UNIVERSITY OF SANTO TOMAS

COLLEGE OF SCIENCE

DETERMINING THE CYTOTOXICITY LEVEL OF THE BLOOD SERUM OF Crocodylus

porosus TO NORMAL AND CANCER CELL LINES USING YELLOW TETRAZOLIUM
BROMIDE ASSAY (MTT ASSAY) AND ITS PROTEIN PROFILE USING SODIUM
DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)

A Research Proposal Submitted to the

Department of Biological Sciences, College of Science,
University of Santo Tomas

In Partial Fulfillment
of the Requirement for the Degree of Bachelor of Science in Biology

Raven Loise Borres

Jan Chester Chan
Ivy Beatrice Cruz
Jan Paolo Racelis

March 2016

CHAPTER 2: REVIEW OF RELATED LITERATURE

The literature review for this study focuses on procedures used to determine the
cytotoxicity level of the blood serum of Crocodylus porosus to normal and cancer cell lines and
what effect will the blood serum bring to both the normal and cancer cell lines. It also supports
the objective on profiling the blood serum and focuses on a number of various instruments and
techniques used to analyze its components and the cytotoxicity level of the Crocodylus blood.
Crocodiles are known to have a strong immune system wherein even though that they
live in harsh environments such as the rivers and lakes, they can still remain infection-free and
have fast wound-healing capabilities when involved in fights. As mentioned by Merchant, et. al.,
crocodilians live with opportunistic bacterial infection but normally suffer no adverse effects.
They are not totally immune to microbial infection, but their resistance thereto is remarkably
effective. They exhibit remarkable ability to heal rapidly and without infection, particularly
considering the septic environment in which they live in. The immune system of crocodilians has
not been well characterized, but there are several reports that describe the antimicrobial efficacy
of alligator serum towards bacteria, viruses, and amoeba. Also, a number of researches were
done in order to prove if there is really a certain activity happening within the blood of the
crocodile. And apparently, activities such as antibacterial, anti-inflammatory, antiviral, and
complement phagocytic activities were recorded by different studies using different techniques
and devices.
Such studies have acclaimed that the crocodile blood extracts possessed considerable
cytotoxic activity, depending upon the experimental variable/s or sample/s used such as the type
of cell lines used, bacterial strains, temperature, and even concentration - for there were also
studies that have proven that there was no or just a small level of activity happening within the

blood of the crocodile, specifically the blood component, serum. Just like the findings of
Kommanee et. al., wherein the blood plasma, which is almost similar to the blood serum, just
with the addition of coagulating properties, of the Siamese crocodile, Crocodylus siamensis
possessed an insignificant cytotoxic potential against a macrophage-like cell, RAW 264.7 using
the yellow tetrazolium bromide assay (MTT Assay). MTT Assay, which is based on the
conversion of MTT into formazan crystals by living cells, which determines the mitochondrial
activity which is related to the number of viable cells. This assay is broadly used to measure the
in vitro cytotoxic effects of drugs on cell lines (Meerloo, Kaspers, & Cloos, 2011). They found
out that the blood serum exhibited almost similar viability of more than 96% to that of nontreated cells which was taken as 100% viability. They have also found out that this crocodiles
serum cannot inhibit the anti-inflammation nitric oxide (NO) production wherein the aberrant
NO expression is thought to cause severe inflammatory diseases. Thus, inhibition of activation of
these cells appears to be an important target for the treatment of inflammatory diseases. Using
the Griess reagent assay to measure the production of NO, other blood components such as the
plasma and crude leukocyte extract were able to reduce the NO production by 46%-59% and
23%-50%, respectively. Thats why they have concluded that the plasma and crude leukocyte of
the Siamese crocodile have a potential in having an anti-inflammatory capacity, and not the
serum component.
But a study performed by Preecharram et. al. concluded that the antibacterial agent in the
serum of Crocodylus siamensis, may represent the first line of an immune system in a freshwater
crocodile. The researchers purified antibacterial agents from the crocodile serum using the anion
exchange chromatography, and these agents exerted potent activity with remarkable broad
spectrum, suggesting applicability for use in therapeutics. The serum was isolated to five peaks

by anion exchange column chromatography P1 and P2. By the liquid growth inhibition assay,
they exhibited the antibacterial activity against all seven tested bacteria namely, Salmonella
typhi, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella
pneumoniae, Pseudomonas aeruginosa, and Vibrio chorelae. The pooled fractions of Gp and
Gp 2/2 inhibited growth of all seven selected bacterial strains whereas Gp 2/1 exhibited
antimicrobial activity against only S. typhi, S. aureus, S. epidermidis, and P. aeruginosa. Next,
they examined the minimal inhibitory concentration against bacterial strains, wherein Gp has a
high activity against Gram negative and Gram positive bacteria. Using the scanning electron
microscopy, the crude serum and its antibacterial fractions were found to deform the cell
membranes, considering the cells to be irregular in shape, with less defined cell walls, outer
membranous blebs, and with ultrastructural damage. And as a conclusion of this particular study,
this system may form the potency of independent innate immunity to fight against pathogens as
the first line of antibodies which causes survival of crocodile from ancient period to the present
time.
Another study conducted by Preecharram et. al., a certain antibacterial compound in
which they named, Crocosin was partially purified and functionally characterized from the
siamese crocodile plasma, a content almost similar to serum. Wherein in this study, crocosin
exhibited antibacterial activity toward Salmonella typhi and Staphylococcus aureus. They also
found out that crocosin exhibited a broad spectrum of antimicrobial activity with high potency,
suggesting a potential use in therapeutics and that it may provide as an early defense mechanism
toward bacterial infection in freshwater. They have also proposed that the antibacterial
mechanism in their study involves the disturbance of the prokaryotic cell membrane structure.
The antibacterial activity was done by disc diffusion method with modifications. Also, high

powered liquid chromatography eluate fractions were assayed for antibacterial activity by a
liquid growth inhibition assay using S. typhi and S. aureus. Pronase digestion was performed for
24 h at 37 degrees Celsius. The reaction was stopped by heating (100 degrees Celsius, 20 min)
and cooling on ice and then tested for antibacterial activity by a liquid growth inhibition assay.
Bovine serum albumin (BSA) was digested with pronase in the same manner to monitor the
efficiency of digestion. Digests were fractioned using 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The digested BSA was separated on SDSPAGE. An absence of any peptide bands indicated that the enzyme completely digested the
protein. Having a conclusion of crocosin, as a compound resistant to pronase digestion and is
thermostable at 100 degrees Celsius for 20 minutes. The results have led the researchers to the
suggestion that crocosin is the compound carrying no amino acids. So, to extract more
information, the primary structure of crocosin was determined by mass spectrometry. Their
findings could not directly indicate that the innate immunity of freshwater crocodile blood is
protein or peptide. Studies have shown that most antimicrobial peptides found thus far, (Mor et
al. 1991; Feder et al. 2000; Krugliak et al. 2000), are known to exert their antimicrobial action
by making the membrane permeable, as may be the case with crocosin. The results also indicated
that crocosin appears to act on the cell surface in a time-dependent manner, progressively
perturbing the permeability of the membrane. And if the crocosin was really a non-proteinous
antibacterial compound, the determination of structure of this compound is required. However,
crocosin appears to be hydrophilic, so it is likely to have a different mechanism from
antimicrobial peptides. The researchers have also indicated that the potential influences of age,
temperature, season and hormones on immunity should be considered. They have also indicated
that further studies are needed.

A study conducted by Merchan et al., indicated that the American alligator (Alligator
mississippiensis) serum complement activity has been shown to have an antibiotic activity
against three enveloped viruses namely, the human immunodeficiency virus type 1 (HIV-1), West
Nile virus (WNV), and Herpes simplex virus type 1 (HSV-1) using cell-based assays. And
according to their findings, the researchers have concluded that the anti-HIV-1 data presented in
this study, along with the wide spectrum of antibacterial and antiparasitic properties of alligator
serum, suggest that alligators have evolved a strong and broad-acting serum complement system.
However, the activities against the HSV-1 and WNV show that other antiviral mechanisms are
active in alligator serum. To the researchers knowledge, their study is the first report of antiviral
activities against enveloped viruses in the serum of lower vertebrates.
Antimicrobial activity of sera from many crocodilian species has been recognized. This
activity was proposed to be mediated, at least in part, by complement. Due to the fact that
complement proteins have different functions in the immune system, they may be involved in
phagocytic process of phagocytes (Aree, Sruntawineti, Chaeychomsri, 2011). In order to
examine the effect of Siamese crocodile (Crocodylus siamensis) serum on phagocytosis, murine
macrophages (RAW 264.7) were treated with 10% (v/v) fresh serum (FS) from C. siamensis and
subsequently incubated with FITC-labeled bacteria at 37C for 2 h. To evaluate the effect of
crocodile blood on macrophage phagocytosis of both gram-positive and gram-negative bacteria,
S. aureus and E. coli were used in the experiments. The amount of phagocytosis was measured
using flow cytometry and presented as the phagocytic level. Treatment of cells with crocodile
serum conferred a two-fold increase in phagocytosis of S. aureus and a four-fold increase with E.
coli over untreated control cells. Together, these results indicated that crocodile serum facilitated
macrophage uptake of both gram-negative and, gram-positive bacteria. The researchers have

suggested that the phagocytosis-enhancing activity was mediated by complement. While

recognition through pattern recognition receptors (PRRs) is a primary feature of phagocytosis,
opsonization promotes phagocytic internalization of diverse types of pathogens. The process of
phagocyte recognition through opsonin involves multiple proteins including C3b, which is
generated from complement activation cascade (Stuart & Ezekowitz, 2005). Crocodilians were
recognized for their resistance to many pathogens (Merchant et al., 2006). Due to the fact that
complement has many functions, raising the possibility that serum complement from crocodiles
may involve in opsonization of pathogens. Therefore, it is of interest to demonstrate the
influence of crocodile serum on phagocytic ability of macrophages, which will provide some
insights into the antimicrobial properties of crocodile that can be beneficial to human health.
Aree, Sruntawineti and Chaeychomsri have concluded that the study suggested that crocodile
serum might be beneficial in limiting the severity of infection. Thus, the findings of the study
indicate that serum from C. siamensis contains complement that mediates phagocytosis of
pathogens. Hence, crocodile serum might be the source of complement for potential therapeutic
use in patients suffered from infection.
A study related to the previous study stated above, is a study conducted by Siruntawinetic
et al., wherein they have obtained a fresh crocodile serum from C. siamensis, and were treated
with Escherichia coli, Pseudomonas aerugenosa, Bacillus subtilis, and Staphylococcus aureus
for 12 hours. The researchers found out that the serum revealed a time-dependent and a
concentration-dependent inhibition of bacterial growth when inoculated with Escherichia coli
and Pseudomonas aerugenosa. For the researchers, crocodiles continuously defend themselves
against infiltration and colonization of microorganisms. Their immune systems consist of an
adaptive and a non-specific, or innate components. According to Siruntawinetic et al.,

complement is an essential part of the innate immune system and involves about 35 soluble and
membrane-bound proteins. The complement system plays an important role in killing and
neutralizing microorganisms.Using the SRBC hemolytic assay (CH50), it revealed that fresh
crocodile serum contained the total classical component activity 78 +/- 2 U/ml and completely
inihibited bacterial growth. The antimicrobial property in the Siamese crocodile blood was
totally eliminated by preincubating at 56 degrees Celcius for 30 min. Therefore the researchers
have concluded that C. siamensis, has an active serum complement system and may be
responsible for the antibacterial property of crocodile serum.
This study will focus on proving if there is really a cytotoxic activity happening in the
blood serum of the saltwater crocodile, Crocodylus porosus when treated to normal and cancer
cell lines. For previous studies in this type of research field, their key crocodile species was
always the Crocodylus siamensis, or some other crocodilian species such as the Alligator
mississippiensis, wherein both blood showed significant results regarding fighting off infections,
inflammatories, bacteria, and even possessing antiviral capabilities.
According to Ekwall, cell culture can be used to screen for toxicity both by estimation of
the basal functions of the cell or by tests on specialized cell functions. In addition, general
toxicity tests, aimed mainly at detection of the biological activity of test substances, can be
carried out on many cell types (e.g. fibroblasts, epithelial). These tests have also provided useful
insight into the pathogeneses of some human disease. The main focus of this study is to
determine the cytotoxicity of crocodilian blood to both normal and cancer cell lines.
Cancer cells are normally highly-specialized cells which have regressed to a much
simpler, more primitive stage and which, unlike the normal parent, divide continuously, although
inefficiently. Because a much higher proportion of cancer cells are undergoing active division,

they are more vulnerable than most normal cells to anticancer drugs. However, normal tissues
with high mitotic indices (e.g. bone marrow, spleen, thymus and intestinal epithelium) are also
more susceptible to anticancer drugs. Also, the selective toxicity on tumours observed for some
of the chemotherapeutic agents, depends more on pharmacokinetic and metabolic factors in
target cells than on the direct proximal action of the agent. The alkylating and intercalating
drugs, which act directly at the level of DNA, and the mitotic poisons have been shown to
produce a fairly uniform response in mammalian replicating cells, i.e. clumping and
fragmentation of the chromatin (Schwartz and Mihic, 1973). Schwartz and Mihic also added that
in many cases, the selectivity of action of these agents depends mainly on the fact that normal
proliferating tissues have distinctive physiological or biochemical characteristics that affect drug
actions.
Although cell culture systems was used in cytotoxicity testing in our study, it should be
mentioned that cell culture systems are also useful to carry out metabolism studies including
biotransformation, interaction with endogenous metabolites, binding to cells, and induction of
metabolism. Since the study uses the terms such as cell cultures and cell lines, then a standard
definition must be set in order to avoid confusion. In 1964, a Terminology Committee of the
Tissue Culture Association was set up in order to recommend a generally acceptable terminology.
The final report of the Committee was accepted at the annual meeting of the Tissue Culture
Association in 1966 (Fedoroff, 1966). This nomenclature was subsequently revised in 1984
(Schaeffer, 1984). Primary cell cultures and cell lines have been defined as follows:

A primary cell culture is 'a culture started from cells, tissues or organs taken directly from

organisms. A primary culture may be regarded as such until it is successfully subcultured for the
first time. It then becomes a cell line'.

A cell line' arises from a primary culture at the time of the first successful subculture. The

term, cell line, implies that cultures from it consist of numerous lineages of cells originally
present in the primary culture. The terms, finite, or continuous, are used as prefixes if the status
of the culture is known. If not, the term line will suffice. Cell lines may be finite or continuous. A
finite cell line is generally diploid and, in this case, no less than 75 per cent of all the cells must
be of the same standard karyotype as the parent species; its lifespan is approximately 40-50
divisions (Fedoroff, 1966; Hayflick and Moorhead, 1961). A continuous cell line derives from
primary cultures or diploid cell lines by transformation processes, which are either spontaneous,
or induced by viruses, chemical or physical agents (Fedoroff, 1977). Available cell lines are
collected by the American Type Culture Collection which provides a catalogue listing of every
cell type with its history and information concerning viability, growth medium, growth
characteristics, plating efficiency, age of culture since origin, morphology, karyology, sterility
tests and virus susceptibility.
Primary cell cultures have morphological and biochemical characteristics that are more
similar to those of the original tissue; however, problems with obtaining reproducible results may
negate these advantages. Nevertheless, primary cultures offer the only possibility for
comparative studies of some specialized tissues taken from different animal species where cell
lines and strains from the same tissues are not available (Ekwall, 1983). On the other hand, cell
lines offer the advantage of being more homogeneous and standardized than primary cultures.
They are well characterized, easy to cultivate and reproducible results are easier to obtain.
However, they may be quite different from the original tissue due to the fact that established cell
lines have undergone a number of transformations. Due to the fact that cell lines are well

characterized and more easily cultured, they are more widely used for general toxicity studies
than primary cell cultures.

According to Moravec, Niles, and Riss, testing the effects of compounds on the viability
of cells grown in culture is widely used as a predictor of potential toxic effects in whole animals.
In vitro cytotoxicity assays measure whether a test compound is toxic to cells in culture, usually
by determining the number of viable cells remaining after a defined incubation period. The
desired approach is to use a convenient and cost-effective method that predicts in vivo toxicity
by measuring a surrogate marker to indicate the viable cell number compared to untreated
controls. The methods of estimating the number of viable cells in culture are usually based on
measuring an indicator of metabolic activity. Several methods have been developed to measure
metabolically active viable cells based on their ability to convert certain classes of chemicals into
forms that can be easily measured. One common method in determining the number of viable
cells is the Yellow Tetrazolium Bromide Assay (MTT Assay), which will be the type of assay
the study is going to utilize.
The MTT Assay was the first convenient 96-well method developed for screening large
numbers of samples. The MTT tetrazolium compound is reduced by viable cells into an intensely
colored formazan precipitate that subsequently is solubilized into a uniformly colored solution
with a second procedural step before 570-nm absorbance is measured using a plate reading
spectrophotometer. The colored product is directly proportional to the number of viable cells. In
addition, The MTT reduction assay is used to determine the level of metabolic activity in
eukaryotic cells, including animal, plant, and fungal cells. If the metabolic rate is constant, the
technique can be employed to count living cells in a sample. Once it is set up, the method is very
robust, and can be automatized to be applied on a large number of samples. (Kupcsik, 2011).

As stated by Meerloo et al., the general purpose of MTT assay is to measure viable cells
in 96-well plates without the need of complex cell counting. As a result, it is the most commonly
use to determine cytotoxicity of several drugs at a different concentration. The principle behind
MTT assay is that the mitochondrial activity of the most viable cells are constant. Therefore an
increase or decrease in the population of the viable cells is most likely related to mitochondrial
activity. In line with this, several study have stated that MTT assay is suitable for the
measurement of drug sensitivity in established dividing cells (usually cell lines) as well as nondividing cells (primary cells). For cell lines, 50% inhibitory concentration (IC50) is achieved
when there is a decrease in cell number. While for primary or non-dividing cells, the drug
sensitivity is measured as enhanced cell kill of treated cells. In the study of Kommanee et al.,
MTT assay was used for their study in antibacterial activity of plasma from Crocodylus against
bacteria. MTT assay assessed the cytotoxicity of the Crocodylus serum to provide to cells of
macrophage RAW 264.7. This RAW 264.7 is an assay of metabolic competence based on
assessment of the mitochondrial performance through colorimetrical measuring of the conversion
of yellow tetrazolium bromide (MTT) to the purple formazan derivative in viable cells. Also for
primary pediatric acute lymphoblastic leukemia (ALL) cells, MTT Assay has been seen to
predict drug sensitivity in vivo. However, despite of the positive results, MTT Assay is not
commonly used as predictive tests for clinics, but rather an in vitro tool to discover potential or
possible antitumor activity of new or combined drugs.

According to Mordacq and Ellington, there are various types of chromatography that
represent powerful and widely-utilized techniques for separating mixtures of biological
molecules into their individual components. In line with this, there is a certain example of
procedure required for the profiling of the blood serum of crocodiles. Polyacrylamide gel

electrophoresis (PAGE), with all of its different modifications, is probably the most widelyutilized procedure in contemporary biochemistry and molecular biology. Of the various classes
of organic molecules within cells, it is the proteins that are without doubt the most variable in
terms of size, structure, and function. Because proteins are the working molecules of the cell,
these large and complex macromolecules are of interest to researchers working in many areas of
contemporary biological research. Many proteins are stable only under a rather limited range of
experimental conditions; if the limits are exceeded, denaturation as a result of intramolecular
alteration and/or fragmentation occurs with the subsequent loss of biological activity.
Particularly, this study focuses on a certain type of PAGE procedure, the SDS-PAGE. As
also mentioned by Mordacq and Ellington, SDS-PAGE is the acronym for the electrophoretic
procedure in which polyacrylamide gel is utilized as the matrix material and the detergent,
sodium dodecyl sulfate, is included in the electrophoresis buffer. The difference between PAGE
and SDS-PAGE is that the latter disrupts the secondary structure of the molecules and binds to
the polypeptides in the same way that it surrounds integral membrane proteins and wrenches
them out of their thermodynamically comfortable positions in the lipid bilayer. The amount of
SDS bound per unit weight of protein is constant (1.4 g of SDS per g of protein) and
consequently, the charge densities (i.e., the z/f values) of all proteins become identical, with the
charge being contributed by the bound SDS rather than the R-groups of the protein. Under these
conditions, electrophoretic mobility becomes a function strictly of molecular weight because of
the molecular-sieving property of the polyacrylamide gel. In other words, smaller molecules will
move through the gel pores faster than larger molecules. Thus, SDS-PAGE not only separates the
different proteins in a mixture, but gives a reasonably accurate estimate of their molecular
weights as well.

In the study conducted by Siruntawineti and Chaeychomsri, the protein profiles of

Siamese crocodile blood (Crocodylus siamensis) were studied by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) in mercaptoethanol-reducing condition at 10%
(w/v) separating gel. It was found that protein profiles of crocodile blood were not difference in
sexes, crocodile captive breeding locations and freeze-drying process. In contrast, the profiles
showed distinction in different compositions of blood. The whole blood revealed 7 bands of
proteins at molecular weight of 119, 91, 67, 62, 59, 45 and 25 kDa, respectively. The serum
protein represented six bands with molecular weight of 225, 121, 67, 62, 45 and 25 kDa,
respectively. The blood cell fractions showed 2 bands of protein at molecular weight of 45 and
25 kDa, respectively. The highest concentration and broad band protein was albumin at 67-kDa
in molecular weight.

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