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Reproductive Toxicology 31 (2011) 534–539

Contents lists available at ScienceDirect

Reproductive Toxicology

journa l homepage: www.e lsev ier .com/ locate / reprotox

ntra uterine growth retardation: Association with organochlorine pesticideesidue levels and oxidative stress markers

ahul Pathaka, M.D. Mustafaa, Tanzeel Ahmeda, Rafat. S. Ahmeda, A.K. Tripathia, Kiran Guleriab,.D. Banerjeea,∗

Environmental Biochemistry and Molecular Biology Laboratory, Department of Biochemistry, University College of Medical Sciences & G.T.B. Hospital (University of Delhi),ilshad Garden, Delhi 110 095, IndiaDepartment of Obstetrics and Gynecology, University College of Medical Sciences & G.T.B. Hospital (University of Delhi), Dilshad Garden, Delhi 110 095, India

r t i c l e i n f o

rticle history:eceived 28 October 2010eceived in revised form 12 January 2011ccepted 14 February 2011

a b s t r a c t

Intra uterine growth retardation (IUGR) is a major complication of pregnancy, affecting ∼5% to 10% ofnewborns. Hexachlorocyclohexane (HCH) is an organochlorine pesticide that consists of eight stereoiso-mers and �-isomer is the only isomer that possesses insecticidal activity. The aim of the present study

vailable online 18 February 2011

eywords:ntra uterine growth retardationrganochlorine pesticides

was to analyze the OCP residues in maternal and cord blood of women and to assess the level of oxidativestress markers as well as to establish correlation with OCP levels. Fifty women delivering neonates withlow birth weight (IUGR) and equal number of women delivering normal birth weight babies (control)were recruited. We have observed higher levels of �-HCH and T-HCH and increased oxidative stressmarkers in IUGR subjects versus control subjects. Significant correlations were also found between HCH

ress my be

xidative stress isomers and oxidative stlevels of HCH isomers ma

. Introduction

Intrauterine growth retardation (IUGR) is a pleiotropic compli-ation of pregnancy, affecting ∼5% to 10% of newborns [1]. It isssociated with substantially increased infant mortality as well ashildhood and adulthood morbidity, including increased risk forardiovascular disease, obesity and diabetes [2]. While the etiologys poorly defined, IUGR may be a consequence of several detri-

ental factors such as aneuploidies, non-aneuploid syndromes,etabolic factors, maternal infection, under nutrition before and

uring pregnancy, drug abuse, hypertension, and placental disor-ers [1,2]. Despite the above suggested risk factors for IUGR, in vastajority of cases (∼40%), the cause still remains idiopathic [3].Increased interest in environmental pollutant-induced oxida-

ive stress, and knowledge of the interactions between free radicals,elated oxidants, and cellular systems, has increased the profilef reactive oxygen species (ROS) in reproductive toxicology [4,5].hen ROS production overwhelms the endogenous protection

fforded by specific degradative enzymes, antioxidant vitamins,nd other radical scavengers, the resulting damage to cellularonstituents is known as oxidative stress. Several studies havemplicated ROS as the agents responsible for impaired repro-

∗ Corresponding author. Tel.: +91 11 22135362; fax: +91 11 22590495.E-mail addresses: banerjeebd@hotmail.com, b.banerjee@ucms.ac.in

B.D. Banerjee).

890-6238/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.reprotox.2011.02.007

arkers in IUGR subjects. In conclusion, our results suggest that higherassociated with IUGR and increased oxidative stress.

© 2011 Elsevier Inc. All rights reserved.

ductive function in organisms experiencing oxidative stress. Inmammals, low concentrations of ROS impair ovarian developmentby negatively affecting steroid hormone biosynthesis, possibly viathe oxidative modification of proteins, lipids, carbohydrates, andDNA [4]. Excessive oxidative stress during pregnancy has beenassociated with various pregnancy complications, such as IUGR[6,7].

According to a Green Peace Report, India is now producing90,000 metric tons of pesticides as the largest industry in thewhole of Asia and twelfth largest in the entire world. The cumu-lative consumption of the pesticide, HCH, in India until 1985 was575,000 tons and since then about 45,000 tons of HCH has been usedannually. Even though usage of technical HCH was finally bannedcompletely in 1977, the Government of India is encouraging itsreplacement with �-HCH, an isomer which has all the hazardousproperties of HCH [8]. Organochlorine pesticides (OCPs) such asHCH and DDT are persistent environment chemicals and are widelyused in India for agriculture and public health programs [9]. Due totheir strongly lipophilic nature and slow degradation rates; thesepesticides tend to bioaccumulate in lipid-rich tissues [10]. As aresult, significant levels of many OCPs have been detected in humanbody tissues like blood, placenta, amniotic fluid, and in secretions

like semen, breast milk, etc. [9–11]. Several in vivo and in vitrostudies have shown that OCPs enhance oxidative stress by produc-ing large number of ROS [12–15]. Moreover, OCPs also possess theability to modulate the endocrine system and thus adversely affecthuman reproduction [16].

ve Toxicology 31 (2011) 534–539 535

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Table 1Demographical parameters of the subjects.

Parameters Controls(n = 50)

IUGR subjects(n = 50)

Age (years) 22.62 ± 3.40 23.04 ± 2.35Residential area

Urban 50 50Rural 0 0

OccupationHousewife 46 50Working 4 0

Socioeconomic statusClass I (upper) 24 12Class II (middle) 17 32Class III (lower) 9 6

Living styleColony 47 48Slum 3 2

Source of water supplyGovt. – directly 35 41Ground – directly 15 9

Dietary habitsVegetarian 32 34Non-vegetarian 28 16Period of gestation 39.3 ± 0.9 37.5 ± 0.9*

Physical examination characteristicsHeight (cm) 153.6 ± 4.3 154.1 ± 4.6Weight (kg) 54.8 ± 6.7 52.1 ± 4.5Body mass index (kg/m2) 23.1 ± 2.2 22.0 ± 2.3Weight gain during pregnancy 8.9 ± 2.3 7.1 ± 2.1*

Placental weight (g) 546.6 ± 72.7 395.0 ± 59.7*

Anthropometric features of the neonateBirth weight (kg) 3.0 ± 0.2 2.05 ± 0.1*

Length (cm) 50.1 ± 1.1 47.8 ± 1.1*

Head circumference (cm) 34.0 ± 0.4 33.3 ± 0.7*

R. Pathak et al. / Reproducti

Therefore, the aim of the present study was to (i) analyze theCP residue levels in the maternal and cord blood of women deliv-ring IUGR and full term babies, (ii) assess different non-enzymaticxidative stress markers in these subjects, and (iii) investigateotential correlations between these parameters.

. Materials and methods

.1. Participant recruitment and collection of samples

The present hospital based case control study was carried out to determine thessociation of OCP levels with IUGR and oxidative stress. Fifty primiparous womenstudy group) delivering IUGR babies (birth weight <10 percentile for gestationalge as per Lubchenco growth chart [17]) were included in this study after theirdmission to Guru Teg Bahadur Hospital, Delhi. Study group was compared withame number of women delivering healthy neonates (control group). Women withnemia, hypertension, toxemia of pregnancy, renal disease, heart disease, diabetes,rinary track infections, metabolic disorders, tuberculosis, smoking, alcohol con-umption or chronic drug intake and having complications during pregnancy and/orelivery were excluded from both the groups. A questionnaire survey of the womenas done to collect general demographical information in order to define the inclu-

ion/exclusion criteria. We have excluded potentially confounding factors such asomen of occupational exposure to pesticides and farming communities from this

tudy. Women confirmed their participation by signing a consent form and this studyas approved by the institutional ethical clearance committee for human research.

ive milliliters sample, each of maternal and cord blood were collected and dividedn EDTA containing and plain vial at the time of delivery. EDTA containing blood

as used for OCPs analysis and serum was separated from plain vial sample. Theerum was used for measurement of oxidative stress parameters. All the tests wereerformed within 12 h of sample collection.

.2. Extraction of OCPs from blood and clean up of the samples

All the chemicals used in the process were of high-purity grade. The HPLC-rade solvents were checked for any contamination before extraction. OCP residuesxtraction was done using hexane and acetone (2:1) according to method of Bush etl. [18]. Cleanup was done by USEPA method using Florisil (Sigma) by column chro-atography. Quantification of organochlorine residue levels was done by Perkin

lmer gas chromatography system equipped with 63Ni selective electron captureetector. Limits of detection were <0.05 pg perchloroethylene with nitrogen. Quan-itative analysis of OCP residues in each sample was done by comparing the peakeights with those obtained from a chromatogram of a mixed organochlorines stan-ard of known concentration. Ten samples of each maternal and cord blood inriplicate were spiked with a mixed standard of organochlorine pesticides, respec-ively, 5 and 25 ng/ml. The average recoveries of fortified samples were exceeding5%. Further, a quality check sample was always run with each set of samples foresticide analysis to maintain accuracy.

.3. Measurement of oxidative stress parameters

In order to detect oxidative DNA damage, DNA from the whole blood was isolatedsing Himedia DNA extraction kit. The amount of DNA was quantified by measur-

ng the optical density. After the digestion with alkaline phosphatase and nuclease,he concentrations of 8-hydroxy-2-deoxy Guanosine (8-OHdG) were analyzed usingnzyme Linked Immunosorbent Assay (ELISA) kit purchased from Caymen Chemi-als, US. 8-OHdG is a product of oxidative damage of DNA by free radicals and servess an established marker of oxidative stress. Hydroxylation of guanosine occurs inesponse to both normal metabolic processes and a variety of environmental fac-ors. Increased levels of 8-OHdG are associated with the aging process as well asith a number of pathological conditions including adverse pregnancy outcomes.alondialdehyde (MDA) level in serum was determined as per method described

y Satoh [19] using thiobarbituric acid reagent (TBA). ROS degrade polyunsatu-ated lipids, forming MDA. This compound is a reactive aldehyde and is one of theany reactive electrophile species that cause toxic stress in cells and form covalent

rotein adducts which are referred to as advanced lipoxidation end products. Theroduction of this aldehyde is used as a biomarker to measure the level of oxida-ive stress in an organism. Serum protein carbonyl content was measured by therocedure of Reznick and Packer [20] using 2,4-dinitrophenyl-hydrazine (DNPH).rotein carbonyls are formed through oxidation of proteins by a variety of mech-nisms. They are sensitive markers of oxidative injury. Ferric reducing ability oflasma (FRAP) was determined as per method of Benzie and Strain [21]. FRAP assay

s presented as a novel method for assessing “antioxidant power”. Ferric to ferrouson reduction at low pH causes formation of a colored ferrous–tripyridyltriazine

omplex. FRAP values are obtained by comparing the absorbance change at93 nm in test reaction mixtures with those containing ferrous ions in knownoncentration.

Total reduced glutathione (GSH) content in red cell hemolysate was measuredy the method of Tietze [22] using 5,5-bis dithionitrobenzoic acid (DTNB). Glu-athione (GSH) is a tripeptide that contains an unusual peptide linkage between

Chest circumference (cm) 32.5 ± 0.8 29.8 ± 0.8*

Mid-arm circumference (cm) 10.5 ± 0.8 8.8 ± 0.5*

n = Number of subjects.* Significant (p < 0.05).

the amine group of cysteine and the carboxyl group of the glutamate side-chain. Itis an antioxidant, preventing damage to important cellular components caused byreactive oxygen species such as free radicals and peroxides.

2.4. Statistical analysis

Significance of odds ratios was tested using Chi-square test to identify the vari-ables such as woman’ age, occupation, socio-economic status, food habits, sourceof water supply and living style and gestation period associated with IUGR. Age,residential area, occupation, living style, source of water supply and dietary habitswere not found to be significant contributing factor towards preterm or IUGR births.Weight gain during pregnancy, gestational age and socio-economic status were sig-nificantly different in control v/s IUGR. As OCPs are not normally distributed, hencenon-parametric Mann–Whitney test was used to compare pesticides levels in casesand controls followed by Dunnett’s test after adjustment of potential confounderssuch as weight gain during pregnancy, gestational age and socio-economic status.The differences between oxidative stress parameters of IUGR and control subjectswere analyzed by one-way analysis of variance (ANOVA). To find out the relation-ship between OCPs and oxidative stress parameters Spearman’s correlation wasused. The values of p < 0.05 were taken to denote significance.

3. Results

3.1. Demographic characteristics

The women who participated in this study were a rela-tively homogenous group, since, they were similar in termsof demographical characteristics such as age, education, paritysocioeconomic status, drinking water supply and living style.Demographical characteristics of the women are listed in Table 1.

3.2. Concentration of OCPs in maternal and cord blood

Level of OCP residues in maternal and cord blood in IUGR casesand controls are summarized in Table 2. Significantly higher levels

536 R. Pathak et al. / Reproductive Toxicology 31 (2011) 534–539

Table 2Comparison of OCP levels (ng/ml) in maternal and cord blood of control and IUGR subjects.

OCP name Mean ± SD (controls) (n = 50) Mean ± SD (IUGR) (n = 50) Adjusted odds ratio (OR)a 95% CI p-Value

Maternal blood�-HCH 3.16 ± 4.05 4.10 ± 3.33 1.04 0.90–1.21 0.540�-HCH 6.26 ± 5.19 7.23 ± 5.99 1.05 0.95–1.16 0.316�-HCH 3.70 ± 4.09 7.09 ± 5.04 1.21 1.05–1.38 0.006*

T-HCH 13.12 ± 7.90 18.43 ± 10.34 1.08 1.01–1.15 0.025*

p,p′-DDE 3.67 ± 1.52 4.24 ± 2.28 1.28 0.97–2.05 0.071p,p′-DDT 1.51 ± 1.20 1.67 ± 1.16 1.19 0.72–1.97 0.491

Cord blood�-HCH 1.38 ± 1.85 1.91 ± 1.95 1.13 0.91–1.66 0.167�-HCH 3.45 ± 2.82 4.36 ± 3.69 1.09 0.91–1.31 0.341�-HCH 1.92 ± 2.79 3.89 ± 3.29 1.44 1.13–1.84 0.003*

T-HCH 6.75 ± 3.72 10.17 ± 6.23 1.22 1.07–1.40 0.003*

p,p′-DDE 2.15 ± 1.10 2.73 ± 1.81 1.45 0.87–2.86 0.062p,p′-DDT 0.80 ± 0.72 0.99 ± 0.83 1.42 0.66–3.06 0.360

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(i) There were significant correlations of �-HCH, �-HCH, T-HCHwith 8-OHdG, MDA and GSH in maternal as well as cord bloodsamples of control + IUGR subjects (Table 4). Significant corre-lation of �-HCH, T-HCH was observed with protein carbonyl

Table 4Correlations between OCP levels in blood (maternal and cord) and oxidative stressmarkers in women subjects (control + IUGR).

OCP 8-OHdG MDA Proteincarbonyl

GSH FRAP

Maternal blood (n = 100)�-HCH 0.06 0.162 0.086 0.011 0.030�-HCH 0.31* 0.298* 0.244 −0.313* −0.259�-HCH 0.27* 0.300* 0.327* −0.339* −0.197T-HCH 0.42** 0.476** 0.419** −0.426* −0.335*

p,p′-DDE 0.11 0.117 0.135 −0.071 −0.110p,p′-DDT 0.03 0.053 0.068 −0.039 −0.123

Cord blood (n = 100)�-HCH 0.13 0.138 0.236 −0.018 −0.050�-HCH 0.34** 0.366* 0.298* −0.477** −0.176�-HCH 0.30* 0.317* 0.236 −0.322* −0.184T-HCH 0.47** 0.530** 0.487** −0.534** −0.245p,p′-DDE 0.04 0.079 0.066 −0.103 −0.025

= Number of subjects.a Variables are adjusted for weight gain during pregnancy, gestational age and so* Significant.

f �-HCH and total (T)-HCH (sum of �-HCH, �-HCH and �-HCH)ere observed in maternal as well as cord blood in IUGR cases

ompared to controls. Although higher levels of �-HCH, �-HCH,,p′-DDE and p,p′-DDT were detected in both maternal and cordlood of IUGR cases than controls, these differences were not sta-istically significant.

.3. Levels of non enzymatic oxidative stress markers

The levels of 8-OHdG, MDA and protein carbonyl were signif-cantly (p < 0.05) increased indicating production of free radicalseading to oxidative DNA damage and peroxidation of membraneipids. The levels of FRAP and GSH were significantly (p < 0.05)ecreased in both maternal and cord blood samples of IUGR cases

n comparison to control subjects (Table 3). GSH is a major antiox-dant molecule present in the erythrocytes. Any decrease in GSHevels will lead to concomitant decrease in FRAP levels.

.4. Correlations between levels of OCPs and non-enzymaticxidative stress markers

Tables 4–6 depict the strength of relationship between maternalnd cord blood levels of OCP residues and oxidative stress param-ters in control + IUGR subjects (Table 4), control subjects (5) andUGR subjects (6).

able 3omparison of levels of oxidative stress markers in maternal and cord blood ofontrols and IUGR subjects.

Control subjects IUGR subjectsMean ± SD Mean ± SD(n = 50) (n = 50)

Maternal blood8-OHdG (pg/mL) 55.51 ± 6.15 75.92 ± 7.59*

MDA (nmol/mL) 2.12 ± 0.24 3.40 ± 0.82*

Protein carbonyl (nmol/mg protein) 0.13 ± 0.02 0.22 ± 0.03*

GSH (�mol/dL) 228 ± 34.08 164 ± 43.11*

FRAP (�mol/L) 636 ± 58.98 474 ± 155.15*

Cord blood8-OHdG (pg/mL) 57.22 ± 6.09 64.46 ± 8.58MDA (nmoles/mL) 2.27 ± 0.09 3.08 ± 1.16*

Protein carbonyl (nmol/mg protein) 0.14 ± 0.03 0.23 ± 0.16*

GSH (�mol/dL) 246 ± 42.19 196 ± 55.30*

FRAP (�mol/L) 520 ± 122.49 463 ± 73.10*

= Number of subjects.* Significantly different from controls (p < 0.05).

nomic status.

p,p′-DDT 0.03 0.023 0.114 −0.168 −0.088

n = Number of subjects.* Correlation is significant at the 0.05 level (2-tailed).

** Correlation is significant at the 0.01 level (2-tailed).

Table 5Correlations between OCP levels in blood (maternal and cord) and oxidative stressmarkers in control subjects.

OCP 8-OHdG MDA Proteincarbonyl

GSH FRAP

Maternal blood (n = 50)�-HCH 0.02 0.240 0.021 0.265 0.262�-HCH 0.21 0.307* 0.071 −0.184 −0.082�-HCH 0.08 0.072 0.073 0.035 −0.171T-HCH 0.07 0.380* 0.047 −0.006 −0.056p,p′-DDE 0.16 0.160 0.048 0.223 0.076p,p′-DDT 0.02 0.166 0.097 0.176 0.142

Cord blood (n = 50)�-HCH 0.01 0.119 0.131 0.217 −0.037�-HCH 0.23 0.109 0.069 −0.326* 0.033�-HCH 0.19 0.281 −0.043 0.091 −0.052T-HCH 0.17 0.359* 0.126 −0.117 0.073p,p′-DDE 0.07 0.184 −0.242 0.082 −0.008p,p′-DDT 0.01 0.081 −0.040 −0.006 −0.377

n = Number of subjects.* Correlation is significant at the 0.05 level (2-tailed).

R. Pathak et al. / Reproductive Tox

Table 6Correlations between OCP levels in blood (maternal and cord) and oxidative stressmarkers in IUGR subjects (n = 50).

OCP 8-OHdG MDA Proteincarbonyl

GSH FRAP

Maternal blood (n = 50)�-HCH 0.15 0.189 0.118 −0.050 −0.082�-HCH 0.37** 0.174 0.178 −0.288* −0.005�-HCH 0.50** 0.486** 0.499** −0.727** −0.313*

T-HCH 0.52** 0.377* 0.344* −0.509** −0.161p,p′-DDE 0.12 0.052 −0.003 −0.081 −0.023p,p′-DDT 0.17 0.173 −0.080 −0.042 −0.082

Cord blood (n = 50)�-HCH 0.174 0.315* 0.344* −0.205 −0.155�-HCH 0.429** 0.385* 0.315* −0.357* −0.205�-HCH 0.340* 0.375* 0.423* −0.339* −0.315*

T-HCH 0.444** 0.506* 0.496* −0.423** −0.339*

p,p′-DDE 0.026 0.111 −0.052 −0.028 0.059p,p′-DDT 0.273 0.052 0.111 −0.262 0.073

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and T-HCH with FRAP in maternal blood. In cord blood �-HCH,T-HCH were correlated with protein carbonyl levels.

(ii) There were only significant correlations of �-HCH, T-HCH withMDA in maternal blood samples and T-HCH with MDA, �-HCHwith GSH in cord blood samples of control subjects (Table 5).

iii) There were significant correlations of �-HCH, �-HCH, T-HCHwith 8-OHdG and GSH, �-HCH, T-HCH with MDA and proteincarbonyl and �-HCH with FRAP in maternal blood samples.Significant correlations were found between �-HCH, �-HCH,T-HCH with 8-OHdG, and GSH, �-HCH, �-HCH, �-HCH, T-HCHwith MDA and protein carbonyl and �-HCH, T-HCH with FRAPin cord blood samples of IUGR subjects (Table 6).

.5. Correlations between oxidative stress markers

Significant correlations were noted between oxidative stressarkers in maternal and cord blood of IUGR and control subjects

Table 7).

able 7orrelations (r) between oxidative stress parameters in maternal and cord blood in

UGR and control subjects.

MDA Proteincarbonyl

GSH FRAP

IUGR maternal blood (n = 50)8-OHdG 0.222 0.155 −0.028 −0.450*

MDA – 0.285 −0.390* −0.641*

Protein carbonyl – – −0.013 −0.460*

GSH – – – 0.440*

IUGR cord blood (n = 50)8-OHdG 0.523* 0.379* −0.301* −0.292MDA – 0.309* −0.472* −0.524*

Protein carbonyl – – −0.184 −0.076GSH – – – 0.313*

Control maternal blood (n = 50)8-OHdG 0.095 0.148 −0.073 −0.136MDA – 0.137 0.075 −0.136Protein carbonyl – – −0.182 0.025GSH – – – 0.351*

Control cord blood (n = 50)8-OHdG 0.221 0.106 −0.110 0.193MDA – 0.291 −0.065 0.203Protein carbonyl – – −0.227 0.026GSH – – – 0.844*

* Correlations are significant at p < 0.05.

icology 31 (2011) 534–539 537

4. Discussion

To the best of our knowledge, this is one of the first report whichshows significant correlation between OCPs and non enzymaticoxidative stress markers in human IUGR cases. In the present studywe have found significantly higher levels of �-HCH in maternal andcord blood of IUGR subjects in comparison to controls. �-HCH is theonly insecticidal isomer of technical HCH and used either as suchor as an ingredient of technical grade HCH [23,24].

This study found a positive association between higher levelsof �-HCH and total HCH and IUGR (Table 2), which is in agree-ment with previous report [24]. Reduction in birth weight byHCH exposure was demonstrated in mice by Sircar and Lahiri[25] and significantly reduced birth weight and length in the off-spring of female day-care workers exposed to Polychlorinateddibenzofurans, 1-(1-phenylcyclohexyl) piperidine and �-HCH wereobserved by Karmaus and Wolf [26]. �-HCH has been also found toalter the level of thyroid, pituitary and sex hormones in females[27]. It has been shown that �-HCH suppresses follicle stimu-lating hormone and transforming growth factor �1 stimulatedprogesterone production [28]. Progesterone is thought to providetropic support for placental and consequently fetal growth sinceit has been reported that complete progesterone withdrawal atmid-pregnancy reduces placental protein synthesis [29]. There-fore, compounds that impair progesterone production, increaseits metabolism or block its action may, alter the homeostasisof pregnancy. �-HCH has also been found to inhibit cytochromeP450 (CYP) enzyme level and steroidogenic acute regulatory pro-tein (StAR) expression in cultured rat granulose cells [28]. StARprotein mediates the intramitochondrial transfer of cholesterolto the CYP 450 enzyme and CYP 450 enzyme catalyzes steroidhormone biosynthesis [30]. Impaired level of sex hormone mayinterfere with the normal pregnancy and can induce fetal abnor-malities. These observations suggest possible mechanisms bywhich �-HCH can be associated with IUGR as observed in ourstudy.

Recent studies have suggested the association betweenincreased oxidative stress and IUGR [6,7]. Significant increase inDNA damage as evident from 8-OHdG levels in IUGR cases wasfound in this study (Table 3). The former studies also confirm theseresults [31,32]. Significantly high level of 8-OHdG, MDA and pro-tein carbonyl and low level of FRAP and GSH in IUGR subjects ascompared to control subjects suggest a higher degree of oxidativestress in IUGR subjects. Thus, IUGR babies may be more vulner-able for the oxidative damage/injury and have diminished abilityto resist oxidative damage than control babies. These observationsare in agreement with previous studies which showed increasedoxidative stress in PTD and IUGR subjects [6,7].

In our study, the significant positive correlations of HCH isomerswith 8-OHdG, MDA and protein carbonyl (Tables 4–6) suggest thatOCP isomers may be associated with increased formation of freeradicals, thus producing oxidative stress in pregnant women. More-over, Hassoun et al. [33,34] have determined that �-HCH inducesoxidative tissue damage in fetal and placental tissues of mice andalso increases lipid peroxidation, superoxide anion production andDNA-SSB in hepatic and extrahepatic tissues. These observationsindicate a possible association of �-HCH with IUGR due to its feto-toxic effects which are a result of free radicals production. Recently,Tope and Panemangalore [35] found an approximately fourfoldincrease in mean values of plasma 8-OHdG levels in the pesticideexposed farm workers compared to control subjects. Lee et al. [36]

also showed that mean concentrations of 8-OHdG in summer andwinter were significantly higher in pesticide sprayers than controls.These results suggest a correlation between pesticide metabolitesand production of oxidative stress which results in higher DNAdamage.

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38 R. Pathak et al. / Reproducti

The dynamic structure of biomembranes and the strongipophilicity of HCH imply that the toxic effects of this xenobioticppear to be membrane-associated. In this context, it has been sug-ested that the disruption of the organization of plasma membranes the primary action of biologically active HCH isomers that mayffect cell survivability. OCPs are reported to reduce membraneuidity, which can reflect drastic changes in membrane function37,38]. We also found higher induction of lipid peroxidation asvident from higher MDA levels in IUGR cases in comparison toontrol subjects (Table 3). The mitochondria and microsomes wereound to be more susceptible to peroxidation. This may be due ton enhanced generation of superoxide radical and H2O2 that accel-rated peroxidation of native membrane lipids [39].

A recent study of our lab reported increased levels of MDA in sca-ies patients after treatment with �-HCH [40]. Samanta and Chainy38] have also observed higher lipid peroxidation in mice and ratestis after treatment with HCH. Oxidative stress causes peroxida-ion of lipids and formation of other reactive intermediates that

ay also result in increased oxidative damage of bimolecules asell as formation of protein carbonyl adducts [41]. An increase inrotein carbonyl levels with higher level of OCPs indicates that nor-al protein metabolism was disrupted, resulting in accumulation

f damaged molecules.Significant negative correlations of HCH isomers with GSH and

RAP were also observed in this study (Tables 4–6). This may beresult of regulation of GSH pools by endogenous enzymes that

an be associated with OCPs [42,43]. The negative correlation ofSH with OCPs may possibly be due to utilization of GSH to formonjugates with electrophilic metabolites of OCPs or due to moreffective oxidation of GSH by glutathione peroxidase. We havebserved higher degree of correlation between some of the HCHsomers and oxidative stress markers in IUGR than control subjectsTables 4–6). This may be due to comparatively higher levels ofhese OCP residues in IUGR cases.

Our observations suggest that all OCPs are not associated withncreased free radicals generation and they require a certain thresh-ld level, in order to exert any effects. In conclusion, our resultsdd to the body of the literature on possible association of OCPsith increased oxidative stress and risk of IUGR. This study mayighlight the potential reproductive risks of environmental chem-

cals on the “course of pregnancy” and women with a risk of IUGRay be benefited by knowing about their OCP burden in special-

zed clinics after consultation with physician/gynecologist. Furtheresearch is needed to corroborate these findings and shed light onhe clear biological mechanisms by which these types of chemicals

ay influence pregnancy outcomes.

cknowledgements

One of the authors, Mr. Rahul Pathak is grateful o Indian Coun-il of Medical Research New Delhi for awarding Senior Researchellowship (Reference No. 3/1/2/3/Env/07/NCD-I). Authors arerateful to Central Pollution Control Board, Delhi and Ministry ofnvironment and Forest (Govt. of India) for financial support. Theyre also thankful to Mrs. Meenu Misra for technical help.

eferences

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