Organochlorine pesticide residue levels and oxidative stress in preterm delivery cases

Rahul Pathak1, Sanvidhan G Suke1, Tanzeel Ahmed1, Rafat S Ahmed1, AK Tripathi1, Kiran Guleria2, CS Sharma3, SD Makhijani3 and BD Banerjee1

Human and Experimental Toxicology 29(5) 351358 The Author(s) 2010 Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233710363334 het.sagepub.com

Abstract A number of studies have focused attention on various biochemical abnormalities evoked due to exposure to organochlorine pesticides (OCPs). The aim of the present study was to analyze the OCP residues in maternal and cord blood of women and assess the levels of different non-enzymatic oxidative stress markers as well as to establish correlation with OCP levels, if any. Thirty women in each group of full-term delivery (FTD; !37 weeks of gestation) and preterm delivery (PTD; <37 weeks of gestation) were enrolled in this study. Levels of OCPs like Hexachlorocyclohexane (HCH), endosulfan, p,p0 Dichlorodiphenyldichloroethylene (DDE) and p,p Dichlorodiphenyltrichloroethane (DDT) were analyzed by gas chromatography. Non-enzymatic oxidative stress was measured by the quantification of malondialhyde (MDA), protein carbonyl, reduced glutathione (GSH) and ferric-reducing ability of plasma (FRAP). MDA and protein carbonyl levels were increased significantly, while the levels of GSH and FRAP were decreased in PTD in comparison to FTD cases. We have observed higher levels of b-HCH and a-endosulfan and increased oxidative stress in PTD than FTD cases. In PTD cases, a significant positive correlation was observed between maternal blood levels of b-HCH and MDA (r .78), b-HCH and GSH (r .65), g-HCH and MDA (r .89), g-HCH and GSH (r .74) and a-endosulfan and MDA (r .54) in PTD cases. We also found significant correlations between cord blood levels of b-HCH and MDA (r .59), b-HCH and GSH (r .69), g-HCH and MDA (r .62) and a-endosulfan and MDA (r .54) in PTD cases. In conclusion, our results suggest that higher levels of some of the OCP residues may be associated with PTD and increased oxidative stress. Keywords organochlorine pesticides, preterm delivery, oxidative stress

Introduction
Organochlorine pesticides (OCPs) like Hexachlorocyclohexane (HCH), endosulfan and Dichlorodiphenyltrichloroethane (DDT) are ubiquitous in environment and organisms. These chemicals, still in use in many developing countries, are resistant to degradation, have long half-lives and are highly lipid soluble. Exposure to OCPs may be associated with adverse reproductive outcomes,1,2 hormonal dysfunction,3,4 oxidative stress5 and immunotoxicity.6 In view of their endocrine disrupting property, ability to accumulate in pregnant women and subsequent transfer to developing fetus, OCPs may have the potential to affect growth and development of the
1 Environmental Biochemistry and Immunology Laboratory, Department of Biochemistry, University College of Medical Sciences & G.T.B. Hospital (University of Delhi), Delhi, India 2 Department of Obstetrics and Gynecology, University College of Medical Sciences & G.T.B. Hospital (University of Delhi), Delhi, India 3 Instrumentation and Bio-Labs, Central Pollution Control Board, Ministry of Environment and Forest, Delhi, India

Corresponding author: BD Banerjee, Environmental Biochemistry and Immunology Laboratory, Department of Biochemistry, University College of Medical Sciences & G.T.B. Hospital (University of Delhi), Dilshad Garden, Delhi 110095, India. Email: b.banerjee@ucms.ac.in

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baby in the womb.1 Moreover, OCPs can hamper hormonal homeostasis during pregnancy by decreasing the amount of available progesterone, which can lead to preterm delivery (PTD).3,4,7 PTD affects approximately 5%7% of live births in developed countries and is found to be significantly higher in developing countries.8 It is a major cause of perinatal morbidity and mortality worldwide and is regarded as a syndrome resulting from multiple causes. The factors contributing to the establishment and persistence of PTD probably include cervical insufficiency, hormonal imbalance, genetic predisposition and altered immune surveillance, suggesting that PTD susceptibility is likely to be heterogeneous and polygenic.9 During the labor hypoxic events, transition from low to high oxygen environment results in generation of reactive oxygen species (ROS) and subsequent oxidative stress. There is circumstantial evidence that perinatal free radical generation and oxidative stress can contribute to PTD.10 Oxidative stress may result in up-regulation of proinflammatory cytokine expression, which may induce uterine contractions and PTD.9 Exposure to environmental chemicals is one of the factors causing excessive oxidative stress during pregnancy by altering lipid and protein oxidation chain as well as glutathione redox system.5,6 OCPs have been shown to enhance oxidative stress by producing large number of ROS in several in vivo and in vitro studies.11-15 However, this information cannot always be extrapolated to predict as to what extent these chemicals will affect humans. Therefore, the aim of the present study was (i) to analyze the OCP residue levels in the maternal and cord blood of women delivering preterm and full-term babies, (ii) to assess different non-enzymatic oxidative stress markers in these subjects and (iii) to establish correlation between above mentioned parameters, if any.

(control group) undergoing spontaneous labor at term. Women with anemia, hypertension, bacterial vaginosis, toxemia of pregnancy, renal disease, heart disease, diabetes, urinary tract infections, metabolic disorders, tuberculosis, smoking, alcohol consumption or chronic drug intake and having complications during pregnancy and/or delivery were excluded from both the groups. A life-style survey of the women was done to collect general demographical information in order to define the inclusion/exclusion criteria. The women who participated in this study were of relatively homogenous group; they were similar in terms of demographical characteristics such as age, weight, parity, food habits, BMI, socioeconomic status, drinking water supply and area of residence. We have excluded potentially confounding factors such as women of occupational exposure to pesticides and farming communities from this study. Women confirmed their participation by signing a consent form and this study was approved by the institutional ethical clearance committee for human research. Ten milliliters sample, each of maternal and cord blood, was collected immediately after delivery and divided in EDTA-containing (Ethylenediaminetetraacetic acid) and plain vial. EDTA-containing blood was used for OCPs analysis. Serum was separated from plain vial sample and was used for measurement of oxidative stress parameters. All the tests were performed within 12 hours of sample collection.

Extraction of OCPs from blood and cleanup of the samples

All the chemicals used in the process were of high-purity grade. The HPLC-grade (High performance liquid chromatography) solvents were checked for any contamination before extraction. OCP residues extraction was done by using hexane and acetone (2:1) according to method of Bush et al.16 Cleanup was done by USEPA method using Florisil (Sigma) by column chromatography. Concentrated samples after evaporation were taken to Central Pollution Control Board, Delhi, for gas chromatography analysis. Quantification of organochlorine residue levels was done by Perkin Elmer Gas chromatography system equipped with 63 Ni selective electron capture detector. Limits of detection were <0.05 pg perchloroethylene with nitrogen. Quantitative analysis of OCP residues in each sample was done by comparing the peak heights with those obtained from a chromatogram of a mixed organochlorines standard of known concentration. Ten samples of each maternal and cord blood in triplicate

Material and methods Participant recruitment and collection of samples

The present hospital-based case control study was carried out to determine the association of OCP levels with preterm labor and oxidative stress. Thirty primiparous women (study group) having preterm labor were included in this study after their admission to Guru Teg Bahadur Hospital, Delhi. All participants went into labor spontaneously with intact membrane. Study group was compared with age-matched primiparous women

Pathak R et al.

353 Table 1. Demographical characteristics of subjects Variable Maternal age (years) Maternal weight (kg) Gestational age (weeks) Newborn weight (kg) BMI Drinking water supply Government source Private source Area of residence Slum Market area Colony Socioeconomic status High Middle Low PTD 24.2 + 49.36 + 34.1 + 2.72 + 19.51 + 29 1 1 17 12 4 25 1 1.24 3.41 1.58 0.51 2.49 FTD 24.3 + 50.3 + 37.4 + 2.81 + 19.62 + 28 2 1 19 10 5 24 1 1.92 2.69 0.41 0.27 2.71

were spiked with a mixed standard of organochlorine pesticides, respectively 5 and 25 ng/mL. The average recoveries of fortified samples were exceeding 95%. Further, a quality check sample was always run with each set of samples for pesticide analysis to maintain accuracy.

Measurement of oxidative stress parameters

MDA level in serum was determined as per method described by Satoh et al.,17 using thiobarbituric acid reagent (TBA). Serum protein carbonyl content was measured by the procedure of Reznick et al.,18 using 2,4-dinitrophenyl-hydrazine (DNPH). Ferric-reducing ability of plasma (FRAP) was determined as per method of Benzie et al.19 Total glutathione (GSH) content in red cell heamolysate was measured by the method of Tietze,20 using 5,5-bis dithionitrobenzoic acid (DTNB).

FTD, full-term delivery; PTD, preterm delivery.

Statistical analysis
Maternal and cord blood levels of various OCP and non-enzymatic oxidative stress marker of both PTD and full-term delivery (FTD) cases were calculated. Data were expressed as mean + standard deviation (X + SD) and 25, 50 (median) and 75 percentile basis. Levels of OCP residue and non-enzymatic oxidative stress marker were compared by the Dunnetts test and correlation coefficient (r) was determined by Pearson test. The values of p < .05 were taken to denote significance.

(sum of a-HCH, b-HCH and g-HCH), b-endosulfan, total (T)-endosulfan (sum of a-endosulfan and b-endosulfan), p,p0 Dichlorodiphenyldichloroethylene (DDE) and p,p DDT were detected in both maternal and cord blood of PTD cases than FTD cases, these differences were not statistically significant.

Levels of non-enzymatic oxidative stress markers

The levels of MDA and protein carbonyl were significantly (p < .05) increased and levels of FRAP and GSH were significantly (p < .05) decreased in both maternal and cord blood samples of PTD cases in comparison to FTD cases (Table 3).

Results Demographic characteristics

The women who participated in this study were a relatively homogenous group, since; they were similar in terms of demographical characteristics such as age, education, parity, BMI, socioeconomic status, drinking water supply and area of residence. Demographical characteristics of the women are listed in Table 1.

Correlations (r) between levels of OCPs and non-enzymatic oxidative stress markers
Table 4 depicts the strength of relationship between maternal and cord blood levels of OCP residues and oxidative stress parameters in whole population of pregnant women (PTD FTD). There were significant correlations of b-HCH, g-HCH and a-endosulfan with MDA (r .71, r .63, r .61), b-HCH and protein carbonyl (r .39), b-HCH and a-endosulfan with GSH (r .45, r .32) and b-HCH and FRAP (r .37) in maternal blood samples (Table 4). In cord blood samples, significant correlations of b-HCH, g-HCH and a-endosulfan with MDA (r .69, r .42, r .54), b-HCH and a-endosulfan with protein carbonyl (r .39, r .31) and GSH (r .44, r .33) and b-HCH and FRAP

Concentration of OCPs in maternal and cord blood

Level of OCP residues in maternal and cord blood in PTD and FTD cases are summarized in Table 2. Significantly higher (p < .05) levels of b-HCH and a-endosulfan were observed in maternal as well as cord blood in PTD cases compared to FTD cases. Although higher levels of a-HCH, g-HCH, total (T)-HCH

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Table 2. Comparison of OCP levels (ng/mL) in maternal and cord blood of FTD and PTD cases FTD maternal blood Mean + SD Range a-HCH b-HCH g-HCH T-HCH a-endosulfan b-endosulfan T-endosulfan p,pDDE p,p DDT 5.87 + 5.31 ND-14.31 4.12 + 3.17 ND-10.33 8.27 + 4.76 ND-21.56 18.26 + 11.34 ND-36.09 2.10 + 1.97 ND-5.68 1.22 + 1.43 ND-5.61 3.32 + 3.27 ND-7.49 4.45 + 6.20 ND-9.33 1.64 + 1.21 ND-3.14 25% 2.12 1.34 4.97 10.13 0.00 0.00 0.00 3.41 0.00 FTD cord blood Mean + SD Range a-HCH b-HCH g-HCH T-HCH a-endosulfan b-endosulfan T-endosulfan p,pDDE p,p DDT 4.76 + 3.29 ND-10.68 1.93 + 1.84 ND-5.99 4.67 + 3.17 ND-11.37 11.36 + 7.05 ND-25.12 1.17 + 1.59 ND-4.27 0.93 + 0.70 ND-4.04 2.10 + 2.11 ND-7. 04 2.67 + 1.42 ND-5.61 1.22 + 1.02 ND-4.11 25% 1.62 0.00 2.30 5.90 0.00 0.00 0.00 1.72 0.00 50% 5.08 2.02 4.79 11.92 0.42 0.00 0.42 2.58 1.30 75% 7.17 2.94 6.95 17.32 1.66 1.05 2.76 3.55 1.65 Mean + SD Range 5.04 + 4.07 ND-12.66 4.52 + 3.41a 0.46-15.12 5.57 + 4.84 ND-19.29 15.13 + 8.99 ND-36.92 2.13 + 2.55a ND-5.54 1.18 + 1.35 ND-4.85 3.31 + 3.54 ND-7.54 3.59 + 3.98 ND-7.65 1.30 + 2.57 ND-4.25 50% 4.94 3.33 7.71 14.47 2.38 1.08 2.93 4.32 1.87 75% 11.42 7.18 11.98 30.69 3.54 1.80 5.30 5.47 2.65 Mean + SD Range 6.65 + 5.33 ND-19.77 9.14 + 5.13a 0.78-28.69 8.46 + 7.07 ND-22.36 24.25 + 15.94 ND-58.05 3.10 + 2.80a ND-6.95 1.43 + 1.26 ND-6.36 4.53 + 3.48 ND-10.98 5.48 + 6.05 ND-15.02 1.93 + 2.48 ND-11.38 PTD maternal blood 25% 1.80 2.87 2.98 9.38 2.20 0.00 2.83 3.58 0.93 50% 5.97 10.25 5.53 21.01 4.40 1.78 6.11 4.91 1.89 75% 11.65 14.58 13.33 40.39 6.50 2.36 7.86 7.42 2.68

PTD cord blood 25% 0.93 2.31 2.14 8.52 0.91 0.00 0.91 2.46 0.00 50% 5.33 4.20 4.42 15.96 3.03 0.80 4.03 3.29 1.39 75% 8.30 7.27 9.75 23.29 4.62 2.21 6.4 4.68 2.04

FTD, full-term delivery; ND, not detected; PTD, preterm delivery. a Significantly different from FTD (p < .05).

(r .33) were found (Table 4). A significant correlation was observed between maternal blood levels of b-HCH and MDA (r .78), b-HCH and GSH (r .65), g-HCH and MDA (r .89), g-HCH and GSH (r .74) and a-endosulfan and MDA (r .54) in PTD cases (Table 5). We found significant

correlations (p < .05) between cord blood levels of b-HCH and MDA (r .59), b-HCH and GSH (r .69), g-HCH and MDA (r .62) and a-endosulfan and MDA (r .54) in PTD cases (Table 5). In FTD cases, significant correlation was found between g-HCH and MDA (r .53) in maternal blood samples

Pathak R et al. Table 3. Comparison of levels of oxidative stress markers in maternal and cord blood of FTD and PTD cases FTD maternal blood Mean (SD) MDA (nmol/mL) Protein Carbonyl (nmol/mg protein) GSH (mmol/dL) FRAP mmol/L) MDA (nmoles/mL) Protein Carbonyl (nmol/mg protein) GSH (mmol/dL) FRAP(mmol/L) 2.12 + 0.12 0.13 + 0.01 236 + 25.72 844 + 42.95 FTD cord blood 2.23 + 0.09 0.15 + 0.01 268 + 20.38 861 + 48.32

355

PTD maternal blood Mean (SD) 3.09 + 0.37a 0.22 + 0.02a 133 + 11.82a 647 + 29.36a PTD cord blood 3.29 + 0.55a 0.23 + 0.03a 141 + 15.12a 647 + 30.66a

GSH, glutathione; FRAP, ferric-reducing ability of plasma; FTD, full-term delivery; MDA, malondialhyde; PTD, preterm delivery. a Significantly different from FTD (p < .05)

Table 4. Correlations (r) between level of OCP isomers and oxidative stress parameters in whole population of pregnant women (PTD FTD) in maternal and cord blood levels OCP residues Maternal blood a- HCH b- HCH g- HCH a- endosulfan b- endo p,p-DDE p,p-DDT Cord blood a- HCH b- HCH g- HCH a- endo b- endo p,p-DDE p,p-DDT MDA 0.26 0.71a 0.63a 0.61a 0.15 0.15 0.14 0.11 0.69a 0.42a 0.54a 0.06 0.00 0.11 Protein Carbonyl 0.03 0.39a 0.05 0.27 0.02 0.12 0.17 0.02 0.39a 0.26 0.31a 0.12 0.18 0.00 GSH 0.17 0.45a 0.02 0.32a 0.10 0.17 0.11 0.05 0.44a 0.20 0.33a 0.10 0.23 0.08 FRAP 0.05 0.37a 0.05 0.28 0.01 0.11 0.07 0.06 0.33a 0.15 0.26 0.13 0.29 0.13

GSH, glutathione; FRAP, ferric-reducing ability of plasma; FTD, full-term delivery; MDA, malondialhyde; OCP, organochlorine pesticides; PTD, preterm delivery. a Correlations are significant at p < .05.

(Table 5). Significant correlations were also noted between MDA and GSH as well as protein carbonyl and GSH in PTD cases (Table 6).

Discussion
Although use of OCPs is restricted in different parts of the world including India, they are still pervasive

environmental contaminants due to their unique chemical properties.1 To the best of our knowledge, this is one of the first reports that shows significant correlation between OCPs and non-enzymatic oxidative stress markers in human PTD cases. In India, PTD accounts for 31% of all neonatal deaths.21 The pathophysiology of PTD is not entirely clear but reproductive hormones, such as progesterone and estrogen, appear to have a role. Progesterone is widely regarded as promoting uterine quiescence. Estrogen, on the other hand, may promote myometrial activation with increased receptivity to uterotonic agents by up-regulating membrane receptors and gap junctions.22 We found higher levels of HCH isomers, endosulfan isomers, p,p0 DDE and p,p0 DDT in PTD cases than FTD cases. b-HCH level was found to be two times higher in cases with PTD than with FTD (Table 2). A possible association between higher levels of b-HCH and PTD is in agreement with previous reports.2,23 b-HCH increases uterine contraction frequency in rats24 and recent reports also show its estrogenic effects in mammalian cells, laboratory animals and fish.25-27 It has also been reported that blood levels of b-HCH in ppb (ng/mL) range have the potential of producing estrogenic effects in mice.26 These observations indicate a possible association of b-HCH with PTD due to its estrogenicity. We have also detected significantly higher levels of a-endosulfan in PTD in comparison to FTD cases, which has not been reported earlier. However, our results are inconclusive due to the small sample size. In accordance with previously reported studies,28,29 our results show high levels of MDA and protein carbonyl in PTD cases than FTD cases in both

356 Table 5. Correlations (r) between level of OCP isomers and oxidative stress parameters in maternal and cord blood in PTD and FTD cases OCP residues MDA Protein Carbonyl 0.14 0.20 0.02 0.19 0.06 0.10 0.20 0.13 0.19 0.28 0.16 0.08 0.05 0.00 0.14 0.21 0.22 0.04 0.05 0.23 0.08 0.24 0.26 0.27 0.17 0.13 0.16 0.07 GSH 0.26 0.65a 0.74a 0.23 0.04 0.07 0.20 0.00 0.69a 0.27 0.29 0.02 0.05 0.10 0.14 0.00 0.14 0.17 0.19 0.00 0.13 0.14 0.14 0.24 0.00 0.10 0.07 0.07 FRAP 0.00 0.10 0.22 0.02 0.23 0.21 0.01 0.10 0.08 0.13 0.05 0.24 0.04 0.26 0.13 0.19 0.14 0.26 0.04 0.11 0.00 0.03 0.14 0.19 0.01 0.09 0.06 0.28

Human and Experimental Toxicology 29(5) Table 6. Correlations (r) between oxidative stress parameters in maternal and cord blood in PTD and FTD cases Protein Carbonyl PTD maternal blood MDA Protein carbonyl GSH PTD cord blood MDA Protein carbonyl GSH FTD maternal blood MDA Protein carbonyl GSH FTD cord blood MDA Protein carbonyl GSH 0.17 0.27 0.01 0.24 GSH 0.65a 0.19 0.35a 0.41a 0.005 0.11 0.15 0.04 FRAP 0.19 0.05 0.14 0.17 0.03 0.20 0.10 0.05 0.01 .10 0.25 0.07

PTD maternal blood a-HCH 0.21 b-HCH 0.78a g-HCH 0.89a a-endo 0.54a b-endo 0.08 p,p-DDE 0.11 p,p-DDT 0.21 PTD cord blood a-HCH 0.22 b-HCH 0.59a g-HCH 0.62a a-endo 0.54a b-endo 0.09 p,p-DDE 0.15 p,p-DDT 0.22 FTD maternal blood a-HCH 0.23 b-HCH 0.14 g-HCH 0.53a a-endo 0.19 b-endo 0.26 p,p-DDE 0.01 p,p-DDT 0.16 FTD cord blood a- HCH 0.04 b- HCH 0.20 g- HCH 0.05 a- endo 0.25 b- endo 0.17 p,p-DDE 0.22 p,p-DDT 0.24

GSH, glutathione; FRAP, ferric-reducing ability of plasma; FTD, full-term delivery; MDA, malondialhyde; OCP, organochlorine pesticides; PTD, preterm delivery. a Correlations are significant at p < 0.05

GSH, glutathione; FRAP, ferric-reducing ability of plasma; FTD, full-term delivery; MDA, malondialhyde; OCP, organochlorine pesticides; PTD, preterm delivery. a Correlations are significant at p < .05.

maternal and cord blood (Table 3). MDA is a common oxidative stress marker due to lipid peroxidation and protein carbonyl is formed as a result of protein modification by various oxidants or covalent linkage of aldehyde products of lipid peroxidation.10 Decreased levels of FRAP and GSH in PTD than FTD cases in both maternal and cord blood (Table 3) indicate diminished ability to resist oxidative damage. FRAP assay also measures the total ability of a subject to resist an oxidative challenge30 and GSH is considered as one of the most important antioxidant agents involved in protection of cell membranes from lipid

peroxidation. Our observations of reduced levels of FRAP and GSH are in agreement with previous studies.31,32 The decreased non-enzymatic antioxidant reserve in mother and fetus would enhance the vulnerability to free radical damage by ROS and may be taken as a support for the prevailing concept of oxidative stress during PTD. The significant positive correlations of b-HCH, g-HCH, a-endosulfan with MDA and/or protein carbonyl suggest that OCP isomers may be associated with increased formation of ROS and thus produce oxidative stress in pregnant women (Table 4). In a recent study, we have reported increased levels of MDA in scabies patients after treatment with g-HCH.33 Similar findings were also observed by Samanta et al.,34 in mice and rat testis after treatment with HCH. Endosulfan has also been reported to induce lipid peroxidation and oxidative stress.14,35 It has been suggested that the disruption of the organization of plasma membrane is the primary action of biologically active OCP isomers that may affect cell survivability and increased lipid peroxidation.33 Oxidative stress causes peroxidation of lipids and formation of other reactive intermediates that may also result in increased oxidative damage of bimolecular as well as formation of protein carbonyl adducts. Significant negative correlations of b-HCH, a-endosulfan with GSH and FRAP were also observed in this

Pathak R et al.

357 to Central Pollution Control Board and Ministry of Environment and Forest for financial support. We also thank Mrs Meenu Mishra for technical help.

study (Table 4). Various response patterns have been observed in GSH levels and this may be a result of regulation of GSH pools by endogenous enzymes that can be associated with OCPs.14,33,36 The negative correlation of GSH with OCPs may possibly be due to utilization of GSH to form conjugates with electrophilic metabolites of OCPs or due to more effective oxidation of GSH by glutathione peroxidese. We have observed higher degree of correlation between some of the OCP residues and oxidative stress markers in PTD than FTD cases (Table 5). These alterations may be due to comparatively high levels of OCP residues and oxidative stress in PTD cases. However, this study only suggests an association between OCPs and PTD with reference to increased oxidative stress, but the exact mechanism of OCPs induced oxidative stress in PTD yet to be established in detail. Based on this study, one could speculate that oxidant radicals that may be initiated with OCPs exposure can cause inflammatory response and subsequent early uterine contractions.9 Only some of the HCH and endosulfan isomers showed a significant correlation with oxidative stress parameters in pregnant women. Such correlation studies might explain that either not all OCPs are associated with increasing number of ROS or they have to reach a certain threshold level, in order to exert their effects. In conclusion, our results suggest that higher levels of some of the OCP residues may be associated with PTD and increased oxidative stress. However, a small sample size in the study limits the sensitivity and statistical power of our data and we recommend that the role of OCPs in PTD cases and increased oxidative stress should be interpreted with caution. This study may highlight the reproductive effects of environmental chemicals or xenobiotics on the course of pregnancy and women with a risk of PTD may be benefited by knowing about their OCP burden in specialized clinics after consultation with physician/ gynecologist. We must emphasize that toxicity depends upon numerous additional factors such as genetic predisposition, dietary habits and contamination with other pollutants. Hence, further studies with large sample size, which examine the relationships between OCPs exposures and PTD along with assessment of oxidative stress, hormonal, genetic and immunological markers, are clearly needed. Acknowledgements
Mr Rahul Pathak is thankful to Indian Council of Medical Research for providing Senior Research Fellowship (Reference No. 3/1/2/3/Env/07/NCD-I) and authors are grateful

References
1. Siddiqui MKJ, Srivastava S, Srivastava SP, Mehrotra PK, et al. Persistent chlorinated pesticides and intra uterine fetal growth retardation: possible association. Int Arch Occup Environ Health 2003; 76: 7580. 2. Torres L, Berkowitz G, Torres SI, Lopez CM, et al. Preterm birth in relation to maternal organochlorine serum levels. Ann Epidemiol 2003; 13: 158162. 3. Liu GH, Yang KD, Liu XP, Qin QF. Effects of residues of organochlorine pesticides on reproductive endocrine in human. Wei Sheng Yan Jiu 2005; 34: 524538. 4. Windham GC, Lee D, Mitchell P, Anderson M, Petreas M, Lasley B. Exposure to organochlorine compounds and effects on ovarian function. Epidemiology 2005;16: 182190. 5. Banerjee BD, Seth V, Ahmed RS. Pesticide induced oxidative stress: perspective and trends. Rev Environ Health 2001; 16: 140. 6. Koner BC, Banerjee BD, Ray A. Organochlorine pesticide-induced oxidative stress and immune suppression in rats. Indian J Exp Biol 1998; 36: 395398. 7. Saxena MC, Siddiqui MK, Bhargava AK, Seth TD, et al. Role of chlorinated hydrocarbon pesticides in abortions and premature labour. Toxicol 1980; 17: 323331. 8. Menon R. Spontaneous preterm birth, a clinical dilemma: etiologic, pathophysiologic and genetic heterogeneities and racial disparity. Acta Obstet Gynecol Scand 2008; 87: 590600. 9. Sakata M, Sado T, Kitanaka T, Naruse K, Noguchi T, Yoshida S, et al. Iron-dependent oxidative stress as a pathogenesis for preterm birth. Obstet Gynecol Surv 2008; 63: 651660. 10. Mocatta TJ, Winterbourn CC, Inder TE, Darlow BA. The effect of gestational age and labour on markers of lipid and protein oxidation in cord plasma. Free Radic Res 2003; 38: 185191. 11. Srivastava A, Shivanandappa T. Hexachlorocyclohexane differentially alters the antioxidant status of the brain regions in rat. Toxicology 2005; 214: 123130. 12. Garca-Fernandez AJ, Bayoumi AE, Perez-Pertejo Y, Romero D, Ordonez C, Reguera RM, et al. Changes in glutathione-redox balance induced by hexachlorocyclohexane and lindane in CHO-K1 cells. Xenobiotica 2002; 32: 10071016. 13. Perez-Maldonado IN, Herrera C, Batres LE, Gonzalez Amaro R, Daz-Barriga F, et al. DDT-induced

358 oxidative damage in human blood mononuclear cells. Environ Res 2005; 98: 177184. Aggarwal M, Naraharisetti SB, Sarkar SN, Rao GS, et al. Effects of subchronic coexposure to arsenic and endosulfan on the erythrocytes of broiler chickens: a biochemical study. Arch Environ Contam Toxicol 2009; 56: 139148. Sohn HY, Kwon CS, Kwon GS, Lee JB, Kim E. Induction of oxidative stress by endosulfan and protective effect of lipid-soluble antioxidants against endosulfaninduced oxidative damage. Toxicol Lett 2004; 151: 357365. Bush B, Snow J, Koblintz R. Polychlorobiphenyl (PCB) congeners, p,p0 -DDE and hexachlorobenzene in maternal and fetal cord blood from mothers in upstate NewYork. Arch Environ Contam Toxicol 1984; 13: 517527. Satoh K. Serum lipid peroxide in cerebrospinal disorder determined by a new colorimetric method. Clin Chem Acta 1978; 90: 3743. Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol 1994; 233: 357363. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: The FRAP assay. Anal Biochem 1996; 239: 7076. Tietze F. Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: application to mammalian blood and other tissues. Anal Biochem 1969; 27: 502522. World health Statistics. Mortality Country Fact Sheet India. 2006; 12, http://www.who.int/whosis/mort/ profiles/mort_searo_ind_india.pdf. Accessed on 12 December 2008. Wood SL, Jarrell JJ, Swaby C, Chan S. Endocrine disruptors and spontaneous premature labor: a case control study. Environ Health 2007; 6: 35. Saxena MC, Siddiqui MK, Seth TD, Krishna Murti CR, Bhargava AK, Kutty D. Organochlorine pesticides in specimens from women undergoing spontaneous abortion, premature of full-term delivery. J Anal Toxicol 1981; 5: 69. Criswell KA, Loch-Caruso R. Lindane-induced inhibition of spontaneous contractions of pregnant rat uterus. Reprod Toxicol 1999; 13: 481490.

Human and Experimental Toxicology 29(5) 25. Toxic Link Information on new POPs. 2007; 5078, http://www.toxicslink.org/docs/newpops.pdf. Accessed on 12 December 2008. 26. Ulrich EM, Caperell GA, Jung SH, Hites RA, Bigsby RM. Environmentally relevant xenoestrogen tissue concentrations correlated to biological responses in mice. Environ Health Perspect 2000; 108: 973977. 27. Wester PW. Histopathological effects of environmental pollutants beta-HCH and methyl mercury on reproductive organs in freshwater fish. Comp Biochem Physiol C 1991; 100: 237239. 28. Winterbourn CC, Chan T, Buss IH, Inder TE, et al. Protein carbonyls and lipid peroxidation products as oxidation markers in preterm infant plasma: associations with chronic lung disease and retinopathy and effects of selenium supplementation. Pediatr Res 2000; 48: 8490. 29. Brucknerova I, Benedekova M, Pechan I, Frankova E, et al. Protection of newborn organism against effect of oxidative stress. Cent. Eur J Public Health 2004; 12: 1820. 30. Friel JK, Friesen RW, Harding SV, Roberts LJ. Evidence of oxidative stress in full-term healthy infants. Pediatr Res 2004; 56: 878882. 31. Lee YS, Chou YH. Antioxidant profiles in full term and preterm neonates. Chang Gung Med J 2005; 28: 846851. 32. Oberoi S, Ahmed RS, Suke SG, Bhattacharya SN, et al. Comparative effect of topical application of lindane and permethrin on oxidative stress parameters in adult scabies patients. Clin Biochem 2007; 40: 13211324. 33. Sahoo A, Samanta L, Chainy GB. Mediation of oxidative stress in HCH-induced neurotoxicity in rat. Arch Environ Contam Toxicol 2000; 39: 712. 34. Samanta L, Chainy GB. Response of testicular antioxidant enzymes to hexachlorocyclohexane is species specific. Asian J Androl 2002; 4: 191194. 35. Omurtag GZ, Tozan A, Sehirli AO, Sener G. Melatonin protects against endosulfan-induced oxidative tissue damage in rats. J Pineal Res 2008; 44: 432438. 36. Dehn PF, Allen MS, Karek J, Thenappan A. Organochlorine insecticides: impacts on human HepG2 cytochrome P4501A, 2B activities and glutathione levels. Toxicol In Vitro 2005; 19: 261273.

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