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Clinical Biochemistry 42 (2009) 746–749

Case Report

Maternal and cord blood levels of organochlorine pesticides: Associationwith preterm labor

Rahul Pathak a, Rafat S. Ahmed a, A.K. Tripathi a, Kiran Guleria b, C.S. Sharma c,S.D. Makhijani c, B.D. Banerjee a,⁎

a Environmental Biochemistry and Immunology Laboratory, Department of Biochemistry, University College of Medical Sciences & G.T.B. Hospital(University of Delhi), Dilshad Garden, Delhi 110 095, India

b Department of Obstetrics and Gynaecology, University College of Medical Sciences & G.T.B. Hospital (University of Delhi), Dilshad Garden, Delhi 110 095, Indiac Instrumentation and Bio-Labs, Central Pollution Control Board, Ministry of Environment and Forest, Parivesh Bhavan, East Arjun Nagar, Delhi 110 032, India

Received 19 September 2008; received in revised form 14 November 2008; accepted 16 November 2008Available online 3 December 2008

Abstract

Objectives: Organochlorine pesticides (OCPs) have been reported to cause adverse reproductive outcomes. Hence, the present study wasconducted to estimate and compare the levels of OCPs in maternal and cord blood of preterm labor and full term labor cases as well as assess theirrole in preterm delivery.

Design and methods: 23 cases each of preterm labor and full term labor were included in the study. Maternal and cord blood OCP levels weredetermined by using a GC-MS equipped with an electron capture detector.

Results: Maternal and cord blood levels of α-HCH, β-HCH, γ-HCH, total-HCH, p,p′DDE and p,p′DDT were found higher in preterm laborthan full term labor cases. However, a statistically significant relation was observed between preterm birth and β-HCH levels only.

Conclusions: Higher blood levels of β-HCH may be associated with risk of preterm labor.© 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Keywords: Preterm labor; Organochlorine pesticides; β-HCH; Endocrine disruption; Estrogenicity

Introduction

Preterm labor is associated with perinatal mortality andattributes to long-term morbidity such as respiratory illness,infections, neurological impairment and developmental delay[1]. Despite substantial basic scientific and clinical investiga-tions, preterm labor continues to be a major clinical and publichealth problem of the highest order, yet mechanisms under-lying the onset of idiopathic preterm labor are poorly under-stood [2]. The pathophysiology of preterm labor is not entirelyclear but balance of reproductive hormones, such as progester-one and estrogen appears to have a role. Progesterone is widelyregarded as promoting uterine quiescence. Estrogen, on theother hand, may promote myometrial activation with increasedreceptivity to uterotonic agents by up regulating membrane

⁎ Corresponding author. Fax: +91 11 22590495.E-mail address: banerjeebd@hotmail.com (B.D. Banerjee).

0009-9120/$ - see front matter © 2008 The Canadian Society of Clinical Chemistsdoi:10.1016/j.clinbiochem.2008.11.007

receptors and gap junctions. Therefore, endocrine disruptors,especially those have estrogenic effect, may induce pretermlabor [3].

Endocrine disruptor chemicals (EDCs) are exogenoussubstances or mixtures that alter function(s) of the endocrinesystem and consequently cause adverse health effects. Thehomeostasis of sex steroids is the main target of EDC effects;hence, reproductive and developmental health is recognized asbeing especially vulnerable to endocrine disruption. EDCs maymimic, block or modulate the synthesis, release, transport,metabolism and binding or elimination of natural hormones.Even though potential EDCs may present in the environment atvery low levels, they may still cause harmful effects [4]. OCPssuch as 1, 1, 1-trichloro2, 2-bis (4-cholorophenyl) ethane (DDT)and hexachlorocyclohexane (HCH) may act as EDCs and inaddition to their endocrine disrupting properties, they can alsoaffect reproduction by induction of DNA damage, and oxidativestress [5–7]. DDT and HCH were previously used for pest

. Published by Elsevier Inc. All rights reserved.

747R. Pathak et al. / Clinical Biochemistry 42 (2009) 746–749

control in crops such as cotton and rice and vector controlprograms such as malaria and locust control in India [8]. DDTand HCH are persistent in the environment and continue to bedetected in the food chain despite their being banned for use inagriculture. DDT is now restricted to vector control programsonly and the production of technical HCH is being phased out.However, due to their lipophilic property and long half livesthese pesticides are biomagnified in the food chain and getaccumulated in human body, especially in fatty tissues. Mothersare exposed to OCPs mainly through food chain contaminationand these pesticides are transferred from mother to fetus duringpregnancy through placenta [9]. Placental transfer of thesepesticides to developing fetuses may affect growth anddevelopment of the baby in the womb [5]. The reproductiveeffects due to persistent exposure of OCPs are a matter ofworldwide concern. Many OCPs have been reported toadversely affect reproductive health and fetal development[10–12] via their function as xenoestrogen. Hormonal home-ostasis (particularly estrogen progesterone balance) is extremelyimportant in maintenance of pregnancy and OCPs can alter thishormonal homeostasis, which may result in termination ofpregnancy [13]. Although several studies have reported thereproductive effects of DDT in humans [12], there are very fewstudies about other organochlorines such as β-HCH. β-HCH isthe most persistent isomer of HCH which constitutes 7–12% oftechnical HCH. β-HCH has been found to be a xenoestrogen invarious in vitro and in vivo studies. β-HCH has negligibleinsecticidal activity but measurable estrogenic effect [14].Hence, in the present study, β-HCH and other OCP residuelevels in maternal and cord blood of women undergoingspontaneous preterm labor with intact membrane were comparedwith those of women with full term babies and the correlationbetween the level of OCPs and preterm labor was examined.

Materials and methods

Chemicals

HPLC-grade hexane and acetone were purchased from E.Merck (Mumbai, India. Florisil was purchased from Sigma-Aldrich Company (St. Louis, Mo, USA). All other chemicalswere of analytical grade and obtained from E. Merck (Mumbai,India).

Cases and controls

The present hospital based case control study was carried outto determine the association of OCP levels with preterm labor.Twenty three primiparous women (study group) having pretermlabor with less than 37 weeks of gestation were included in thisstudy after their admission to Guru Teg Bahadur Hospital,Delhi. All went into labor spontaneously with intact membrane.Study group was compared with same number of women(control group) undergoing spontaneous labor at term. Womenwith anemia, hypertension, bacterial vaginosis, toxemia ofpregnancy, renal disease, heart disease, diabetes, urinary trackinfections, metabolic disorders, tuberculosis, smoking, alcohol

consumption or chronic drug intake and having complicationsduring pregnancy and/or delivery were excluded from both thegroups. A life style survey of the women was done to collectgeneral demographical information in order to define theinclusion/exclusion criteria. The women who participated inthis study were of relatively homogenous group; they weresimilar in terms of demographical characteristics such as age,weight, parity, food habits, BMI, socioeconomic status,drinking water supply and life style. We have excludedpotentially confounding factors such as women of occupationalexposure to pesticides and farming communities from thisstudy. Women confirmed their participation by signing aconsent form and this study was approved by the institutionalethical clearance committee for human research.

Samples

One milliliter, each of maternal and cord blood sample wascollected in EDTA containing vial at the time of delivery. Allthe tests were performed within 12 h of sample collection.

Extraction of OCPs from blood samples

All the chemicals used in the process were of high-puritygrade. The HPLC-grade solvents were checked for any con-tamination before extraction. OCP residues extraction wasdone by using hexane and acetone (2:1) according to methodof Bush et al. [15]. Cleanup was done by USEPA methodusing Florisil (Sigma) by column chromatography. Concen-trated samples after evaporation were taken to Central Pollu-tion Control Board, Delhi, for gas chromatography analysis.Quantification of organochlorine residue levels was done byPerkin Elmer GC-MS equipped with 63Ni selective electroncapture detector. Quantitative analysis of OCP residues in eachsample was done by comparing the peak heights with thoseobtained from a chromatogram of a mixed organochlorinesstandard of known concentration. Ten samples each ofmaternal and cord blood in triplicate were spiked with amixed standard of OCPs. The average recoveries of fortifiedsamples exceeded 95%. Further, a quality control check of thesamples was always run with each set of samples to maintainaccuracy.

Statistical analysis

All the values were expressed as mean±standard deviationand percentile basis. The data were analyzed by ANOVA andfollowed by Dunnett's test for multiple comparisons. Correla-tion between individual OCPs in maternal and cord blood wastested using Pearson co-efficient of determination. The valuesof pb0.05 were considered to denote significance.

Results

OCP levels of study group and control group in maternal andcord blood are listed in the Tables 1 and 2 respectively. Inpreterm labor cases, maternal and cord blood had higher and

Table 1Comparison of OCP levels (ng/mL) in maternal blood of full term and preterm labor cases

Full term labor (n=23) Preterm Labor (n=23)

Mean (SD) 25% 50% 75% Mean (SD) 25% 50% 75%

α-HCH 5.70±3.43 1.35 7.14 8.57 5.73±3.53 3.36 5.14 7.84β-HCH 6.55±3.21 2.54 6.39 9.31 9.83±4.13 5.87 7.62 16.38 a

γ-HCH 6.34±4.44 3.97 6.96 7.75 7.94±5.50 4.69 6.98 7.84T-HCH 8.59±8.11 18.29 20.07 25.14 23.50±9.95 17.23 20.09 32.85p,p′DDE 3.70±2.63 0.00 3.58 4.36 3.77±3.32 0.00 3.32 4.51p,p′ DDT 1.66±1.18 0.00 1.87 2.86 1.71±1.85 0.00 1.26 2.32a Significantly high from full term labor (pb0.05).

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statistical significant levels of β-HCH than full term labor cases.Although, concentrations of α-HCH, γ-HCH, Total-HCH, p,p′DDE and p,p′DDT was also higher in preterm labor cases ascompared to full term labor cases in maternal as well as cordblood, however, the differences were not statistically signifi-cant. Pearson coefficients show a statistically significant andpositive correlation between maternal and cord blood levels ofall the OCPs (Table 3). It can therefore be suggested thatmaternal blood can be used to predict cord blood levels.

Discussion

We found significantly higher concentration of β-HCH instudy group than control group and these results are in agreementwith previous reports, [11,12] which showed associationbetween high β-HCH levels and preterm labor. Preterm laborcontinues to be a major clinical and public health challenge ofthe highest order. Incidence of various adverse reproductiveoutcomes is becoming more common and especially pretermbirth is a major contributor of infant mortality in India andconstitutes 31% of neonatal deaths [16]. The underlying cause ofpreterm labor after prolonged rupture of the membranes isclearly different from that of preterm labor with intact mem-brane. Local infection leads to the onset of labor by a method,which bypasses the increase in the estrogen to progesterone ratiothat normally occurs before labor. Hence, we have selected casesof preterm labor with intact membrane only.

β-HCH by itself is neither being produced nor marketed asinsecticide. It is a constituent of technical HCH used as organo-chlorine insecticide or chemical intermediate to manufactureenriched HCH (lindane) [17]. Li et al. [18] estimated global

Table 2Comparison of OCP levels (ng/mL) in maternal and cord blood of full term and pre

Full term labor (n=23)

Mean (SD) 25% 50% 75%

α-HCH 3.30±3.29 0.50 3.53 4.8β-HCH 3.59±1.84 1.12 4.02 4.9γ-HCH 2.60±2.56 1.02 1.54 3.2T-HCH 9.49±6.95 5.66 11.34 12.4p,p′DDE 1.98±1.86 1.12 2.31 3.2p,p′ DDT 0.93±1.85 0.00 0.00 1.8a Significantly high from Full term labor (pb0.05).

emissions of β-HCH from the usage of technical HCH between1945 and 2000 at 850,000 tons, of which 230,000 tons wereemitted into the atmosphere over the same period. Its chemicalstructure seems to confer the greatest physical and metabolicstability. β-HCH is the predominant isomer in soils and animaltissues because its configuration favors storage in biologicalmedia and affords its greater resistance to hydrolysis andenzymatic degradation [19].

A possible association between maternal and cord bloodlevels of β-HCH in preterm labor was observed in this study.However, the mechanism behind this association is not clear,but it has been shown that β-HCH increases uterine contrac-tion frequency in a concentration-dependent manner in rats [7].β-HCH may be the most toxicologically significant HCHisomer as evidenced by recent reports of its estrogenic effects inmammalian cells, laboratory animals and fish [20–23]. It hasbeen reported that blood levels of β-HCH in ppb (ng/mL) rangehave the potential of producing estrogenic effects in mice [20].β-HCH with mixture of other OCPs has the ability to generatean estrogenic microenvironment through estrogen receptor(ERα) activation [21]. β-HCH produces moderate uterotrophiceffects in the rodent uterus [20]. Moreover, β-HCH exposureat low concentration results in induction of vitellogenesis andhermaphroditism in freshwater fish [22]. Adverse effects of β-HCH were also seen in an in-vivo rat bone marrow chromo-somal aberration study [23]. These observations lend support tothe role of β-HCH in reproductive toxicity and a possibleassociation with preterm labor due to its estrogenicity.

In summary, our results suggest an association of pretermlabor with higher blood levels of β-HCH. However, a smallsample size in the study limits the sensitivity and statistical

term labor cases

Preterm labor (n=23)

Mean (SD) 25% 50% 75%

6 3.32±2.68 0.95 3.49 5.327 5.48±3.50 2.68 6.55 7.51 a

5 3.38±3.24 2.29 5.67 6.551 12.18±6.13 10.65 13.01 16.211 2.61±2.23 0.55 2.36 3.579 1.05±2.56 0.00 1.00 1.54

Table 3Pearson correlation coefficient (r) for OCPs in maternal and cord blood in fullterm and preterm labor cases

Full term labor Preterm labor

α-HCH 0.608 a 0.827 a

β-HCH 0.949 a 0.619 a

γ-HCH 0.717 a 0.840 a

Total HCH 0.871 a 0.917 a

P,P'DDE 0.794 a 0.931 a

P,P'DDT 0.597 a 0.857 a

a Correlation is significant at the pb0.01 level.

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power of our data. Hence, we are unable to conclude the role ofthese OCPs in preterm labor cases and these results must beinterpreted with caution. Larger studies on OCPs exposure andrisk of preterm labor are further required to address theseuncertainties.

Acknowledgments

The authors are grateful to Mrs. Meenu Mishra for technicalhelp and Central Pollution Control Board (CPCB), Ministry ofEnvironment and Forest, East Arjun Nagar, Delhi, 110032 forproviding financial assistance for this study.

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