes its threshold value, the compensatory mechanism fails and leads to metabolic stress. Chaos theory provides a different perspective from the mechanistic explanation of metabolic stress, suggesting it to be a non-linear phenomenon determined by the bodys adaptive mechanism. Metabolic stress occurs only when the adaptive mechanism fails. Chaos theory also predicts that disease states will render biological systems both more predictable and less adaptable. Adaptive mechanisms fail when our systems fall sick. During normal immune response, the antioxidant metabolism of the adaptive mechanism maintains the state of disequilibrium until it reaches it s threshold, thereby minimizing the chances of metabolic stress. But when an existing condition or repeated oxidative stress pushes the immune system over the antioxidant threshold, the normal state of disequilibrium fails and metabolic stress follows. This nonlinear account of metabolic stress satisfactorily explains oxidative stress induced immune suppression described in chapter 4. Now we must ask, can pollution induced generation of free radicals cause squalene metabolic stress in the bodys protective coat. The discussions in chapters 7 10 strongly suggest that rising levels of oxidative stress in our environment due to ozone depletion, increased background radiation and accumulation of xenobiotics

77 put tremendous pressure on the squalene metabolism in the protective coat. An incident of metabolic stress on the protective coat may be transient, but it may also be sudden and intense, - especially considering the huge surface area of the coat [more than 400sq.m]. We introduced part three of this book as a search for a model with which to study the impact of pollution in the evolutionary mechanism of the body. Bearing in mind that evolution is no more than an extension over many generations of the greater adaptive mechanism of the body, we have proposed that metabolic stress puts pressure on the adaptive mechanism implying that squalene metabolic stress would also put pressure on our evolutionary mechanisms. Thus, the evolutionary presence of squalene in skin makes squalene metabolism in suitable model to study the long term impact of pollution in the body. Conclusion The existence of two squalene pools in the body is a special characteristic of squalene metabolism. They are maintained by some force of disequilibrium. Environmental pollution may create metabolic stress in squalene metabolism, disrupting the disequilibrium and resulting in chaotic fluctuation of squalene and other isoprenoids. Such fluctuation may produce gradual but significant changes in our body, mainly in the protective coat defense system. We have already suggested that the high concentration of squalene in skin and fat cells is probably due to the accumulated evolutionary requirements of thousands or millions of years. In contrast, the present change in our environment is both sudden and enormous, with potentially disastrous results. Squalene metabolism in skin may serve as a useful model to study the impact of pollution in the evolutionary dynamics of health and disease. 78

Epilogue Geological and evolutionary evidence shows that life on this planet has developed a profound dependence upon antioxidant isoprenoids such as squalene for protection against the rigors of the environment. Homo sapiens the most sophisticated product of evolution has not outgrown that dependence. The thick coating of squalene on our skin is an example. It appears that without squalene we could not have shed the furry outer coat still worn by our fellow primates. This simple biochemical has played a crucial role in the protection of living systems for billions of years. Now however, we have inadvertently changed the environment in ways that are taxing our protective systems, perhaps pushing them to their very limit. Our voracious industrial societies have produced and propagated countless carcinogens and other pollutants, diminished atmospheric protection against ultraviolet radiation, spread new sources of terrestrial radiation and exposed our bodies to previously unknown chemicals that increase the chances of oxidative stress in the body. The greatest threat to terrestrial life is the speed at which these changes are taking place. The marvelous ability of biological systems to adapt is now profoundly challenged. We cannot reverse the damage we have done. Nor can we realistically enumerate the various threats and devise a pharmaceutical remedy for each one although sometimes it seems that is exactly what we are attempting. What we can do is to study our inner protective mechanisms and find ways to help them resist the oxidative pressure of this sudden and potentially catastrophic environmental change. Squalene and its metabolism in the skin may serve as a model for such studies. 79 Glossary Acetone [Ch3Coch3] Compound with solvent properties and characteristic odor; obtained by fermentation or synthetically Acetyl Co-A A compound from which many other vital substances are derived; derived from sugar in our cells Acyltransferase Enzyme involved in the antioxidant activity of glutathione Adaptive Mechanism Control mechanism of the body that adapts to new situations, environmental changes

or generalized threats. Adipocyte [Fat cell] Chief constituent cell of adipose [fat] tissue Adipose tissue Bodys fat deposits where triglycerides are stored until needed to provide energy; also provides heat insulation for the body. Afferent Signal pathway from the periphery to the center of the body; for example touch provokes an afferent signal Aids [Acquired immune deficiency syndrome] A deficiency in the immune system due to infection by the human immunodeficiency virus [HIV] Alkylphenol Toxic industrial detergent that promotes endocrine disrupting effects Allergen Agent that may trigger an allergic reaction in the body; for example, dust particles, pollen, etc Allergy Altered response of immune system to an allergen Alpha Particle Elemental particle composed of two protons and two neurons; alpha particles have very strong ionizing power but cannot penetrate the body easily Alpha-Tocopherol Chemical name of vitamin E Amaranth Plant found in North and South America and in Asia; rich in squalene Amifostine Agent used in cancer treatment to protect normal tissue from the damaging effect of chemotherapeutic agents. Amyloidosis A common complication of several diseases [leprosy, tuberculosis], often associated

with immune disorder Anaplasia Process of transformation of less cancerous cells to highly cancerous cells; degree of malignancy 80

Angiotensin converting enzyme [ACE inhibitor] Potent antihypertensive drug used to treat high blood pressure by converting angiotensin to angiotensin 11 Angiotensin 11 Potent blood pressure increasing agent, used by the body when kidney blood flow decreases; patients experiencing high blood pressure often suffer increased levels of level of angiotensin and angiotensin 11 in the blood Ankylosing spondylitis A polyarthritis involving the spine, characterized by progressive, painful stiffening of the joints and ligaments that almost exclusively affects young men Antibody Protein molecule produced by specialized cells when encountering an antigen; antibodies remember and act specifically against the antigen which originally provoked their synthesis Anti-carcinogen One of three types of cancer fighting agents; those that prevent chemical precursors forming carcinogens, those that prevent carcinogens from reaching or acting on target sites and those that suppress the expression of neoplasia in cells already exposed to carcinogens Antigen presentation Process by which lymphocytes are made aware of a particular antigen Antioxidant Molecules able to counteract the damaging effects of free radical induced oxidation by donating an electron to neutralize free radicals Antioxidant metabolism Cellular and tissue synthesis, maintenance and recycling of antioxidants AOM [Azoxymethane] Chemical that can cause colonic aberrant crypt foci a precancerous condition of colon cancer Apoptosis control system Hypothetical control system that regulates programmed cell death Archae

An ancient form of life, formerly considered a type of bacteria but now considered a separate and distinct evolutionary branch, found in deep sea hot springs and other unusual habitats; along with bacteria and eukarya, one of the three domains of life while archae resemble bacteria in morphology and genetic organization, they resemble eukarya in their method of genetic replication. Asthma Lung disorder characterized by airway obstruction and recurrent shortness of breath due to spasmodic contraction of the lungs airways. Atherosclerosis Thickening and hardening of blood vessels resulting in narrowed lumen and obstruction of blood flow Atom Smallest particle of an element; a positively charged nucleus orbited by negatively charged electrons 81 Atopic dermatitis An allergic, inflammatory skin disorder resulting in an itchy rash Auto-antibody Antibody formed in response to and reacting against a constituent of an individuals own tissues Beta Carotene Group of antioxidant carotenoids found in plants Biomembrane Membranous envelope of a living organism such as that enclosing a cell or organelle Biotransformation Chemical alteration of a substance by or in a biological system Bronchi The larger air passages of the lungs starting at the point where the trachea branches into two Bronchiole Smaller airway of the lungs connecting bronchi to air-sacs [alveoli] Cachexia Profound and marked general ill health and malnutrition Cancer Malignant cellular tumor

Carbohydrate Compound of carbon, hydrogen and oxygen, e.g. cellulose, sugar, starch Carbon Dioxide [CO2] Odorless, colorless gas produced by oxidation [burning] of carbon; naturally formed in animal tissue and eliminated by the lungs, or produced by the burning of fossil fuels Carbon Monoxide [CO] Odorless, colorless gas produced by burning carbon or organic fuels in a low oxygen environment, when inhaled, prevents blood from absorbing oxygen and quickly leads to death Carcinogen Substance able to transform normal cell into cancer cell Carotenoids Plant derived substances belonging to the isoprenoid antioxidant family, such as vitamin A Catalase Enzyme that catalyzes decomposition of hydrogen peroxide: found in most cells Cataract Partial or complete opacity of the lens of the eye, impairing vision or causing blindness Cell Cycle Proliferation Proliferation of cells by division into two identical new cells Cell Fundamental structural and functional unit of living organisms, consisting of a nucleus and cytoplasm enclosed in a plasma membrane Cellular Defense System System that protects cells from environmental toxins, free radicals, drugs and various other noxious agents 82

Cellular Homeostasis Dynamic balance of the internal environment of the cell Dynamic balance of the internal environment of the cell Cellular Oxidative Distress Inability of cellular microenvironment to maintain oxidant-antioxidant balance due to intense generation of free radicals inside the cell Cellular Redox System System enabling cells to carry out oxidation-reduction reactions and consisting of special redox molecules to keep the oxidant-antioxidant ratio in balance Cellular Pertaining to structure or system of a cell CFC [Chlorofluorocarbon] Type of hydrocarbon containing both chlorine and fluorine, used as refrigerants, blowing agents, cleaning fluids, solvents and for fire extinguishing CFCs are known to cause ozone depletion Chaos Theory [Non-linear Dynamics] Study of systems that respond in a nonlinear way to initial conditions or perturbing stimuli; fractal [non-linear] representations of chaotic systems often reveal similar but nonidentical patterns across varying scales of time and space Chemical Quenching Reaction in which a free radical is chemically incorporated with a neutralizing antioxidant by the sharing, rather than the exchange of an electron Chlorophyll Green pigment in plants that harnesses light energy making water and carbon dioxide react to produce oxygen and glucose Chloroplast Chlorophyll-bearing bodies of plant cells Cholesterol Regulatory Mechanism Mechanism that controls the synthesis and distribution of cholesterol in the body Cholesterol Waxy substance used in construction of cell membranes and synthesis of steroid

hormones; also a precursor of bile acids; mostly manufactured in the liver but also partially absorbed from diet Chromanol Ring Aromatic ring structure; main backbone of vitamin E Chronic Fatigue Syndrome [CFS] Long term [six months or more] affliction characterized by persistent or recurrent fatigue, diffuse musculoskeletal pain, sleep disturbances and subjective cognitive impairment Chronic Radiation Injury Harmful effects of long term exposure to ionizing or non-ionizing radiation Cirrhosis of the Liver Damage, scarring and subsequent hardening of the liver Clonal Evolution Development of genetically identical cells descended from a single ancestral cell by division and multiplication, during which daughter cells acquire new properties through natural selection; used especially regarding development of cancer mass 83 Co-Enzyme Q Ubiquinone, a quinone with isoprenoid side chains found in mitochondria and involved in energy production Colon Cancer Tumors or cancer of the large intestine Colonic Aberrant Crypt Foci Change in normal structure of the epithelial coat of the large intestine; a precancerous sign Control Group A group of subjects used in a test but not undergoing test conditions; used to produce normal data for comparative purposes Control System Operating system that navigates and commands internal body processes Coronary Artery Either of two arteries that carries oxygenated blood to the muscular tissue of the heart

Coronary Heart Disease Disease resulting from a blocked coronary artery, usually due to atherosclerotic plaque Corticosteroid Group of hormones that regulate various functions of the body including the energy metabolism, healing and stress response; corticosteroids are involved in growth, development and bodily vitality Cosmic Radiation High energy radiation of particles originating from extraterrestrial space Crohns Disease A chronic inflammation in the digestive tract; similar to but more severe than ulcerative colitis Cysteine A sulfur-containing amino acid; scarcest of the three constituents of glutathione Cytochrome P450 Type of enzyme used in biotransformation of many foreign compounds Cytokine system Component of the immune system that regulates messaging among immune cells Cytokine Protein mostly secreted by immune cells to regulate immune function by inducing inhibitory and excitatory activity of other immune cells Cytoplasm Substances other than nucleus, mitochondria and chloroplasts that make up the cell body Cytoprotection Process by which chemical compounds protect cells from harmful agents Cytoprotective Therapy Use of substances that protect normal tissue from harmful effects of disease processes or aggressive therapies used to combat them, such as anticancer radiation or chemotherapy Cytotoxin

Substance toxic or harmful to cell and its function DDE An organochlorine pesticide ethylene metabolite of DDT 84

DDT A polychlorinated pesticide resistant to destruction by light and oxidation and believed to be carcinogenic Dementia An acquired organic mental disorder with significant loss of intellectual abilities Depleted Uranium Radioactive by-product of nuclear weapon manufacturing and reactor use Dibenzofuran Group of ether compounds related to dioxin and PCBs, used by petrochemical, pulp/paper and many other industries Differentiating Action Drug action pushing a cancer cell towards its normal behavior and function; eg. Vitamin A can push nerve cancer cell to become a normal nerve cell Dioxin Man made chemical found to act as a persistent carcinogen Disease progression Progressive damage to the body tissue by a disease process DNA Synthesis Cellular production of DNA Dolichol Derivative of the mevalonate pathway used for carbohydrate synthesis Down-Regulation Process on cell surfaces that decreases interaction with incoming biochemicals by reducing the number of available receptors Drug Resistance Inherent or acquired ability of a disease process to resist effectiveness of a therapeutic chemical or drug Efflux

Pumping mechanism of cancer cells by which they expel anticancer drugs; movement of drug from interior to the exterior of the cell Electrical Channel Special doors in a cell wall guarded by proteins and charged with a specific voltage; only specific ions are allowed to enter through such channels, hence sodium channel, calcium channel etc Electron Imbalance The lack of an electron in an atoms outer electron ring, such that it seeks a replacement; usually the result of free radical damage Electron Transfer Reaction Chemical reaction within a special molecule involving the transfer of electrons from one molecule to another Electron Negatively-charged particle orbiting the nucleus of an atom; the number or orbits and the number of electrons in the outer orbit determine all the atoms physical and chemical properties except mass and radioactivity Emit To liberate, to give off 85 Endogenous Anti-Oxidant Antioxidant synthesized within the body, including glutathione [GSH], coenzyme Q10 catalase, superoxide dismutase [SOD] and squalene Endogenous Produced within or caused by factors within an organism Endogenous Antioxidant Metabolism Metabolism of antioxidants synthesized within the body e.g. glutathione and squalene Endometrial Cancer Tumors or cancer of the inner lining of the uterus Endometrium Mucous membrane lining of the uterus

ENE Reaction Hydrogen atom donation and reception in a biochemical reaction Entropy Tendency of all physical systems to fall into disorder Environmental Pollutant Substance present in high enough concentrations to produce adverse effects on the environment Enzymatic Transformation Change of one substance to another by enzyme induced chemical reaction; e.g. transformation of acyclic squalene to cyclic squalene by the enzyme squalene cyclase Enzyme Protein produced in a cell and able to accelerate a chemical reaction without being altered by the reaction Epidemiology Branch of medical science dealing with the incidence, distribution and control of disease in a population; the sum of factors controlling the presence or absence of a disease or pathogen Epidermal Fat Fat present in the epidermis, including cholesterol, triglycerides and various methyl sterols; a very significant portion about 12% of epidermal fat in human beings is squalene Epidermis Outermost layer of skin, varying in thickness from 0.07mm to 1.4mm in human beings Epioxide Substance formed after oxidation; e.g. squalene epioxide formed by reaction of oxygen with squalene under the influence of the enzyme squalene epioxidase Epithelial Of or relating to the epithelium Epithelium Cellular covering of the outer and inner body surfaces, including the lining of vessels and small cavities Estrogen Receptor Protein combination on the surface of a cell that attracts and hooks estrogen for use in cellular activity

86

Eukarya Cells of more complex living organisms, containing a true nucleus enveloped by a nuclear membrane Evolution Developmental process by which an organ or organism becomes more and more complex through differentiation of its parts Evolutionary Mechanism Mechanism of a species that adapts to a changing environment Evolutionary Pressure Pressure exerted by the threat of extinction on an organism or a species Excitatory Cytokine Cytokine causing increased proliferation of immune cells Excitatory Stimulatory Exfoliative Dermatitis A scaly dermatitis often associated with the loss of hair and nails, thickening of skin in the palms and soles and intense itching Exogenous Antioxidant Antioxidant not synthesized within the body, usually derived from dietary sources Exogenous Originating outside or caused by factors outside the organism Farnesyl Small isoprenoid derived from mevalonate and a precursor of squalene and coenzyme Q10 Fat Transport The circulation of non-water soluble fats through the water based blood circulation in combination with proteins, called lipoproteins Fatty Acid An acid containing only carbon, hydrogen and oxygen which combine with glycerin to form fat Feed Back Control System

Systems used by the body to maintain balance in various metabolic and other processes Fibromyalgia Common rheumatic syndrome not affecting the joints and characterized by muscle tenderness and pain Force of Disequilibrium Force by which various parts of a normally balanced system are maintained at different levels Free Radical Molecule or atom containing an unpaired electron in its outer orbit Free Radical Biology Branch of medicine that studies and explains disease processes resulting from or contributed to b y free radicals in the biological system Frequency [Of light and other electromagnetic radiation] the number of waves per second; higher frequency waves have more energy 87 Gene Functional unit of hereditary material determining characteristics such as blood type, eye color, etc Genetic Control System Biological control system dealing with functioning of genes Genetic Evolution Evolution of energetic trends and character Geranyl Small isoprenoid of the mevalonate pathway; a precursor to farnesyl ad involved in protein isoprenylation Glomerulonephritis An inflammatory kidney condition; a suspected autoimmune disorder Glutathione S-Acyltransferase

Enzyme involved in glutathione metabolism Glutathione Intracellular antioxidant found in almost all organism; the major component of the cellular redox system that helps maintain thiol homeostasis within a cell Glycerol Type of sugar alcohol Good Cholesterol [HDL] High Density Lipoprotein Protein fat complex [lipoprotein] that transports cholesterol from tissue to liver for excretion in the bile Granulomatous Disorder A genetic defect in which phagocytes ingest but fail to digest bacteria, resulting in recurring bacterial infections Hepatic [liver] failure Severe inability of the liver to function normally Hepatitis Inflammation of the liver Hereditary Blueprint Biological description [genetic information] of an organism passed from parent to child in genes carried in its cellular nucleus Hexachlorobenzene Agricultural fungicide and seed treatment agent Hexachloro Biphenyl Biphenyl group of xenobiotics related to PCBs [PolyChlorinated Biphenyls] High Density Lipoproteins [HDL; Good Cholesterol] Protein fat complex [lipoprotein] that transports cholesterol from tissue to liver for excretion in the bile HIV [HI virus; Human Immunodeficiency Virus] Virus believed to cause AIDA [acquired immune deficiency syndrome] Histamine A neurotransmitter that plays an important role in the regulation of several physiological processes, including dilation of capillaries, contraction of most smooth

muscle tissue, induction of increased gastric secretion [ it most important use], and acceleration of heart rate 88

HMG Co-A Reductase -A Reductase Enzyme that helps conversion of acetyl coenzyme A into mevalonate; the rate limiting enzyme of the mevalonate pathway controlling the entire isoprenoid metabolism Homeostasis Maintenance of normal stability in physio-logical states of an organism, enabling us to adapt to changing environmental conditions; maintained by the internal control systems of the body such as genetic control system, immune control system, etc; these systems operate through negative feedback and positive feedback Hopanoid Class of organic compounds derived from cyclic squalene Hydrogen Peroxide [H2O2] Strong oxidizing agent Hydrophobic Averse to water Hydroxyl [OH,OH+] Ion consisting of one atom of hydrogen and one of oxygen, either neutral or positively charged Hydroxylation Introduction of hydroxyl into ion or radical usually by replacement of hydrogen Hyper activation Over stimulation Hypercholesterolemia Abnormally high levels of cholesterol in the blood Hyperplasia Abnormal, non cancerous increase in number of cells in a tissue or organ Hypoxia Decreased cellular oxygen content Hypoxia reperfusion Injury

Cellular damage caused by sudden flow of oxygen to oxygen deprived tissue Immune Cell Cells of the immune system principally macrophages, T cells and B cells but including many others Immune Defense System The coordinated system that protects the body from microorganisms and other foreign agents Immune Response Coordinated response of the body to invasion, such as bacterial or viral infection Immune Suppression [Immunosuppression] Prevention or diminution of the hosts immune response Immunodeficiency Imbalance of immune response due to too much or too little immune activity Inactivate Transformation of molecule from functioning to nonfunctioning state Inactive Squalene [Stable Squalene] Obsolete term describing the portion of linear squalene that is not metabolized in the mevalonate pathway and which maintains its antioxidant properties 89 Industrial Carcinogen Industrial cancer causing chemical substance Infarction Sudden pathological fall in blood supply to an area resulting in cell death and loss of function of that particular area Inflammation Protective response of tissue to injury and potential destruction; attempt to destroy, dilute or block the injurious agent and the injured tissues; classical signs of inflammation pain include warmth, redness, swelling and loss of function Inflammatory Bowel Disease

Types of chronic intestinal inflammation, including ulcerative colitis and Crohns disease Inhibitor Agent that slows or prevents a biological process Inhibitory Cytokine Cytokine causing decreased proliferation of immune cells Insulin Resistance Diminished response of blood sugar levels to insulin Internal Metabolic Process Operating system of a metabolic process such as enzymatic control of a metabolic pathway, feedback inhibition and hormonal influence Intima Innermost coat of an organ Intracellular Anti-oxidant Antioxidant present inside the cell Ion Atom or molecule with one electron more or less than normal, resulting in an acquired positive or negative charge Ionization Threshold Amount of stimulation required for an electron within a molecular system to break free Ionization Break up of a substance into ions Ionizing Radiation Radiation that can split atoms and molecules in the body Iron Chemical element and essential constituent of hemoglobin, cytochrome, and other components of the respiratory enzyme system Irradiate To expose to radiation

Isoniazid Therapy of choice for tuberculosis Isoprene [Isoprene unit] Building block of isoprenoids; an isoprene unit contains five carbon atoms and a double bond Isoprenoid Metabolism Chemical process that synthesizes three secondary isoprenoids in the mevalonate pathway; geranyl, farnesyl and squalene 90

Isoprenoid Synthesis Pathway See Squalene synthesis pathway Isoprenoid Group of molecules with isoprene units Isoprenylation [ Protein isoprenylation] Modification of proteins on the surface of a cell by the attachment of one of two isoprenoids; farnesyl diphosphate or geranyl diphosphate Keratin Principal protein constituent of epidermis, hair and nails LDL [Low Density Lipoprotein; Bad Cholesterol] Protein fat complex [lipoprotein] that transports cholesterol from the liver through blood into other tissues, where it leads to plaque buildup LDL Receptor Protein complex on the surface of a cell that attracts and hooks LDL; prevalent in the cell membrane of liver cells Leptin Hormone secreted by fat cells Leukemia Progressive, malignant disease of blood forming organs Light Harvesting Complex [LHC] Group of carotenoids antioxidant isoprenoids present in a plants chloroplasts that gather light and make photosynthesis efficient Light Harvesting Compound Same as LHC Linear Dynamics Conventional branch of physics, in which an effect is proportional to its cause [ in contrast to chaos theory or nonlinear dynamics]; a branch of mathematics dealing with systems that obey laws of proportion. Lipase An enzyme that is produced by tongue glands and the pancreas, initiating digestion of

dietary fats Lipid Fat or fat like substance including fatty acids, neutral fats, waxes, isoprenoids and steroids, insoluble in water Lipid Bilayer Double layer of fats [lipids] forming a biomembrane Lipid Peroxidation Chain Reaction Domino effect in which free radical damage to a lipid [fat] molecule renders it radical itself and similarly affects neighboring molecules; analogous to a multi-vehicle highway pile up in a rapid sequence of single steps Lipid Peroxidation Chemical reaction in which a lipid molecule consisting of mainly unsaturated fat is oxidized injury that triggers inflammation Lipophilic Having an affinity for fat Lipoprotein Combination of fats within a protein coat by which lipids are transported in the blood 91 Liquid Chromatography Method of separating mixtures into their constituent substances Long Chain Hydrocarbon Carbon compound with a long chain of interlinked carbon atoms, e.g. petroleum Lumen Cavity of a tubular organ Lycoma Tumor Metabolic Stress resulting from excessive application of lycopene in the skin Lycopane Reduced form of lycopene

Lycopene Red carotenoids pigment of tomatoes and various berries and fruits Lymph Gland Bodily tissue containing lymphocytes and other immune cells that filter micro organisms and toxins from the body Lymphocyte White blood cells formed in the bodys lymphoid tissue Lymphoma General term for various cancerous diseases of the lymphoid tissue Macrophage Immune cell that envelops and digests incoming pathogens by the process of phagocytosis; the major cellular constituent of the mucosal defense system; macrophages also interact with lymphocytes to facilitate antibody production Mass In relating to cancer; autonomous accumulation of cancer cells Mast Cell Type of white blood cell involved in allergic reaction MCG/GM Measure of concentration of one substance within another; microgram [one millionth of a gram] per gram Media Outside wall of an artery Melanin Pigments in skin and hair Melanoma Type of skin cancer; malignant cell growth originating from cells normally forming melanin and spreading widely to lymph nodes, liver, lungs, and brain Metabolic Pathway Process in which a substance is produced in a series of chemical transformations from another substance

Metabolic Pool Portion of a substance that, in contrast to another portion of the same substance, is destined for different biological activity Inability of the metabolism to respond to a situation in which demand for a substance exceeds its production 92

Metabolism Sum of all chemical and physical processes by which living organisms produce organic substances; also, the transformation by which energy is made available for the uses of the organism [catabolism] Metabolite Any biochemical product of metabolism Methyl Bromide Chloroform like volatile and toxic chemical used as fumigant, powerfully destructive of ozone layer Methyl Group [CH3] An organic chemical group Methylation Introduction of the methyl group into a chemical compound Mevalonate Pathway Metabolic sequence of biochemical reactions leading from glucose to cholesterol; Mevalonate Mother molecule of all isoprenoids in the biological system; derived from glucose by several complex enzymatic process Mevalonic Acid [C6H12O4] Precursor of squalene in the biosynthetic pathway forming cholesterol MG/KG Measure of concentration of one substance within another; microgram [one millionth of a gram] per kilogram Migrate The departure of a group of cell or tissue from its natural location to another Mitochondria Energy production factories within a cell that burn nutrients to produce electrons and generate electricity which is converted into chemical energy and stored in the cell for future use

Mitosis Formation of two new nuclei from a single parent nucleus, each having the same number of chromosomes as the parent Mixed isoprenoid Substance composed of isoprene units attached to some other organic group, in contrast to pure isoprenoid; e.g. vitamin E, which contains a chomarol group attached to three isoprenoid side chains Modulate To adjust; to influence the fate of a chemical or physical function Molecular Oxygen [O2] Oxygen in the form of two combined atoms present in air and surface water; ultraviolet radiation can break molecular oxygen into separate oxygen atoms [oxygen radicals] which in turn can combine with molecular oxygen to form ozone Molecule Smallest amount of a substance which can exist alone; an aggression of atoms forming a specific chemical substance Mucosa Mucous membrane coating the lumen of intestines, lung, nose mouth etc 93 Mucosal Defense System Immune system of skin and mucosae Myocardial infarction Sudden death of part of the heart muscle due to interrupted blood supply, a heart attack Myopathy Disorder of muscle tissue or muscles Nanometer Billionth of a meter Natural Defense System Term used in this book to denote the immune system and the antioxidant defense system [ cellular protective system]

Negative Feedback Modulating process of biological control systems; too much or too little activity of a particular sort may initiate negative feedback and return the activity to normal levels; a source of homeostasis Nerve Fiber Sheath [Myelin] Electrical insulator covering nerve fibers enabling faster, more efficient transmission of impulses Neuron Principal cells of nervous tissue able to transmit and receive nervous impulses Neutrophil Immune cell involved in phagocytosis NNK [4 Methlynitro-samino -1-3-Pyridyl-1-Butanone] A potent carcinogen used in laboratory experiments Non-Ionizing Radiation Radiation unable to penetrate and ionize deep tissue Non-Linear Behavior Behavior that does not follow simple, predictable patterns; biological behavior that does not obey proportionality; see chaos theory Non-Linear Dynamics [Chaos Theory] Study of systems which respond disproportionately [nonlinearly] to initial conditions or perturbing stimuli; nonlinear systems may exhibit chaos classically characterized as sensitive dependence on initial conditions; chaotic systems are neither ordered in a mechanistic way nor random; their behavior over time is displayed in phase space in which constraints are described as strange attractors; these representations usually reveal fractal patterns self-similarity across time scales; biological systems often display nonlinear dynamics and chaos Non Specific An immune response to a pathogen that is not tailored to its specific propertied and or weaknesses Normal Chaotic Fluctuation Fluctuation normally considered abrupt [in linear dynamics] but within normal range of nonlinear [chaotic] dynamics; e.g. daily fluctuations of heart rate

Nucleus The heart of a cell, containing genes 94

Oleic Acid An unsaturated fatty acid; the most widely distributed and abundant fatty acid in nature Oncogene Cancer promoting gene; gene that transmits the cancer character as cancerous cells multiply Oncologist Specialist in the study of tumors Organ Somewhat independent body part with a specialized function Organelle Specialized functional structure within a cell Organochlorine A pesticide Oxidant-antioxidant Balance Appropriate ratio of free radicals to antioxidants under which cells, and tissue can operate optimally Oxidation Removal of electrons Oxidation- Reduction Chemical reaction in which electrons are removed [oxidized] from a substance and transferred to those being reduced [ reduction] Oxidative Injury Free radical induced damage to a cell Oxidative Phosphorylation Complex electrochemical process by which mitochondria derive energy from nutrients and oxygen Oxidative Stress Overproduction of free radicals causing tissue damage

Oxidized Cholesterol [oxLDL] Bad Cholesterol subjected to lipid peroxidation; the worst type of cholesterol Oxygen Chemical element constituting about 20% of atmospheric air; essential for respiration in plants and animals Oxyradical Oxygen derived free radical Ozone [O3] Bluish explosive gas or blue liquid; an antiseptic and a disinfectant; an irritant, toxic to the respiratory system Ozone Layer Outermost layer of the planets atmosphere, composed principally of ozone, that filters a portion of the suns harmful ultraviolet radiation P53 Gene Natural anticancer defence; tumor suppressor gene located on the short arm of human chromosome 17 and coding for the phosphoprotein P53 Pathogen Any specific agent causing or threatening the body with disease 95 Pathology Structural and functional manifestations of disease PCBs See polychlorinated biphenyls Peer Review Journal Medical research journal in which research articles are subjected to the approval of a select committee of established scientists Pemphigus Vulgaris A chronic, relapsing sometimes fatal skin disease characterized among other symptoms by serum autoantibodies directed against antigens in the intracellular zones of the

epidermis Pentamethyleicosane [PME] Ancient, acyclic isoprenoid present in primitive bacteria Phagocyte Bacteria-eating immune cell Phagocytosis Process of engulfing microorganisms and other foreign particles by immune cells Phenobarbital A nonselective central nervous system depressant; a barbituric acid derivative Phenol [C6H5OH] An antiseptic and disinfectant Photooxidation Oxidation of organic molecules by light energy Photosynthesis Light induced formation of carbohydrates from carbon dioxide and water in the chlorophyll tissue of plants Physical Quenching Neutralization of free radicals by antioxidants donation of an electron without chemical reaction PI Electron System Special arrangement of electrons I a carbon-carbon double bond that activates nearby hydrogen atoms; each isoprene unit has one double bond and therefore one pi electron; all pi electrons in an isoprenoid molecule combine to make one pi electron system, each one stabilizing the molecules hydrogen atoms Plasma Fluid part especially of blood, lymph, or milk, apart from suspended material Polarized To be separated into polar opposites such as off and on; positive and negative, hydrophilic and hydrophobic Polychlorinated Biphenyls [PCBs]

Highly lipophilic industrial products and compounds that accumulate in fat stores, many of which are potential environmental pollutants Positive Feedback Process into which many biological control systems fall as they lose homeostatic control; leads to instability, disease and death; positive feedback is occasionally a normal process, for example in sexual intercourse 96

Pravastatin Drug acting as a competitive inhibitor of HMG Co-A reductase Precambrian Era Geological time that spanned from 3.8 billion to 570 million years ago. The great Precambrian era is divided into two parts; during the archean period from 3.8 to 2.5 billion years ago archae and cyanobacteria dominated life. The proterozoic era from 2.5 billion To 570 million years ago includes over 85% of living history Prednisolone [C21H28O5] Glucocorticoid used in systemic corticosteroid therapy of inflammatory diseases Prednisone A synthetic anti-inflammatory glucorticoid derived from cortisone; biologically inert and converted to prednisolone in the liver Premature Apoptosis Apoptosis forced upon a young and active cell; an offensive strategy for example of HIV Pro-Carcinogen Normally noncancerous chemical transformed into a carcinogen within the body Proliferation Increase in number, as in cell proliferation [[multiplication] Pro-oxidant Antioxidant molecule that has lost its stability and becomes a free radical Prostate Cancer Tumors or cancer of the prostate a male gland surrounding the neck of bladder and the urethra Protective Coat Term used in this book to encompass those bio-surfaces of the body exposed to the outside environment skin, mouth, intestine, nose, throat and lung Proton Stable elementary particles possessing the smallest known positive charge and found in

the nuclei of all elements Pure Isoprenoid Isoprenoid composed exclusively of isoprene units Pyrophosphate Inorganic salt of phosphoric acid containing phosphate groups Quaternary Carbon Group Carbon atom within an organic compound in which each of its four bonds are connected directly to another carbon atom Quench To neutralize a free radical Quinone Benzene derivative in which two hydrogen atoms are replaced by two oxygen atoms; ubiquinone [Coenzyme Q10] is quinone with an isoprenoid tail Ras Oncogene Family of most commonly found oncogenes in human cancerous tumors 97 Reactive Arthritis Also known as Reiters syndrome. An aberrant reaction of the immune system to the presence of bacterial infections in the genital, urinary, or gastrointestinal systems and leading to inflammation in the joints and eyes Receptor Protein molecule within or on the surface of a cell that recognizes and binds with specific molecules, producing a specific effect in the cell Redox Molecule Type of organic molecule able to participate in reduction oxidation reaction Redox Reaction See oxidation reduction Reduction Addition of electron

Reduction oxidation Electron transfer from one molecule to another; gain [ reduction] and loss [oxidation] of electrons Reperfuse Restoration of blood flow to oxygen deprived tissue or organ Respiratory Tract Disease Disease in airway tubes; e.g. asthma Rheumatic Fever Probable autoimmune disease following streptococcal infection and involving inflammation of joints and damage to heart valves Rheumatic Heart Disease The most important manifestation of and sequel to rheumatic fever Rheumatoid Arthritis Chronic inflammatory destruction of joints considered by some to be an autoimmune disorder Rhinitis Inflammation of the mucous membrane of the nose Ribosome Organelle [ functional unit within a cell] rich in RNA and proteins; site of protein synthesis Risk Factor Contributing cause or circumstance of disease or potential disease RNA Transcription Synthesis of RNA from its complementary DNA strand Roentgen German term for X-ray inventor of x-rays Sarcoidosis Disease of unknown etiology involving chronic inflammatory Granulomatous lesions in the lymph nodes and other organs

Scleroderma Hardening of the skin Sebum Fatty, lubricating secretion of skins sebaceous glands 98

Serum Sickness A hypersensitivity response to the injection of large amounts of antigen Sex Hormone Steroid substances that differentiate male and female beings; testosterone for males and estrogen for females Simvastatin Drug derivative of lovastatin and potent competitive inhibitor of HMG Co-A reductase the rate limiting enzyme in cholesterol biosynthesis Singlet Oxygen Molecular oxygen with one missing electron Spleen Large gland like organ situated in the upper left part of the abdominal cavity Splenomegaly Enlargement of the spleen Squalane Natural emollient found in skin; squalene with an added hydrogen atom Squalene Isoprenoid hydrocarbon consisting of six isoprene units; in linear form an excellent antioxidant; in cyclic form a principal constituent of the sterol nucleus of cholesterol Squalene Metabolism Metabolic process involving production and storage of squalene in the body Squalene synthase Enzyme that converts farnesyl to squalene Squalene synthesis pathway Metabolic pathway that synthesizes squalene from mevalonate via production of geranyl and farnesyl; part of the isoprenoid metabolism Squamous Cell Carcinoma A type of skin cancer on the increase due to ozone depletion

Statins Group of drugs that lower production of cholesterol in the body by inhibiting the enzyme HMG Co-A reductase Sterol Solid steroid alcohol widely distributed in animal and plant lipids; fundamental building block of cholesterol Strychnine Alkaloid found in the seeds of nux vomica; a convulsant and poison Subcutaneous Beneath the skin Submucosal Beneath the mucosa Superoxide Dismutase [SOD] Intracellular antioxidant present mostly in a cells mitochondria Superoxide Highly reactive compound produced when oxygen is reduced by a single electron Synapse Specialized junctions from which neurons communicate with target cells 99 Synthesis Creation of compound by the union of elements; manufacture Systemic Lupus Erythematosus A probably autoimmune disease characterized by antinuclear and other antibodies in plasma Terminator Antioxidant that can stop [terminate] a lipid peroxidation chain reaction Terrestrial Radiation

Earthbound radiation from radioactive materials in rocks and sediments Testicular Cancer Cancer of the Testicle Theophylline Drug that stimulates the heart and central nervous system, dilates bronchi and blood vessels and causes diuresis Threshold [In metabolism] Limit at which biochemical processes undergo drastic change Tissue Aggregation of similar cells which together perform certain special functions TNF-Alpha Tumor necrosing factor; an excitatory cytokine; increased release of this cytokine can cause insulin resistance Topical Designed for or involving local application to a bodily part Total Suspended Particulate Matter [TSP] Solid or liquid atmospheric particles [diameter 10 micrometers] from sources including diesel exhaust, wood- stoves and power plants; may be formed in the atmosphere from reaction of So2, NOx and other gaseous pollutants Toxic Shock Syndrome A staphylococcal infection of blood Transient Temporary Triglyceride Compound consisting of three molecules of fatty acid and the usual storage form of lipids in animals; a neutral fat Triterpene Group of isoprenoid compounds having thirty carbon atoms, such as squalene Tuberculosis

A lung infection caused by a species of mycobacterium Tumor necrosis Factor [TNF-Alpha] Type of stimulatory cytokine secreted by immune and fat cells; increased release can cause insulin resistance Tumor Neoplasm New abnormal growth of tissue Tumor Suppressor Gene Gene causing suicide of a cell to prevent malignant transformation Ulcerative Colitis Chronic inflammatory disease of the mucous membranes of the colon leading to ulcers 100

Uranium 238 238 Radioactive uranium of atomic mass 238 Urticaria Vascular reaction of the skin to allergy, characterized by redness, heat and pain in the affected area UV Radiation Portion of the electromagnetic spectrum immediately below the visible range and extending into the x-ray frequencies UV-A Radiation Ultraviolet spectrum from 320 to 400 nm wavelength; contributes to aging of the skin UV-B Radiation Ultraviolet spectrum from 280 to 320 nm wavelength; contributes to sunburn, aging of the skin and development of skin cancer Vasculitis Inflammation of any vessel Vesicle Membranous, usually fluid filled pouch Vitamin Group of chemically unrelated organic substances occurring in many foods in small amounts and necessary in trace amounts for the normal metabolic functioning of the body Wavelength Length of a wave from trough to trough or crest to crest; the wave length of light is measured in nanometers White Blood Cells Cells in plasma without red cells; all immune cells are white cells Xenobiotic Chemical substances foreign to the biological system