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Toxicology 307 (2013) 74– 88

Contents lists available at SciVerse ScienceDirect

Toxicology

j our nal ho mep ag e: www.elsev ier .com/ locate / tox ico l

ersistent organochlorinated pesticides and mechanisms of their toxicity

zra J. Mremaa,b,∗, Federico M. Rubinoa, Gabri Brambillaa, Angelo Morettoc,ristidis M. Tsatsakisd, Claudio Colosioa

Department of Health Sciences of the University of Milan, International Centre for Rural Health of the Occupational Health Unit of the University Hospital San Paolo, and Laboratoryor Analytical Toxicology and Metabolomics, Via San Vigilio 43, 20142, Milan, ItalyDepartment of Environmental and Occupational Health, Muhimbili University of Health and Allied Sciences, P.O. Box 65015, Dar es Salaam, TanzaniaDepartment of Biomedical and Clinical Sciences, Luigi Sacco of the University of Milan and International Centre for Pesticides and Health Risk Prevention (ICPS) of the Universityospital Luigi Sacco, Via GB Grassi 74, 20157 Milan, ItalyCentre of Toxicology Sciences and Research, Division of Morphology, Medical School, University of Crete, Voutes, Heraklion, 71003 Crete, Greece

r t i c l e i n f o

rticle history:eceived 22 October 2012eceived in revised form4 November 2012ccepted 27 November 2012vailable online 3 December 2012

eywords:uman exposuredverse health effectrganochlorinated pesticidesechanisms of action

a b s t r a c t

Persistent organic pollutants comprised of organic chemicals like polychlorinated biphenyls, dibenzo-p-dioxins, dibenzofurans and organochlorinated pesticides which have many characteristics in common.Once released in the environment they resist physical, biological, chemical and photochemical breakdownprocesses and thus persist in the environment. They are subject to long transboundary air pollution trans-port. They accumulate in the food chain due to their lipophilicity, bioaccumulation and biomagnificationproperties. Human exposure occurs through inhalation of air, ingestion of food and skin contact. Becausemost of them bioaccumulate and remain preferentially in fat, their long-term effects are still a matter ofpublic health concern. They are condemned for health adverse effects such as cancer, reproductive defects,neurobehavioral abnormalities, endocrine and immunological toxicity. These effects can be elicited via anumber of mechanisms among others include disruption of endocrine system, oxidation stress and epi-genetic. However most of the mechanisms are not clear thus a number of studies are ongoing trying toelucidate them. In this review, the underlying possible mechanisms of action and their possible roles in

adverse developmental and reproductive processes are discussed and where possible a linkage is made tosome existing epidemiological data. Both genomic and nongenomic pathways are used to describe theseeffects. Understanding of these mechanisms will enable development of strategies to protect the pub-lic by reducing these adverse effects. This review is limited to persistent organochlorinated pesticides(OCPs) such as dichlorodiphenyltrichloroethane (DDT) and its metabolites, hexachlorobenzene (HCB),beta-hexachlorocyclohexane (�-HCH) and endosulfan.

Abbreviations: •NO, nitrogen oxide radical; 16�-OHE, 16�-hydroxyestrone;-OHE, 2-hydroxyestrone; 6 CpG, Cytosine Guanine base pair; DDT,ichlorodiphenyltrichloroethane; GABAA, gamma butyric acid type Aeceptor; HCB, hexachlorobenzene; HO• , hydroxyl radical; o,p′-DDD,,1-dichloro-2-(o-chlorophenyl)-2-(p chlorophenyl)ethane; o,p′-DDE,,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethylene; o,p′-DDT, 1,1,1-richloro-2-(o-chlorophenyl)-2-(p chlorophenyl)-ethane; O2

•− , superoxideadical; p,p′-DDD, 1,1-dichloro-2, 2-bis (4-chlorophenyl)ethane; p,p′-DDE,,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene; p,p′-DDT, 1,1,1-trichloro-,2-bis(p-chlorophenyl) ethane; PCBs, polychlorinated biphenyls; PCDDs,olychlorinated dibenzo-p-dioxins; PCDFs, polychlorinated dibenzofurans; PI3K,hosphatidylinositol 3-kinase; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; �-HCH,eta-hexachlorocyclohexane.∗ Corresponding author at: Department of Health Sciences of the University ofilan, Via San Vigilio 43, 20142 Milan, Italy. Tel.: +39 02 81843469;

ax: +39 02 49538671; mobile: +39 327 2262702.E-mail addresses: ezra.mrema@unimi.it, ezrajm@yahoo.com (E.J. Mrema).

300-483X/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.tox.2012.11.015

© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Persistent organochlorinated pesticides are a structurally het-erogeneous class of organic compounds composed primarily ofcarbon, hydrogen and several chlorine atoms per molecule. Theyshare similar properties with other persistent organic pollutants(POPs) such as polychlorinated biphenyls (PCBs) and polychlori-nated dibenzo-p-dioxins/furans (PCDDs/Fs). Once released in theenvironment, they break down very slowly in air, water, soil andin living organisms and are thus subject to long trans-boundaryair pollution (LTAP) transport where they are carried over long dis-tances via the atmospheric transport and human activities. Even thebody burden of the flocks of migratory birds is a sizeable contribu-tion to LTAP (WHO, 2003). As OCPs bio-magnify through the food

chain, consumers of food of animal origin such as fish, meat, milkand dairy products end up with high levels of exposure and dueto slow biodegradation, OCPs accumulate in the body: for exampleDDT can remain in the body for 50 years, lifelong sequestrated into

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he soft tissue compartments (mainly the adipose tissues), fromhich they partition to the bloodstream into plasma or serum lipids

nd are actively secreted into breast milk as the main eliminationathway in mammals.

Human exposure begins during early prenatal and continuesuring the breast-feeding neonatal periods, which are criticaltages for the development and differentiation of sensitive bodyrgans and systems. In fact, these chemicals cross the placenta tohe foetus and are secreted into breast milk (Perera et al., 2005).

In adults the dietary exposure route accounts for more than 90%f total organochlorine compounds (OCs) burden for the generalopulation, while workers are exposed mainly through inhalationnd skin contact. OCs are rapidly absorbed in the small intestinend enter circulatory system where they are distributed throughouthe body and accumulate in body tissues with high lipid contents,ith a continuous exchange between blood and tissues. As such

hese chemicals have been detected in human tissues such aslood (whole blood, cord blood, serum and plasma), adipose tis-ues (in autopsies and living subjects), breast milk, muscles and hairAppenzeller and Tsatsakis, 2012; Covaci et al., 2002; Dewailly et al.,999; Jakszyn et al., 2009; Mrema et al., 2011; Rappolt and Hale,968; Tsang et al., 2011; Tsatsakis and Tutudaki, 2004; Tsatsakist al., 1998, 2008; Tutudaki et al., 2003).

Exposure to these persistent chemicals has been associated withealth effects including cancer (Aronson et al., 2000; Cohn et al.,007; Mathur et al., 2002; Recio-Vega et al., 2011), reproductiveefects (Nicolopoulou and Stamanti, 2001) and behavioral changesZala and Penn, 2004). These effects are believed to be related toheir ability to disrupt the functions of certain hormones, enzymes,rowth factors, neurotransmitters and to induce key genes involvedn metabolism of steroids and xenobiotics (Gourounti et al., 2008).

The mechanism of action involves toxicokinetic and toxicody-amic processes, at several levels between chemical exposure andhe endpoints at individual level. Variation within humans canffect the endpoint at any toxicodynamic and/or toxicokinetic stepsMortensen and Euling, in press). Direct, non/genomic mechanismsf toxicity involve perturbation of the homeostatic redox level(s) ofiological compartments (oxidative stress) and consequent cellulareath through apoptosis. Indirect, genomic mechanisms of toxic-

ty involve permanent modification of the transcription of genelements by interference at the epigenetic level of DNA functioning.

Since little is known on the relationship among events thatesult in OCPs’ toxicity in humans (Mortensen and Euling, in press),

number of studies are currently ongoing to investigate mecha-isms responsible for OCPs’ toxicity. A better knowledge on howhese compounds influence the development and progression ofiseases is a crucial step towards addressing the emerging dis-ases and hence improving public health. In this review, we providen overview on available information of possible mechanisms ofoxicity of some selected OCPs and where possible a linkage is

ade to epidemiological data. We summarize findings of epidemi-logical studies and both in vitro and in vivo experimental studiesTables 1–3).

The main OCPs for which we review available informationre dichlorodiphenyl-trichloroethane (DDT) and its metabolites,exachlorobenzene (HCB), beta-hexachlorohexacyclohexane (�-CH) and endosulfan. These chemicals have been employed ingriculture and in public health as insecticides and biocides foreveral decades but were mostly banned between the ‘1970 and990s’. Only a few active substances, essentially DDT is still used

n tropical countries to control malaria vectors through indooresidual spraying (Bouwman et al., 2011) and lindane for treat-

ent of lice and scabies (Engeler, 2009). So, most exposures

erive from past uses and, in certain cases, are still significantecause of the long persistence and biomagnification of theseompounds.

y 307 (2013) 74– 88 75

2. Mechanisms of OCPs toxicity

2.1. Endocrine activity

The endocrine system controls, balances and produces hor-mones and modulates their actions in the body through a networkof activation and repression pathways at multiple levels of synthe-sis, secretion, transport, binding, action or elimination of naturalhormones. Steroid hormones are involved in cell development,metabolism, protein synthesis, behavior, reproduction and devel-opment. These responses are elicited through binding of hormonesto their specific receptors such as nuclear receptors (NRs). Amongthe members of NRs are estrogen receptors (ERs), androgenreceptor (AR), progesterone receptor (PR), glucocorticoid receptor(GR), constitutive androstane receptor (CAR), rodent pregname Xreceptor (PXR), mineralocorticoid receptor and thyroid hormonereceptors (TRs). Aromatic hydrocarbon receptor (AhR) is a key reg-ulator of the cellular response to xenobiotic exposure (Swedenborget al., 2009). NRs and AhR are involved in the induction of xenobioticmetabolizing enzymes.

In the absence of ligands NRs and AhR stay in the cytoplasm orin the cell nucleus in a complex form (Picard, 2006). In the presenceof ligands, receptor-ligand binding occurs, followed by translo-cation of NRs/AhR to the nucleus. In the nucleus, a complex ofaryl receptor nuclear translocator (ARNT) protein and co-activatorsis formed. The complex binds to the specific AhR-DNA recogni-tion site and finally induces transcription of target genes. Theligand binding triggers conformational changes that lead to dissoci-ation of the repressive complex, the recruitment of transcriptionalco-activators and receptor dimerization (Hankinson, 2005). NRshomodimerize or heterodimerize with retinoid X receptor and AhRdimerizes with ARNT leading to gene transcription of NR or AhRtarget genes (Claessens and Gewirth, 2004; Swanson, 2002). NRscontrol transcription through binding to promoter regions of DNA.

Several chlorine atoms in the molecular structures of OCPsimpart their highly lipophilic character and fairly rigid confor-mation (Fig. 2). The chlorine atoms are poorly reactive towardsnucleophilic displacement and elimination reactions and thus theirbiotransformation and biodegradation reactions are limited and aremostly confined, albeit to poor yield, only to anaerobic environmentof sludges, but seldom in mammalian organisms. As a consequence,their interaction with biological systems is mostly limited to ago-nistic or antagonistic binding to the intracellular receptors forwhich natural hydrophobic substances, such as steroid derivatives,are the endogenous ligands. Agonistic binding leads to recruit-ment of coactivators and thus increases transcriptional activitywhile antagonistic binding prevents coactivator recruitment and/orattracts corepressors, leading to decreased transcriptional activityof the receptors.

Many POPs including OCPs are known or suspected to beendocrine active. When present in the body they may interfereat several control points in the hormone signaling pathways. Asa result, the response cascade of natural hormones can either beinhibited or excessively enhanced, at the wrong time, in the wrongtissue (Swedenborg et al., 2009). Endocrine activity of OCPs can bedue to direct binding with hormone receptors due to their confor-mational similarity with the receptor-binding portions of naturalhormones, mainly of the steroid and diphenylether (thyroxine)structural groups. This is the case of aromatic polychlorinated sub-stances such as DDT and its structural cognates, endosulfan andlindane. Other compounds indirectly alter hormonal pathway bydirectly inhibiting enzyme activities responsible for biosynthesis

of the precursors of steroid hormones. In particular, the DDT ana-log, mitotane, a pharmaceutical drug which is used to suppresscortisol production also causes a sharp raise in plasma cholesterollevels.

76 E.J. Mrema et al. / Toxicology 307 (2013) 74– 88

Table 1Summary for selected epidemiological studies showing the relationship of DDT/DDE exposure and health-related effects.

Author Effects assessed OCP measured OCP level(�g/L serum)

OCP level (�g/glipid serum)

Results ptrend

Cohn et al. (2007) Breast cancer DDT Tertiles Tertiles OR, 95% CI<8.09 <1.1 1.00 0.018.09–13.9 1.1–1.8 2.8 (1.1–6.8)>13.9 >1.8 5.4 (1.7–17.1)

McGlynn et al. (2006) Liver cancer DDT – Quintiles OR, 95% CI<0.265 1.00 0.0490.265–0.382 1.3 (0.7–2.5)0.383–0.521 1.4 (0.7–2.6)0.522–0.787 1.4 (0.7–2.7)>0.787 2.0 (1.1–3.9)

McGlynn et al. (2006) Liver cancer DDT adjusted for DDE level – Quintiles OR, 95% CI<0.265 1.00 0.00240.265–0.382 1.5 (0.8–2.7)0.383–0.521 1.7 (0.9–3.3)0.522–0.787 1.4 (1.0–4.3)>0.787 2.0 (1.7–8.6)

McGlynn et al. (2008) Testicular germ tumors DDE – Quartiles RR, 95% CI≤0.157 1.00 0.00020.158–0.250 1.01 (0.75–1.36)

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.1.1. Interference of OCPs in estrogen hormone metabolism17�-Estradiol is the main estrogen hormone, responsible for

evelopment of female sex characteristics such as growth ofhe breasts and suppression of body hair growth. Biotransfor-

ation of 17�-estradiol proceeds via two common metabolicathways yielding catechol estrogen 2-hydroxyestrone (2-OHE),

weak estrogen; and 16�-hydroxyestrone (16�-OHE), a fullyotent estrogen. Enzymes CYP1A1, CYP1B1, CYP1A2 and catechol--methyltransferase (COMT) are responsible for this metabolism.6�-OHE, but not 2-OHE increases unscheduled DNA synthesis,ncontrolled cell division and increased anchorage-independentrowth. Interrupting these pathways might lead to formation ofore potent products as exemplified by some xenoestrogens.

xposure of cell cultures of MCF-7 to concentrations as low as0−5 M of various OCPs such as DDT, lindane, chlordecone andndosulfan blocked the 2-OHE pathway and increase levels of 16�-HE in estrogen positive (ER+) cells (Bradlow et al., 1995). This

nterference in estrogen biotransformation led to increasing 16�-HE/2-OHE ratio relative to the ratio in the untreated control cellultures. This increasing ratio has been associated with breast andther cancers of animals (Telang et al., 1992) (Fig. 1); possiblyecause of 16�-OHE has a strong estrogenic activity (Bradlow et al.,995). Levels of 16�-hydroxylation have been found increased bybout 50% in human breast cancer as compared to control casesSchneider et al., 1982).

CYP1A1 metabolizes estradiol and environmental contami-ants into the corresponding hydroxylated derivatives through theighly reactive intermediates of arene-oxide connectivity (Hayest al., 1996). The expression of CYP1A1 is regulated by the arylydrocarbon receptor (AhR), a ligand-dependent transcription

actor that activates the transcription of target genes, such asYP1A1, CYP1A2, CYP1B1 and oncogenes (Androutsopoulos et al.,009b). DDT and its metabolites are capable of up regulatingYP1A activity (Van Tonder, 2011). According to Navas et al. (2004)YP1A inducers have defined structures which make them capablef binding AhR. DDT has two aromatic rings linked by sp3 carbonnd thus each of the rings can rotate along its single bond with the

2 carbon. This allows some degree of conformational mobilitynd in principle the possibility to bind AhR but the rings cannot lien the same plane, as in the estrogen molecules (Fig. 3). Lack of this

olecular recognition motif greatly limits the affinity of AhR for

0.251–0.390 1.00 (0.73–1.38)>0.390 1.71 (1.23–2.38)

DDT. In fact, the concentration of DDT employed in the Bradlowexperiments is 10−5 M (10 �M), while the physiological concen-tration of steroid hormones such as estradiol is of at least four tosix orders of magnitude lower (low-picomolar). On the other handDDT and its metabolites as well as many other OCPs are knownphenobarbital-type cytochrome P450 inducers as they have beenshown to induce cytochrome P450 2B (CYP 2B) in rat liver or cul-tured rat hepatocytes (Campbell et al., 1983; Li et al., 1995; Lubetet al., 1990, 1992; Yoshioka et al., 1984). The enzyme activationupon exposure to these pesticides can thus lead to alterations inthe endogenous levels of hormones and consequently compromisehormone signaling. In addition to xenobiotics metabolism, CYP1A1has been shown to participate in the activation of dietary polyphe-nols (Androutsopoulos et al., 2008, 2009a) and it is inhibited byvarious polyphenolics (Androutsopoulos et al., 2010, 2011).

2.1.2. DDT and DDT metabolitesDDT is produced as a technical mixture of 3 isomeric forms, the

most prevalent being the p,p′-isomer; o,o′- and o,p′-being present inminor and variable amounts. DDT was initially used in public healthcontrol of lice and malaria mosquitoes during the Second WorldWar and afterwards it was used as pesticide in agriculture. GloballyDDT production was ∼1.8 million tonnes since 1940s (ATSDR, 2002)and more than 40,000 tonnes were used in agriculture annually(Geisz et al., 2008). It is estimated 600,000 tonnes of DDT wereused domestically in USA 30 years prior to its ban. Over 80% ofthe quantity of the pesticide used in 1970–1972 was applied tocotton crops and the remainder was used on peanut and soybeancrops. The global production was reported to drop to 3314 tonnesin 2009; the production is meant for use in control of malaria andleishmaniasis (UNEP, 2010a).

DDT and its metabolite DDE exhibit hormonal activity in vari-ous tissues with mechanisms involving the steroidogenic pathway,receptor mediated changes in protein synthesis or antiandrogenicand estrogenic actions. Most of their endocrine effects result fromthe ability to mimic 17-�-estradiol. o,p′-DDT is a well character-ized DDT isomer; its intracellular mechanism of action is mediated

through the ER pathway (Steinmetz et al., 1996) described in Sec-tion 2.1. It has been shown to bind and activate the ER, to promotethe expression of estrogen-dependent genes and to induce prolif-eration of ER-competent cells such as breast cancer cells (MCF-7)

E.J. Mrema et al. / Toxicology 307 (2013) 74– 88 77

Table 2Summary of selected in vitro, in vivo and epidemiology studies on the endocrine effects of endosulfan.

Author Study type Study sample/materials Effects investigated Observed effects

Soto et al. (1994) In vitro MCF-7 cell culture Cell proliferation potential ofpesticides

Endosulfan was 106 times less potent than17�-estradiol. Effects observed at doses of10 �M endosulfan or greater.

Soto et al. (1995) In vitro MCF-7 cell culture Relative binding affinity ofpesticides

Endosulfan hPR receptor binding affinity was105 less than 17�-estradiol.

Wade et al. (1997) In vitro MCF-7 cell culture Cell proliferation potential ofpesticides

Endosulfan was 105 less potent as compared to17�-estradiol. Effect observed at the highestsoluble dose.

Sinha et al. (1999) In vitro Rat testicular cells in culture Cell viability and lactatedehydrogenase leakage

Endosulfan produced cytotoxic change. Effectsobserved at 20 �M of endosulfan at or above.

Sinha et al. (2001b) In vitro Rat testicular cells in culture Activity of polyol pathwayenzymes

Increased aldose reductase activity anddecreased sorbitol dehydrogenase activity ator >20 �M of endosulfan.

Grunfeld andBonefeld-Jorgensen(2004)

In vitro MCF-7 BUS cells Human ER� and ER� mRNAexpression

Endosulfan decreased human ER� mRNAlevels weakly with no effect on human EB�expression.

Li et al. (2006) In vitro MCF-7 cell culture Activation of MAPK, PI3-K,PKC, PKA and CaMKIV

At 20–75 �M endosulfan activated P13-K andMAPK. The effect observed at 10 nM of17�-estradiol and DES.

Sinha et al. (1995) In vivo Adult Druckrey ratsadministered with repeatdose of endosulfan (0, 2.5, 5or 10 mg/kg, 5 days per weekfor 70 days)

Testicular enzyme activities,sperm count, spermmorphology, andintra-testicular spermatidcount

Increased testicular enzyme activities at alldoses tested; sperm count decreased in adose-dependent manner; decreased spermatidcounts and reduced daily sperm production atthe two highest test doses was reported.

Sinha et al. (1997) In vivo Weanling Druckrey ratsadministered with repeatdose of endosulfan (0, 2.5, 5or 10 mg/kg, 5 days per weekfor 90 days)

Testicular enzyme activity,testes weights, sperm count,sperm abnormality,spermatid count and dailysperm production

No observed effects on testes weights;increased levels of testicular enzyme activity;sperm count decreased in a dose-dependentmanner; decreased spermatid count andsperm production rate.

Raizada et al. (1991) In vivo Ovariectomized Wistar ratsadministered with repeatdose of endosulfan(1.5 mg/kg daily for 30 daysand injected daily withestradiol 1 �g/rat i.p.)

Organs weights, glycogencontent of organs, andhistopathological changes inorgans

No effects on weights of uterus, cervix, vaginaand pituitary; no effects on glycogen content ofthe organs examined; no histopathologicalchanges reported.

Shelby et al. (1996) In vivo Mice administered withrepeat dose of endosulfan onpostnatal days 17–19 at adose of 10 mg/kg

Competitive binding ofendosulfan to mouseestrogen receptor anduterine growth

No competitive inhibition of estrogen receptorobserved; no increase in uterine wet mass;DES and estradiol significantly affected uterinegrowth.

Wade et al. (1997) In vivo 18 days rats injected with3 mg/kg/day endosulfan dailyfor 3 days

Uterine estrogen receptorbinding

Estradiol binding inhibited at high doses(105 M endosulfan); no change in uterineweights, uterine peroxidase activity ornumbers of uterine estrogen and progesteronereceptors.

Saiyed et al. (2003) Cohort Male children lived in thevillage sprayed withendosulfan and children wholived in 20 km away fromvillage

SMR assessment; serumtestosterone, LH, and FSH;and endosulfan residues inblood

Sexual maturity delayed in the exposedpopulation; controlled for age, only serum LHlevels differed between the populations.

Ibarluzea et al. (2004) Case–control Breast cancer women (cases)and non-cancer women(controls)

Relationship between cancerdiagnosis and the adiposepesticides

Endosulfan residues were not associated withincreased risk of breast cancer

Abbreviations: PI3-K, phosphatidylinositol-3-kinase; PKC, protein kinase C; PKA, protein kinase A; CaMKIV, calcium calmodulin-dependent kinase IV; AI, apoptotic index;M hormr

inaiwaaWogeao

h

CF-7, Michigan Cancer Foundation-7; SMR, sexual maturity rating; LH, luteinizingeceptor; hPR, human progesterone; DES, diethylbestrol.

n vitro (Soto et al., 1995). At 10 �M concentration, o,p′-DDT ago-ized both ER�- and ER�-mediated transcription a potency that ispproximately 0.1% that of estradiol (Lemaire et al., 2006). A signif-cant dose-dependent increase in cell number in MCF-7 cell culture

as observed starting at 10−8 M and maximum response achievedt 10−6 M (Steinmetz et al., 1996). Enantiomer-specific estrogenicctivity has been suggested (Hoekstra et al., 2001; McBlain, 1987;ang et al., 2009). At 5.26 × 10−5 M (S)-(−)-o,p′-DDT produced half

f the maximum effect (EC50) whereas (R)-(+)-o,p′-DDT had negli-ible estrogen activity (Hoekstra et al., 2001). The (+)-enantiomerxhibited estrogen-like response only at an environmentally unre-

listic concentration of 10−3 M, and thus estrogenic activity of,p′-DDT is due to the (−)-enantiomer.

Transient cotransfection assay of monkey kidney CV1 cells withuman AR expression vector and a mouse mammary tumor virus

one; FSH, Follicle Stimulating Hormone; i.p., intraperitoneal injection; ER, estrogen

promoter with a luciferase reporter vector revealed that androgen-induced AR transcriptional activity was inhibited by p,p′-DDE or bythe potent antiandrogen, hydroxyflutamide at 0.2 �M (63.6 ppb)concentration. This level of p,p′-DDE is lower than 140 ppb foundamong residents living in DDT-treated homes (Bouwman et al.,1991). In utero exposure to DDE has been shown to decreaseanogenital distance (a morphologic quantitative indicator of fem-inization of male offspring) in male rat offspring, to increaseretention of male nipple and to cause an alteration in expression ofandrogen receptor (Kelce et al., 1995) suggesting that these abnor-malities might be mediated at the level of the androgen receptor.

In addition to anti-androgen activity, p,p′-DDE can activate theER and induce cell proliferation (Kelce et al., 1995). 17�-Estradiol(1 × 10−8 M), o,p′-DDT (1 × 10−5 M) and �-HCH (1 × 10−5 M) sim-ilar showed gene expression profile of estrogen-responsive genes

78E.J.

Mrem

a et

al. /

Toxicology 307 (2013) 74– 88

Table 3Summary of recent studies on the apoptotic effects of OCPs (DDT, DDE, methoxychlor, �-HCH and �-HCH).

Author Study type Study sample/material Effect investigated Observed effects

Shi et al. (2009) In vitro Rat Sertoli cells from testes of 18 to20 days old male Sprague-Dawleyrats. The cells were incubated with10, 30, 50 or 70 �M p,p′-DDE for24 h

Effects of p,p′-DDE on apoptosis, FasL,caspase-3, caspase-8 mRNA, procaspase-3,procaspase-8 and NF-�B activation.

Apoptotic cell death observed at >30 �M; FasL mRNA levels higher in a 50 �M group;caspase-3 mRNA levels higher in 30 and 50 �M group; caspase-8 mRNA increased indifferent doses; FasL mRNA induced in 50 �M group, NAC attenuated this effect.Procaspase-3 and -8 significantly reduced over 30 and 10 �M, respectively; NF-�Bactivation enhanced with increase of dosage.

Shi et al. (2010) In vivo 20-Day old male rats Prepubertalrats administered with 0, 20, 60,100 mg/kg b.wt of p,p′-DDE for 10days of treatment

Effects of p,p′-DDE on body weights, organsweights, apoptosis, GSH-Px activity, SODactivity, MDA level, Fas, FasL; caspase-3 and-8; procaspase-8, NF-�B p65 proteins,caspase-3 and -8 activities in rat testis.

No change in body weights and testis weights; selective degeneration of germ cells atthe seminiferous tubules observed at >20 mg/kg b.wt; all tested doses induced MDAincrease and decrease SOD and GSH-Px activity; mRNA level of Fas, FasL, caspase-3and -8 elevated in 100 mg/kg b.wt group; p,p′-DDE induced increase in FasL andreduction of procaspase-8; NF-�B p65 activated in 60 and 100 mg/kg b.wt groups;caspase-3 and 8 activities increased in 100 mg/kg b.wt group.

Shi et al. (2011) In vitro Rat Sertoli cells from testes of18–20 days old male rats treatedwith 10, 30, 50, 70 �M �-BHC totest the viability of cells. 50 �Mwas found to be the maximumtolerant dose. Thus10, 30 and50 �M �-BHC (�-HCH) were usedin subsequent experiments

Effect of �-BHC on apoptosis, ROS production,LDH leakage rate, SOD activity, MDA level,JNK/p38 MAPK protein levels, FasL,procaspase-3 and -8 protein levels, NF-�Bactivation, NF-�B p65 protein levels.

Apoptotic rate increased in 30 and 50 �M �-BHC group and attenuated by NAC; ROSproduction increased in 30 and 50 �M group and neutralized by NAC; LDH leakagerate enhanced in 30 or 50 �M group, SOD activity lower in all groups, higher MDAlevel in all group; dose dependent phosphorylation of JNK1 and JNK2 observed, p38MAPK phosphorylation not high in all groups; FasL increased in 30 and 50 �M groupand attenuated by NAC; procaspase-3 and -8 reduced significantly at >30-�M; NF-�Bactivation enhanced with dosage and neutralized with NAC; NF-�B p65 increased inthe 30, 50 �M groups and attenuated by NAC.

Song et al. (2008) In vitro Rat Sertoli cells from testes of 18days old male Sprague-Dawleyrats. The cells were treated with10,30, 50 or 70 �M p,p′-DDE

Effects of p,p′-DDE on LDH leakage, ROSproduction, SOD activity and MDA level, ��m,Bax family, cytochrome c translocation,procaspase-9 and -3 cleavage; effect of NAC onp,p′-DDE-induced apoptosis.

Apoptotic cell death induced at >30 �M, LDH leakage enhanced in 30 or 50 �M groups;apoptotic characters exhibited in 30 or 50 �M treated cells and blocked with NAC; ROSproduction increased in 50 �M group; lower SOD activity in all groups; higher MDAlevel in 30 or 50 �M groups; loss of �� m induced with 10, 30 or 50; Bax and Baksignificantly increased in cells treated with 30 or 50 �M. NAC had little effect on thesechanges; Bcl-w protein level declined significantly in 50 �M group and neutralized byNAC; 30 or 50 �M induced cytochrome c translocation to cytosol, NAC rescued thistranslocation effectively. Procaspase-9 and -3 significantly decreased at over 30 �M.

Pérez-Maldonadoet al. (2004)

In vitro andin vivo

Human PBMC from volunteers(without DDT exposure) for in vitrostudy. DDT exposed andunexposed children for in vivo

Effects of DDT or its metabolites on inductionof apoptosis in humans both in vitro and in vivo.

o,p′-DDT, p,p′-DDT, p,p′-DDE and p,p′-DDD induced apoptosis of human PBMC in vitroat 20 �g/mL; blood level of DDT, DDE and DDD blood and the % apoptosis were higherin the exposed children than in the unexposed children.

Song et al. (2011) In vitro Rat Sertoli cells from testes of 18days old male Sprague-Dawley ratstreated with various 10, 30, 50 or70 �M p,p′-DDE

Effect of NAC on p,p′-DDE-induced cellularviability reduction, ROS generation, MAPKsphosphorylation; effect of p,p′-DDE on mRNAlevels of cytochrome c, bax, bak and bcl-w;effect of NAC, antinomycin D, SB202190 orSP600125 on p,p′-DDE-induced apoptosis.

Viability of cells reduced with 30, 50 or 70 �M treatments, NAC attenuated the cellularviability reduction induced by 50 �M; ROS production significantly increased by50 �M treatment and neutralized with NAC; p38 and JNKs phosphorylation elevatedafter10, 30 or 50 �M treatments and inhibited by NAC; cytochrome c, bax and baklevels increased in 30 or 50 �M group and inhibited by antinomycin D; no significantchanges of bcl-w observed; apoptotic cell death induced by 50 �M and attenuatedwith NAC or SB202190 or antinomycin D; SP600125 did not have any effect.

Saradha et al.(2009)

In vivo Adult male Wistar rats (80–90 daysold) administered with a singledose of lindane (5 mg/kg b.wt)

The effects of lindane: on the levels ofcytochrome c, caspase-3 and-9, Fas and FasL;on localization of caspase-3 and Fas; onimmunolocalization of FasL; on levels of NF-�Bp65 in the cytoplasmic and nuclear extracts ofthe testis, localization of NF-�B p65, germ cellin the testis of rats by TUNEL assay.

Cytosolic cytochrome c and procapase-9 elevated as early as 6 h. A time-dependentelevation in procaspase-3, Fas, FasL levels observed. A time-dependent increaseobserved in colocalized expression of caspase-3 and Fas. This was the case forimmunoreactivity of FasL. NF-�B p65 level in cytoplasmic extract and in nuclearextracts decreased and increased, respectively, in a time-dependent manner. TheTUNEL-positive cells found in the peripheral region near the basement membrane ofthe seminiferous tubules. The number of apoptotic cells increased in atime-dependent manner following exposure to a single dose of lindane.

Vaithinathan et al.(2010)

In vivo Adult male Wistar rats (80–90 daysold) administered with a singledose of methoxychlor(50 mg/kg b.wt)

The effects of methoxychlor: on the levels ofcytochrome c, caspase-3 and-9 (in adult rattestis), Fas, FasL, NF-�B (in testis cytoplasmicextract); on localization of caspase-3 and Fas;on immunolocalization of FasL and NF-�B; onlocalization of NF-�B p65 in the rats testis; ongerm cell in the testis of rats by TUNEL assay.

Cytosolic cytochrome c and procaspase 9 elevated as early as 6 h. A time-dependentelevation in procaspase-3, Fas, FasL observed. NF-�B decreased in a time-dependentmanner. NF-�B p65 changed from cytoplasm to nucleus of germ cells. Atime-dependent increase in co-localization of Fas and Caspase 3 and inimmunoreactivity of FasL observed. TUNEL-labeled germ cells increased in theperipheral regions in time-dependent manner after a single dose of methoxychlor. Thenumber of TUNEL labeled germ cells increased progressively.

Abbreviations: Caspases, cysteinyl aspartate-specific proteinases; GSH-Px, Glutathione Peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde; NF-�B, Nuclear factor kappa B; LDH, lactate dehydrogenase;�� m = mitochondrial membrane potential; MAPKs (s), mitogen-activated protein kinases; NAC, N-acetyl-l-cysteine; JNKs, c-Jun NH2-terminal kinase; PBMC, peripheral blood mononuclear; SB202190, p38 inhibitor; SP600125,JNK inhibitor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; ROS, reactive oxygen species; b.wt, body weight.

E.J. Mrema et al. / Toxicology 307 (2013) 74– 88 79

Fig. 1. Bifunctional pathways to breast cancer (BCA). Abbreviations: OHE, hydroxyestrone; ER, estrogen receptor; GJI, gap junction inhibition; 17�-HSD, 17 beta-hydroxysteroiddehydrogenase. 17�-Estradiol metabolites affect cell proliferation and BCA development either directly via receptor-independent mechanisms involving structural/functionala henotd

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ource: Adapted from Davis et al. (1998) with some modifications.

i.e. TFF1, PR, ER, BRCA1 and CCND1) in MCF-7 cell line, while p,p′-DE (1 × 10−5 M) was a weak transcriptional inducer (Silva et al.,010).

In adult mice p,p′-DDT induced a dose-dependent effect onstrogen receptor activity in liver, brain, thymus and prostate of aransgenic mouse strain (ERE-tkLUC). The same effect was caused

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y o,p -DDT, except in the liver. This study showed that DDT isomersodulate ER activity also in nonreproductive organs (Di Lorenzo

t al., 2002). At a dose known to interfere with animal fertility50 �g/kg), o,p′-DDT significantly affected ER response. Treatment

Fig. 2. Molecular structures of phenobarbital (a proto

ypic growth regulation. These mechanisms upregulate aberrant proliferation and

of rats with 50 or 100 mg/kg body weight p,p′-DDT (repeateddoses for ten successive days) induced decrease in the number andmotility of epididymal spermatozoa in a dose dependent mannerand DDT induction of testosterone metabolism causing reducedtestosterone levels (Ben Rhouma et al., 2001). The same treatmentled to increased thyroid hormones and to a decrease in serum

T4 levels and hypothyroidism (Tebourbi et al., 2010). Given thevery high doses used in the experiments, the results of this studyare hardly comparable with effects forecasted at human environ-mental exposure levels. In fact, a cross-sectional study conducted

typic CYP2B inducer) and some OCPs reviewed.

80 E.J. Mrema et al. / Toxicolog

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mong 781 young men did not find association between DDEevels (median: 2.7 �g/g serum lipid; range 0.1–56.1 �g/g serumipid) and anogenital distance (Longnecker et al., 2007). Also aested case–control study by Torres-Sánchez et al. (2008) among 37oung men showed that higher DDE levels (10th–90th percentile:.21–5.89 �g/g serum lipid) significantly reduced the anogenitalistance. A study by Bornman et al. (2010) on urogenital malforma-ions in male newborns suggests increased rates of malformationsmong those whose mothers lived in DDT-treated homes. Althoughhese studies do not provide sufficient information for determi-ation of a quantitative risk to humans, biological plausibility istrengthened by animal findings (WHO, 2011).

Cohn et al. (2007) conducted an epidemiological study andhowed an association, with a dose response relationship, betweenDT exposure and development of breast cancer when exposure isrepubertal. This was the case for the younger women (<14 yearsf age) in second and third tertile of exposure who had DDT expo-ure in their childhood [2nd tertile: 8.09–13.90 �g/L (∼1.1–1.8 �g/gipid), OR = 2.8, 95% CI, 1.1–6.8; 3rd tertile: >13.90 �g/L, OR = 5.4,5% CI, 1.7–17.1]. McGlynn et al. (2006) found significant increas-

ng risk of liver cancer among individuals with values in theighest versus the lowest quintile of serum DDT concentrationgeometric mean = 0.49 �g/g lipids; OR = 3.8; 95% CI, 1.7–8.6; p forrend = 0.0024) when analysis was adjusted for age, sex, hepatitis

surface antigen status, places of residence and DDE level. Theowest and the highest quintile of serum DDT concentrations were0.265 and >0.787 �g/g lipids, respectively. However there was novidence of significant association between liver cancer and serumDE concentration (p = 0.51). McGlynn et al. (2008) showed anssociation between testicular germ cell carcinomas and DDE expo-ure (upper quartile: >0.39 �g/g lipid, RR = 1.71; 95% CI, 1.23–2.38;able 1).

Several epidemiological data showed an association betweenDT exposure and miscarriage or preterm birth (Dewailly et al.,993; Gladen et al., 2003; Karmaus and Zhu, 2004). To understandhe involvement of the AhR/CYP1A1 pathway in the mechanismf action of DDT and DDE, Wójtowicz et al. (2011) used humanlacenta explants cultures originated from normal pregnancy andotreated them with 3.2 ng/mL TCDD and 1, 10 or 100 ng/mLapprox. 0.18, 1.81, 18.10 �g/g lipid) of p,p′-DDT o,p′-DDT, p,p′-DDEr o,p′-DDE for 24, 48 or 72 h (these concentrations are knowno occur in serum of pregnant women). Immunoblot analyseshowed that the isomers of DDT and DDE inhibited the expres-ion of CYP1A1 most effectively at 48 and/or 72 h after treatment.n a separate experiment to study the expression of AhR, the pla-enta explants were cultured in the presence of these isomers at00 ng/mL for 1, 3, 6, 14, 24, 48 and 72 h. The level of AhR pro-

ein decreased gradually with time starting after 3 h of treatmentn all treatment experiments, with exception of that with o,p′-DDE,

here the decrease was delayed at 24 h after the start of the exper-ment. The author argued that as CYP1A1 is involved in metabolism

y 307 (2013) 74– 88

and detoxification in the human placenta, any malfunction of thisenzyme will disrupt the placental detoxification machinery. Thismay lead to an increased susceptibility of the foetus to environ-mental toxins and may be a risk factor for recurrent pregnancy loss.A cross sectional study conducted by Tsatsakis et al. (2009) among aGreek rural population, revealed a significant association betweenCYP1A1*2A polymorphism and miscarriages (p = 0.012). The inves-tigated population had the median concentrations of 6.3, 2.9, 81.5,3.1, 7.1, 5.3, 3.2 and 19.7 pg/mg for ˛-HCH, HCB, lindane, o,p′-DDE,p,p′-DDE, o,p′-DDD, p,p′-DDD + o,p′-DDT and p,p′-DDT, respectively.The significant associations were also evident for other ailmentssuch as chronic obstructive pneumonopathy (p = 0.045), peripheralcirculatory problems (trend p = 0.042), arteritis (p = 0.022), allergies(trend p = 0.046), hemorrhoids (trend p = 0.026) and allergic der-matitis (p = 0.0016). Thus it was suggested that CYP1A1 may playa significant role in the incidence of several diseases among thepopulation with long exposure to organochlorinated pesticides.

2.1.3. EndosulfanEndosulfan is synthesized via Diels–Alder addition of

hexachloro-cyclopentadiene and cis-butene-1,4-diol in xylene.The annual production is estimated to range between 18,000 and20,000 tonnes worldwide (UNEP, 2010b). In USA, 400 tonnes areestimated to be used annually for domestic purpose. The mainuses include control of pests in vegetables, fruits, cereal grains andcotton, ornamental shrubs, trees, vines and ornamental plants. Itwas also used to control ectoparasite on beef and lactating cattle.In Africa it is commonly used in cotton production at a dose ratebetween 1000 and 2000 g/ha while in India it is used to controlpests on cashew plantations at the dose rate of 1200 g/ha. Theestimated annual production in India is 9500 tonnes, of which4500–5000 tonnes are consumed domestically.

Most studies investigating endosulfan estrogenicity in vitroshow that its potency is 105–106 times lower than that of estradiol(Table 2; Arcaro et al., 1998; Soto et al., 1994, 1995; Wade et al.,1997). Briz et al. (2011) showed that endosulfan directly interactswith ERs in primary cultures of cortical neurons (CN) and cerebel-lar granule cells (CGC). It inhibited the binding of [3H]-estradiol tothe ER in both CN and CGC with a fairly high IC50 of 21.7 ± 4.2 and35.0 ± 8.3 �M, respectively. These IC50 values are very close to thatof �-endosulfan with recombinant human steroid receptors partic-ularly hPR (20 �M) (Scippo et al., 2004). Exposure of endosulfan at10 �M for 5 h increased Akt phosphorylation and activated ERK1/2through a mechanism involving GABAA and glutamate receptorsin CN. The observed alterations on ER-mediated signaling and ERlevels in neurons were suggested to contribute to the neurotoxicityof endosulfan.

In vitro assay showed that endosulfan, dieldrin and lindaneact as antagonists of androgen receptors (Andersen et al., 2002;Li et al., 2008; Nativelle-Serpentini et al., 2003). Endosulfan anddieldrin were shown to inhibit the aromatase enzyme (CYP19); therate-limiting enzyme of estrogen synthesis from the non-aromaticprecursor androstenedione. Like other organochlorines, endosul-fan and dieldrin alter the estradiol metabolism by inducing CYP1enzymes (Badawi et al., 2000; Bradlow et al., 1995). Endosulfanand dieldrin were shown to inhibit androgen response at 20 �Mand thus acted as very weak antiandrogens. At 50 �M endosulfanreduced the aromatase activity in human placenta microsomes to87% of the control level (i.e. 1 �M of 4-Androsten-4-ol-3,17-dione)and hence exhibited weak aromatase-inhibiting effect (Andersenet al., 2002).

In vitro assay shows that dieldrin (5 �M) and endosulfan (1 �M)

significantly increase cell proliferation and ER transactivation generesponse in MCF-7 cells (Andersen et al., 2002). The maximumresponse occurred at 25 �M for both dieldrin and endosulfan. How-ever, only endosulfan was shown to potentiate the 17�-estradiol

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nduced proliferation when tested together with a concentrationf 17�-estradiol causing a sub maximum response. Other authorsave also shown the ability of endosulfan to promote MCF-7ell proliferation in vitro (Grunfeld and Bonefeld-Jorgensen, 2004;barluzea et al., 2004; Soto et al., 1994). However, there are no epi-emiological studies linking exposure to endosulfan and increasef cancer risk in humans. In vitro studies examining the effects ofndosulfan on androgen-responsive systems showed that endosul-an exhibited cytotoxic effects on testicular cells at a concentrationf 20 �M or greater (Sinha et al., 1999, 2001a). These doses arenvironmentally irrelevant. In vivo assays have not demonstratedstrogenic effects (Gellert, 1978; Raizada et al., 1991; Shelby et al.,996; Wade et al., 1997). Endosulfan caused alterations in testicularunctions at very high doses. At doses of 2.5, 5 and 10 mg/kg admin-stered orally to adult Druckrey rats for 5 days per week for 70 days,ndosulfan was shown to increase testicular enzyme activities at allested doses. Sperm count decreased in a dose-dependent mannerSinha et al., 1995). Similar study conducted for 90 days using wean-ng Druckrey rats yielded similar results where spermatid countnd sperm production rate decreased (Sinha et al., 1997). Repeatedose of 3 mg/kg/day of endosulfan administered to immature ratsor 3 days caused no change in uterine weights, uterine peroxidasectivity, or numbers of uterine estrogen and progesterone recep-ors. Only at a high dose (10 �M) endosulfan inhibited estradiolinding (Wade et al., 1997) as was in the case for in vitro studiesiscussed above.

Exposure of adult rats to 2.5, 5 or 10 mg/kg endosulfan for 10eeks (5 day per week) or 3, 6 or 1000 mg/kg b.wt/day endosul-

an have shown to cause reduction in intratesticular spermatidounts, sperm abnormalities, and changes in the marker enzymes ofesticular activities, such as lactate dehydrogenase, sorbitol dehy-rogenase, g-glutamyl transpeptidase, and glucose-6-phosphateehydrogenase (Khan and Sinha, 1996; Sinha et al., 1995). More-ver exposure of pregnant rats to endosulfan at 1 mg/kg/day fromay 12 through parturition has shown to decrease spermatogene-is in offspring (Sinha et al., 2001a). It should be noted that theseoses are orders of magnitude higher than those to which humansre usually exposed in the living environment and at the workplace.

Limited human data exist on the effect of endosulfan. A cohorttudy conducted by Saiyed et al. (2003) among male school children10–19 years old) showed that endosulfan exposure was associ-ted with delayed male sexual maturity and interfered with theale sex-hormone synthesis. Sexual maturity rating (SMR) scoring

or development of pubic hair, testes, penis, and serum testos-erone level was positively related to age and negatively relatedo aerial exposure to endosulfan (AEE; p < 0.01). Serum LH levelsere significantly positively related to AEE after controlling for

ge (p < 0.01). Serum endosulfan levels were significantly higherp < 0.001) in the exposed population (mean 7.47 ± 1.19 ppb) thanontrols (1.37 ± 0.40 ppb). Moreover, a big proportion of exposedroup (78%) had detectable serum endosulfan as compared to 29%f the controls. Although these findings match those expected onhe basis of experimental evidences, nevertheless, it was not clearf endosulfan was the only chemical that could be linked to thebserved associations.

Several cellular and molecular mechanisms of endosulfan toxic-ty have been proposed. These include mitochondrial dysfunction,nduction of oxidative stress, modulation of activities of stress-esponsive signal transduction pathways, activating protein-1AP-1) and antioxidant response element (ARE)-mediated tran-cription. Recently a model has been proposed in which endosulfans shown to increase extracellular signal regulated kinases (ERK)

/2 and p38 activities and, in turn, these proteins activateitogen-activated protein kinases (MAPKs) and increase c-Jun

hosphorylation. Phosphorylated c-Jun, in turn, increases AP-1ctivity, which results in activation of ARE-mediated transcription

y 307 (2013) 74– 88 81

(Song et al., 2012). However, at the moment no firm conclusion canbe drawn on these proposed mechanisms.

2.1.4. HexachlorobenzeneHexachlorobenzene, a fully chlorinated aromatic hydrocarbon,

was widely used as fungicide. Its half life in human is estimatedto be in the range of 6 years (To-Figueras et al., 1997). The mostsensitive target organs are the liver, the ovary and the centralnervous system (ATSDR, 2002). HCB is well known to induce por-phyria through a free-radical generation mechanism (Mazzettiet al., 2004), due to its rather unique chemical characteristicsof lipophilicity and possibly to conversion into the redox-activetetrachloro-1,4-benzoquinone. HCB is a weak agonist of the AhRprotein (Hahn et al., 1989). Unlike estrogen, HCB does not havea significant affinity for the ER thus suggesting other intracellularpathways than the one taken by endogenous estrogen in mediatingthese actions.

HCB can elicit tumorigenic activity through AhR-dependent andindependent pathways. In vivo administration of HCB has shownto induce alterations in insulin-like growth factors (IGFs) signalingpathway in mammary gland and mammary tumors in rat only whenco-administered with N-nitroso-N-methylurea (Randi et al., 2006).Since the latter compound is a strong mutagen, HCB plays the role ofa tumor cocarcinogen in rat mammary gland, as an inducer of cellproliferation and of c-Src kinase activity in MCF-7 breast cancercells.

Garcia et al. (2010) used ER� (+) MCF-7 and ER� (−) MDA-MB-231 cell lines to investigate ability of HCB to promote cellproliferation and alter Insulin/IGF-I signaling pathway. At 0.005 and0.05 �M, HCB significantly increased the proliferation of only MCF-7 cell lines, with no effect at 0.5 and 5 �M. In estrogen-depletedmedium, HCB stimulated proliferation of MCF-7 cells only at thelower concentrations showing that the effect of HCB on cell prolifer-ation was not dose responsive. Thus HCB stimulates proliferation inestrogen-sensitive cells in an ER�-dependent manner. At 0.005 and0.05 �M, HCB increased the level of IGF-IR and IR in ER� (+) MCF-7cell line. However at 0.5 and 5 �M, HCB did not alter the level ofthese receptors but increased CYP1A1 gene expression and inducedapoptosis in MCF-7 cell lines, suggesting HCB effect on apoptosis isAhR-dependent. At 0.005 and 0.05 �M of HCB did not induce theexpression of CYP1A1 mRNA. On the other hand 0.005 �M of HCBled to c-Src activation signifying this effect is not AhR-dependent.At 0.005, 0.05, 0.5 and 5 �M, HCB increased IRS-1 phosphoryla-tion with no effect in IRS-1 protein levels. IGF-I receptor is knownto regulate proliferation, survival and differentiation in mammarygland. Insulin-like growth factor-insulin receptor (IGF-IR) path-way is involved in development of breast cancer and IR contentis known to increase in human breast cancer whereas IRS-1 is themain intracellular substrate activated by IGF-I and insulin in humanbreast cancer cells. c-Src can promote growth of tumor cells, par-ticipating in or augmenting mitogenic signaling pathways that areinitiated by extracellular growth factors or intracellular oncogenes(Biscardi et al., 2000). As per authors’ point of view, these resultsgive a clue to the molecular mechanism of HCB in breast cancerdevelopment.

To investigate the effects of HCB, Pontillo et al. (2011) showedthat at 0.05 �M HCB produced an early increase of c-Src, humanepidermal growth factor receptor (HER1) activation, signal trans-ducers and activators of transcription (STAT) 5b, ERK1/2, but notAkt, in ER� (−) MDA-MB-231 cell lines in a dose-dependent manner(0.005, 0.05, 0.5, and 5 �M).

Young rats (65 days of age) were administered 100 mg/kg b.wt of

HCB three times a week (total dose of 1800 mg/kg b.wt over 45 dayperiod) (Pena et al., 2012). When the test animals reached oestrousphase they were sacrificed. In mammary glands, HCB increased c-Src and HER1 activation, c-Src/HER1 association; and STAT5b and

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RK1/2 phosphorylation. Moreover HCB enhanced ER� phosphory-ation and ER�/c-Src physical interaction. In tumors, HCB induced-Src and HER1 activation, c-Src/HER1 association, as well as Aktnd STAT5b phosphorylation. Additionally, HCB increased ER� pro-ein content and decreased p-ER� levels and ER�/c-Src association.CB increased serum 17-� estradiol and prolactin contents andecreased progesterone, FSH and LH levels in rats without tumors.his might be a consequence of a negative feedback on the pituitaryormones secretion, by E2. The opposite effect was observed in ratsith tumors. These results indicate that HCB induces an estrogenic

ffect in mammary gland, increasing c-Src/HER1 and ER� signalingathways. HCB stimulates c-Src/HER1 pathway, but decreases ER�ctivity in tumors, appearing to shift them towards a higher malig-ancy phenotype. The enhanced c-Src and HER1 activation could bessociated to the increase in the hyperplasia found in the mammaryland and the higher malignancy observed in tumors (Pena et al.,012). The synergism between c-Src and HER1 serves to upregu-

ate the mitogenic activity of HER1 downstream effectors involvedn tumorigenesis (Biscardi et al., 2000). However, the very highoses tested significantly affect the relevance of this informationor human environmental and occupational risk assessment.

.1.5. ˇ-HexachlorocyclohexaneComplex and variable mixtures of hexachlorocyclohexane

HCH) products are produced by photochemical chlorination ofenzene. HCH exists in 9 stereoisomeric forms, which include (+)-nd (−)-alpha- (�), beta- (�), gamma- (�), delta- (�), epsilon- (�),eta- (), eta- () and theta- (�) isomers. Technical-grade HCH isomprised of 60–70% �-HCH, 5–12% �-HCH, 10–15% �-HCH, 6–10%-HCH, and 3–4% �-HCH. Purified HCH isomers, as well as technical-rade HCH are used either as fungicides or in the synthesis of otherhemicals.

Lindane (�-isomer) was used as seed treatment for barley, corn,ats, rye, sorghum and wheat to protect seeds for sowing fromolds and ants during storage before sowing and in the ground

efore sprouting. The estimation of global production between950 and 2000 was 600,000 tonnes and the majority was used ingriculture. In the past, �-HCH was also used in veterinary productso control mites, lice and other pests, but recent data suggest thato products are currently registered, at least in the United States

or this use. It was also used as an insecticide to treat fruit, vegeta-les, forest crops, animals and animal premises. It is also currentlysed in treatment of scabies and lice in humans in form of lotion,ream or shampoo, especially in tropical countries.

Another important HCH isomer is the beta-isomer (�-HCH). Thissomer has all chlorine atoms in an equatorial position, and thus (a)

hile it lacks aromatic character, its molecular shape is more sim-lar to that of strictly planar hexachlorobenzene than those of allther isomers and (b) it lacks any axial chlorine atom(s) which cane the site for 1,2-elimination by chemical mechanisms (Fig. 4).

ig. 4. 3D molecular shapes of: (A) beta-hexachlorocyclohexane (�-HCH or,2,3,4,5,6-HCH [1�,2�,3�,4�,5�,6�]); (B) hexachlorobenzene (HCB) (Plots by ACD-abs/3D, Advance Chemistry Development, 2007).

y 307 (2013) 74– 88

This is one reason why this isomer shows a higher bioaccumula-tion (half-life in human body of about 7.2 years; Jung et al., 1997)and a higher bioaccumulation factor of 5274 (WHO, 1992). Sincethis isomer is eliminated very slowly from human body, it makes asignificant contribution to the total HCH body burden. �-HCH waswidely used on cotton plants during 1960s and 1970s.

�-HCH (1 �M) has mitogenic activity in MCF-7 cell lines in vitro(Coosen and van Velsen, 1989) but not in ER�-MDA-MB231 celllines which express a non-functional ER (Steinmetz et al., 1996).This result indicates that a functional ER is necessary to elicit themitogenic activity.

In vivo assay with mouse xenograft showed that 10−8 M �-HCHsignificantly increased MCF-7 cell number and at 10−5 M maximalresponses were achieved (Steinmetz et al., 1996). Moreover �-HCHincreased pS2 gene mRNA level. Like HCB, �-HCH does not com-pete with estradiol for binding to the ER (Coosen and van Velsen,1989; Hatakeyama et al., 2002; Steinmetz et al., 1996). Its chemicalstructure characteristics may not allow binding the classical ligandbinding domain and thus fail to activate ER in the nucleus, but itshows affinity to the alternative pocket and consequently triggersthe Src/Ras/ERK pathway (Silva et al., 2010). Although unable tobind the ER, �-HCH is capable of activating ER target genes and theproliferation of estrogen-responsive cell lines (Silva et al., 2007;Steinmetz et al., 1996) by other chemical and biochemical mecha-nisms.

Treatment of MCF-7 cells with 1 × 10−5 M �-HCH resulted instrong activation of the Src kinase, ERK1 and ERK2. Thus �-HCHhas been shown to induce rapid and sustained Src/ERK signalingin MCF-7 cell lines lasting for at least 30 min, while this path-way was not significantly affected by p,p′-DDE (Silva et al., 2010).Hatakeyama et al. (2002) observed similar activation of ERK1/ERK2by �-HCH.

In epidemiological study Mussalo-Rauhamaa et al. (1990) foundthat �-HCH frequently occurs in breast fat of breast cancer patientswith mean concentration of 0.13 ± 0.06 mg/kg fat. Controlling forage and parity, �-HCH remained a significant risk factor (OR, 10.51;95% CI, 2.00–55.26). The cutoff point for the residue level in breastadipose tissue was >0.1 mg/kg fat.

2.2. Oxidative stress, apoptosis and OCPs’ toxicity

Apoptosis is the mechanism whereby damaged or unnecessarycells are naturally eliminated without causing damage to the sur-rounding tissue (Ameisen, 1996). This effect is accomplished by apartial, yet exhaustive digestion of cell structures (DNA, structuralproteins, enzymes and lipids) prior to rupture of the cell membraneand release of products in the extracellular medium for clear-ance. When apoptosis is excessive or inappropriate, incomplete orinsufficient, defections trigger pathological-related condition suchas immunodeficiency, autoimmunity diseases, cancer (Alison andSarraf, 1995; Gougeon et al., 1996) and reproductive anomalies,such as those due to abnormal spermatogenesis (Allan et al., 1992).

Oxidative stress, i.e., a shift of the cell redox balance towardsa more oxidative (more negative) value of the electrochemi-cal potential with respect to that of the relevant cell phase, isone main trigger in the induction of apoptosis (Kannan et al.,2000; Pérez-Maldonado et al., 2005). Many pesticides share asa possible mechanism of toxicity the ability to trigger apopto-sis through alterations in redox homeostasis generated by adecrease of antioxidant defenses and by accumulation of reac-tive oxygen species (ROS). An over-production of ROS leadsto processes such as oxidative modifications of redox signaling

protein, oxidative DNA damage, endoplasmic reticulum stressand alterations in mitochondrial function which in turn trig-ger the activation of specific signaling cascades. Activation ofstress-activated protein kinases (SAPKs) such as c-Jun N-terminal

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inases (JNK) and of transcription-dependent p53 signalingascades act as important sensors of electrophilic (and of oxidized)ubstances among which xenobiotic metabolites and induce apop-otic cell death. Pesticides also induce the activation of survivalesponses such as DNA repair mechanisms, mitogen-activated pro-ein kinase/phosphatidylinositol 3-kinase (MAPK/PI3K) signalingascades and up-regulation of antioxidant defenses in an attempto cope with and counteract the deleterious effects of higher lev-ls of intracellular reactive species which in turn trigger cell deathathways. In most cases apoptotic and survival signaling cascadesre activated simultaneously in response to exposure to pesticides.able 3 summarizes some studies on apoptotic effects of someCPs.

DDT derivatives have been shown to induce neural cell deathy apoptosis through the activation of mitogen-activated proteininases (MAPKs) which play significant roles in controlling cellurvival, proliferation, differentiation and cellular responses toarious harmful signals (Shinomiya and Shinomiya, 2003). �-HCH,,p′-DDE and other OCPs can induce elevation of ROS such as O2

•−,O• and •NO (Samanta and Chainy, 1997; Song et al., 2008; Sujathat al., 2001).

Shi et al. (2009) hypothesized the signaling pathways involvedn p,p′-DDE-induced apoptosis: in his model ROS generation playsritical role in the initiation Sertoli cells apoptosis through twoechanisms. The first one is mitochondria-mediated pathway

nvolving elevation of ROS, decrease in mitochondrial trans-embrane potential along with the cytochrome c release fromitochondria into the cytosol and activation of the caspase-9 and

3. Other mechanism involves elevation of ROS, which resultedn activation of NF-�B, expression of FasL and triggered FasL-ependent pathway (i.e. FasL/caspase-8/-3 signaling module). Inheir investigation, Song et al. (2008) found at levels above 30 �M,,p′-DDE induced apoptotic cell death of cultured rat Sertoli cellsn a pro-oxidant and mitochondria dependent manner by acti-ating the intrinsic programmed cell death pathway. Moreover,p′-DDE elevated the apoptotic rate of Sertoli cells in vitro (at30 �M) and germinal cells in vivo (at >20 mg/kg b.wt) by mech-nism suspected to involve FasL-dependent pathway (Shi et al.,009, 2010).

Exposure to p,p′-DDE can enhance ROS production and oxida-ive stress, and then induce activation of NF-�B and expressionf Fas-FasL. As a result, an intrinsic program of apoptotic deaths stimulated in a target cell resulting to the activation of cas-ase 8. Ultimately, apoptosis of Sertoli cells and germinal cells isediated by caspase 3, thereby disturbing the spermatogenesis

Shi et al., 2010). Song et al. (2011) reported the induction of Ser-oli cell apoptosis by p,p′-DDE at above 30 �M through oxidativetress-mediated p38 MAPK and mitochondria-related pathway.ethoxychlor, which was intended to replace DDT, has been shown

o induce testicular apoptosis in rats following oral exposure to sin-le doses of 50 mg/kg b.wt where apoptosis was induced throughnvolvement of Fas-FasL and mitochondria-dependent pathwaysVaithinathan et al., 2010). It is necessary to remark that the dosesested are unrealistically high if compared to those typical of occu-ational and environmental exposures.

Pérez-Maldonado et al. (2004) showed that o,p′-DDT, p,p′-DDT,,p′-DDE and p,p′-DDD can induce apoptosis of human periph-ral blood mononuclear cells in vitro at 20 �g/mL. This level is00-fold higher than the mean blood total DDT level (0.05 �g/mL)bserved in children in their pilot study (Pérez-Maldonado et al.,004). The significant level of ROS, as detected by flow cytometry,as induced at 60 and 80 �g/mL (Pérez-Maldonado et al., 2005).

marginal influence of DDT and DDE on percentage of apoptosisas observed (p = 0.06) confirmed by a weak positive association

etween apoptosis and DDT exposure. In the follow-up study Pérez-aldonado et al. (2006) found significant association between the

y 307 (2013) 74– 88 83

percentage of apoptotic cells and the blood levels of only DDE ofexposed children recruited in 2003 (p = 0.010) and those recruitedin 2004 (p = 0.040). The association between DDT or DDE exposureand DNA damage was significant (p = 0.004 and p = 0.005 respec-tively), whereas the association between DDT or DDE and oxidativeDNA damage and that of oxidative damage and apoptosis were notsignificant.

An isomer of DDD, o,p′-DDD, marketed under the name Mitotaneis used as an antineoplastic medication in the treatment of adreno-cortical carcinoma. Mitotane alters steroid peripheral metabolism,directly suppresses the adrenal cortex through the controlleddestruction of adrenal tissue, which leads to a decrease in cortisolproduction (Maher et al., 1992; Wu et al., 2006).

Exposure of Wistar rats to a single dose of lindane (�-HCH;5 mg/kg b.wt) induced testicular apoptosis through the involve-ment of Fas-FasL and mitochondria-dependent pathways (Saradhaet al., 2009). Shi et al. (2011) showed that concentrations >30 �Mof �-HCH induced apoptotic cell death in rat Sertoli cells associatedwith increased expression of FasL levels which could lead to theFas activation. These two genes are known to induce apoptosis.Moreover �-HCH treatment induced an increase of nuclear factorkappa B (NF-�B) p65. The latter can directly stimulate the expres-sion of these genes. Oxidative stress has been reported to enhanceNF-�B activation (Nakamura and Omaye, 2008). �-HCH has beenshown to induce activation of caspase-8, that plays the role intransduction of death signal (Said et al., 2004) and caspase-3, thatinitiates cell apoptosis (Khan et al., 2000).

Moreover, Shi et al. (2011) showed that �-HCH induces increasein apoptotic rate of Sertoli cells by possible mechanisms ofROS/JNK/FasL pathway. In vitro exposure to �-HCH in rat Sertolicells can enhance ROS and oxidative stress, and then induce activa-tion of JNKs and NF-�B and expression of FasL. Significant increaseof FasL protein expression was observed with 30–50 �M �-HCHtreatment. This increase could lead to activate the Fas system. Uponengagement of FasL to Fas, an intrinsic program of apoptotic deathis stimulated in a target cell leading to the activation of caspase-8.Finally, apoptosis of Sertoli cells is mediated by caspase-3, therebydisturbing the spermatogenic process (Shi et al., 2011; Fig. 5). Theseresults led the authors hypothesize that ROS generation may playa critical role in the initiation of �-HCH-induced apoptosis by acti-vation of the JNKs, translocation of NF-�B, expression of FasL andfurther activation of caspase cascade.

Yu et al. (2008) studied the individual effect of p,p′-DDE and�-HCH as well as the effect of their mixture on JNK and MAPKpathway in rat Sertoli cells. The latter were exposed to these OCPsat the final concentration of 10, 30 and 50 �M/L for 24 h in eachcase. In both treatments, the expression of JNK and c-jun was ele-vated in a dose dependent manner after 24 h of exposure. Lianget al. (2008) showed that at these levels p,p′-DDE, �-HCH and theirmixture could induce apoptosis in rat Sertoli cells which was associ-ated with activation of caspase-3 mediated by cleavage of caspase-8and caspase-9. As stated for the case of p,p′-DDE above, these lev-els are also unrealistically high if compared even with the highestoccupational exposures.

As for specific mechanisms, OCPs, which are themselves fairlyun-reactive from the chemical point of view, are able to causeor trigger oxidative stress. However, little is known or hypothe-sized at the molecular level. Only the porphyrinogenic pesticidehexachlorobenzene is known for its ability to directly gener-ate oxidative stress due to its unique property to act as anelectron sink either as such or, more likely, through biotransforma-tion products such as tetrachloro-1,4-benzoquinone (van Ommen

and van Bladeren, 1989). That HCB is able to trigger apoptosisthrough the mitochondrial pathway has been demonstrated in theliver (Giribaldi et al., 2011) and in the thyroid (Chiappini et al.,2009).

84 E.J. Mrema et al. / Toxicology 307 (2013) 74– 88

Fig. 5. �-HCH-induced signaling pathways leading to apoptosis. In this proposed model, ROS generation might be responsible for initiating the �-HCH-induced apoptosisof Sertoli cells. The increase of ROS could result in activation of JNKs and NF-�B, and expression of FasL, then triggered FasL-dependent pathway (FasL/caspase-8/caspase-3s sor th

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ignaling module). N-Acetyl-cystein (NAC) can inhibit triggers by protecting the sen

ource: Adapted from Shi et al. (2011) with some modification.

.3. Epigenetics and OCPs’ toxicity

Epigenetics is an emerging field of study of heritable changeshat influence chromosomal stability and gene expression whileot directly affecting changes in DNA sequence, such as by pointutations or chromosome disruption and re-assembly (Rodenhiser

nd Mann, 2006). One main mechanism of epigenetic modulationf genetic expression is methylation at 5-position of cytosines inepeated CpG sequences, a modification which suppresses expres-ion of downstream DNA sequences. Thus suppression of specificenes or re-activation of silenced genes determines the destiny ofndividual cells towards quiescence or proliferation, thus openinghe way to uncontrolled cell proliferation and cancer. Moreover,hen epigenetic changes occur during certain stages of develop-ent, they become permanent and can be inherited by offsprings

nd disturbance of epigenetic modulation is thus thought to be anmportant mechanism in many diseases (Ozanne and Constancia,007).

Epigenetic effects have been hypothesized as a possible mecha-ism of POPs toxicity (Porta, 2006). For example, pregnant rats werereated with methoxychlor at the fairly high dose of 50–150 mg/kguring the sex-determining time window of pregnancy (days7–E10). Gonads were collected from embryos in gestating moth-rs at E16 and from postnatal males showed a reduced germell to testis area at developmental age and an almost doubledraction of apoptotic germ cells, although males were fertile androduced normal offspring (Cupp et al., 2003). In a further experi-ent (Anway et al., 2005) exposition of pregnant rats to 100 mg/kg

roduced male offsprings with reduced sperm capacity and fer-ility and the compromised fertility trait was passed through thedult male germ line for four generations. Altered patterns of DNAethylation was demonstrated to occur in the germ cells of gener-

tions two and three.In vitro and animal studies have suggested that exposure to

ndocrine active compounds, such as POPs, may adversely affectNA methylation patterns (Anway and Skinner, 2006; Anway et al.,006). It has been proved that chemicals can alter the epigeneticarks and that epigenetic marks can be found in patients with

he disease of concern or in diseased tissue (Baccarelli and Bollati,009).

DDT has been shown to affect DNA methylation in experimentalnimals as shown by Shutoh et al. (2009). DDT was found to alter the

iol residue of the signalling proteins.

methylation pattern in the hypothalamus of young rats (3 weeks ofage) exposed at a dosage of 0.06 mg/kg/day. The 6 CpG islands wereconsiderably hypomethylated as compared with controls. Thus theauthors speculated that under low level of oxidation stress, the DNAmethylation machinery malfunctions and this leads to incompletemethylation of specific gene regions. Following pyrosequencingmethylation analysis of the rat livers from the rats exposed tohigh dose of mixture of twelve OCPs (1.9 mg/kg/day) showed nodecrease in the methylation of CpG sites which is contrary to othertested chemical mixtures (Desaulniers et al., 2009).

To our knowledge only two epidemiological studies have beenconducted to investigate exposure to POPs and DNA methyla-tion levels in a human population. The first study was carried onGreenlandic Inuit and suggested a significant inverse linear rela-tionship between DNA methylation and plasma concentrations ofDDT, DDE, �-HCH, oxychlordane, �-chlordane and mirex (Rusieckiet al., 2008). This relationship was replicated by Kim et al. (2010)when they investigated the relationship between POPs exposureand DNA hypomethylation among healthy Koreans. These twostudies demonstrated epigenetic changes related to environmen-tal exposure although the two populations differ in magnitude ofexposure, Greenland Inuit people being more exposed than Koreanpopulation.

3. Conclusions

The general population is exposed to OCPs through several ways,food being the major route of exposure. A few groups of the humanpopulation may show much higher levels of exposure than othersdue to the fact that they live in areas with a historical uncon-trolled use of these pesticides in agriculture (typical examples beingsome areas of Central Asia) or that their diet mainly consists ofbio-accumulating organisms, such as large marine mammals (thetypical example is that of Arctic Inuits). In both cases the hazardis transmitted trans-generationally starting with breastfeeding ofnewborns and consequently the impairment of fertility can deter-mine a demographic decline of some populations.

A number of adverse health effects such as endometriosis, infer-

tility, immunotoxicity, neurotoxicity and spontaneous abortions,breast cancer, prostate cancer and neurodegenerative disordershave been suggested as a result of this exposure. OCPs may exertthese effects through various mechanisms. Animal and in vitro

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tudies using cell cultures are most widely used to evaluate toxicityf these chemicals. OCPs act as agonists on ER� and/or antag-nists on ER� and also as probably antagonists on androgeniceceptors. These effects may contribute to the tumor promotingffects of these pesticides, which are observed in animals treatedith high doses, which are not representative even of worst-caseuman exposure levels. Thus, p,p′-DDE and HCH have been showno exhibit antiandrogenic effects by binding to ARs and compet-ng with natural androgens, a characteristic which supports theirstrogenic effect. Although HCB and �-HCH elicit estrogen-likeesponses, they do not compete with estradiol for binding to the ERike estrogen does. This suggests other intracellular pathways thanhe one used by endogenous estrogen in mediating these actions.hey can elicit responses through AhR-dependent and independentathways.

Many OCPs modulate or trigger apoptosis by redox signalinghich involves alterations in antioxidant defenses and accumu-

ation of ROS leading to oxidative stress. When not regulated,poptosis can contribute to several diseases as immunodeficiency,utoimmunity and cancer. However, limited information exists onull mechanistic events involved in the induction of cell death orurvival by these pesticides.

Although not extensively explored, epigenetics has been con-idered to be a potential mechanism of POPs toxicity. Few studiesave addressed OCPs and epigenetic modification as evident inuman studies where only two studies have been conducted tolucidate the involvement of OCPs in health effects at epige-etic level. However some of these studies have shown OCPs mayperate at epigenetic level. Thus more studies (both animals anduman) are needed to document evidence of involvement of pes-icides at epigenetics level in eliciting adverse health effects touman.

It is also important to remark that a very common charac-eristics of laboratory studies is that they have been carried outesting unrealistically high doses, and this makes the extrapolationo environmental and occupational human exposures very critical.owever they produce hypotheses that deserve an investigation

hrough specifically addressed epidemiological and experimentaltudies. This is particularly important because most of the epidemi-logical studies we have evaluated are not conclusive, and oftenesults of different epidemiological studies are conflicting.

However, despite the not fully conclusive picture, it is evidenthat, having in mind the toxic potential, the bioaccumulation andiomagnification properties, there is a need to perform specific

nterventions addressed at reducing exposure, especially of vul-erable population subgroups. Thus stringent control in food safetyystem should be put in place to prevent the occurrence of OCPs inood and feedstuffs.

The restriction of the use and the ban of most of OCPsn most developed countries limits the possible health threatshey might pose. Cessation of direct application on animals orresence of OCPs in their feed, housing or pasture will helpo control them. Removal of the contaminated items from theuman food chain can be opted when acceptable limits have beenxceeded. Withdrawal times can be appropriate means for grow-ng (meat-type) animals, which do not deliver a product on aaily basis. Finally, continuing research is needed on these con-aminants, their body burden, potential health effects and wayso reduce their bioavailability in food. Moreover, effective poli-ies are needed to control their manufacture and release into thenvironment.

onflict of interest

The authors declare that there are no conflicts of interest.

y 307 (2013) 74– 88 85

References

Alison, M.R., Sarraf, C.E., 1995. Apoptosis: regulation and relevance to toxicology.Hum. Exp. Toxicol. 14, 355–362.

Allan, D.J., Harmon, B.V., Roberts, S.A., 1992. Spermatogonial apoptosis has threemorphologically recognizable phases and shows no circadian rhythm duringnormal spermatogenesis in the rat. Cell Prolif. 25, 41–50.

Ameisen, J.C., 1996. The origin of programmed cell death. Science 272, 1278–1279.Andersen, H.R., Vinggaard, A.M., Rasmussen, T.H., Gjermandsen, I.M., Bonefeld-

Jørgensen, E.C., 2002. Effects of currently used pesticides in assays forestrogenicity, androgenicity, and aromatase activity in vitro. Toxicol. Appl.Pharmacol. 179, 1–12.

Androutsopoulos, V.P., Papakyriakou, A., Vourloumis, D., Spandidos, D.A., 2011.Comparative CYP1A1 and CYP1B1 substrate and inhibitor profile of dietaryflavonoids. Bioorg. Med. Chem. 19, 2842–2849.

Androutsopoulos, V., Wilsher, N., Arroo, R.R., Potter, G.A., 2009a. Bioactivation of thephytoestrogen diosmetin by CYP1 cytochromes P450. Cancer Lett. 274, 54–60.

Androutsopoulos, V.P., Tsatsakis, A.M., Spandidos, D.A., 2009b. Cytochrome P450CYP1A1: wider roles in cancer progression and prevention. BMC Cancer 9,187.

Androutsopoulos, V.P., Papakyriakou, A., Vourloumis, D., Tsatsakis, A.M., Spandidos,D.A., 2010. Dietary flavonoids in cancer therapy and prevention: substrates andinhibitors of cytochrome P450 CYP1 enzymes. Pharmacol. Ther. 126, 9–20.

Androutsopoulos, V., Arroo, R.R., Hall, J.F., Surichan, S., Potter, G.A., 2008. Antiprolif-erative and cytostatic effects of the natural product eupatorin on MDA-MB-468human breast cancer cells due to CYP1-mediated metabolism. Breast Cancer Res.10, R39.

Anway, M.D., Cupp, A.S., Uzumcu, M., Skinner, M.K., 2005. Epigenetic transgen-erational actions of endocrine disruptors and male fertility. Science 308,1466–1469.

Anway, M.D., Leathers, C., Skinner, M.K., 2006. Endocrine disruptor vinclozolininduced epigenetic transgenerational adult-onset disease. Endocrinology 147,5515–5523.

Anway, M.D., Skinner, M.K., 2006. Epigenetic transgenerational actions of endocrinedisruptors. Endocrinology 147, S43–S499.

Appenzeller, B.M., Tsatsakis, A.M., 2012. Hair analysis for biomonitoring of environ-mental and occupational exposure to organic pollutants: state of the art, criticalreview and future needs. Toxicol. Lett. 210, 119–140.

Arcaro, K.F., Vakharia, D.D., Yang, Y., Gierthy, J.F., 1998. Lack of synergy by mixturesof weakly estrogenic hydroxylated polychlorinated biphenyls and pesticides.Environ. Health Perspect. 106, 1041–1046.

Aronson, K.J., Miller, A.B., Woolcott, C.G., Sterns, E.E., McCready, D.R., Lickley, L.A.,Fish, E.B., Hiraki, G.Y., Holloway, C., Ross, T., Hanna, W.M., SenGupta, S.K., Weber,J.P., 2000. Breast adipose tissue concentrations of polychlorinated biphenylsand other organochlorines and breast cancer risk. Cancer Epidemiol. BiomarkersPrev. 9, 55–63.

ATSDR, 2002. Toxicological Profile for DDT, DDE and DDD. U.S. D.H.A.H. Services.Baccarelli, A., Bollati, V., 2009. Epigenetics and environmental chemicals. Curr. Opin.

Pediatr. 21, 243–251.Badawi, A.F., Cavalieri, E.L., Rogan, E.G., 2000. Effect of chlorinated hydrocarbons

on expression of cytochrome P450 1A1, 1A2 and 1B1 and 2- and 4 hydrox-ylation of 17beta-estradiol in female Sprague-Dawley rats. Carcinogenesis 21,1593–1599.

Ben Rhouma, K., Tebourbi, O., Krichah, R., Sakly, M., 2001. Reproductive toxicity ofDDT in adult male rats. Hum. Exp. Toxicol. 20, 393–397.

Biscardi, J.S., Ishizawar, R.C., Silva, C.M., Parsons, S.J., 2000. Tyrosine kinase signallingin breast cancer: epidermal growth factor receptor and c-Src interactions inbreast cancer. Breast Cancer Res. 2, 203–210.

Bornman, R., de Jager, C., Worku, Z., Farias, P., Reif, S., 2010. DDT and urogenitalmalformations in newborn boys in a malarial area. BJU Int. 106, 405–411.

Bouwman, H., Cooppan, R.M., Berker, P.J., Ngxongo, S., 1991. Malaria control andlevels of DDT in serum of two populations in Kwazulu. J. Toxicol. Environ. Health33, 141–155.

Bouwman, H., van den Berg, H., Kylin, H., 2011. DDT and malaria prevention:addressing the paradox. Environ. Health Perspect. 119, 744–747.

Bradlow, H.L., Davis, D.L., Lin, G., Sepkovic, D., Tiwari, R., 1995. Effects of pesticideson the ratio of 16 alpha/2-hydroxyestrone: a biologic marker of breast cancerrisk. Environ. Health Perspect. 103, 147–150.

Briz, V., Molina-Molina, J.M., Sanchez-Redondo, S., Fernandez, M.F., Grimalt, J.O.,Olea, N., Rodriguez-Farre, E., Sunol, C., 2011. Differential estrogenic effects ofthe persistent organochlorine pesticides dieldrin, endosulfan, and lindane inprimary neuronal cultures. Toxicol. Sci. 120, 413–427.

Campbell, M.A., Gyorkos, J., Leece, B., Homonko, K., Safe, S., 1983. The effectsof twenty-two organochlorine pesticides as inducers of the hepatic drug-metabolizing enzymes. Gen. Pharmacol. 14, 445–454.

Chiappini, F., Alvarez, L., Lux-Lantos, V., Randi, A.S., Kleiman de Pisarev, D.L., 2009.Hexachlorobenzene triggers apoptosis in rat thyroid follicular cells. Toxicol. Sci.108, 301–310.

Claessens, F., Gewirth, D.T., 2004. DNA recognition by nuclear receptors. EssaysBiochem. 40, 59–72.

Cohn, B.A., Wolff, M.S., Cirillo, P.M., Sholtz, R.I., 2007. DDT and breast cancer in

young women: new data on the significance of age at exposure. Environ. HealthPerspect. 115, 1406–1414.

Coosen, R., van Velsen, F.L., 1989. Effects of the beta-isomer of hexachlorocyclo-hexane on estrogen-sensitive human mammary tumor cells. Toxicol. Appl.Pharmacol. 101, 310–318.

8 icolog

C

C

D

D

D

D

D

E

G

G

G

G

G

G

G

G

H

H

H

H

H

I

J

J

K

K

K

K

6 E.J. Mrema et al. / Tox

ovaci, A., Tutudaki, M., Tsatsakis, A.M., Schepens, P., 2002. Hair analysis: anotherapproach for the assessment of human exposure to selected persistentorganochlorine pollutants. Chemosphere 46, 413–418.

upp, A.S., Uzumcu, M., Suzuki, H., Dirks, K., Phillips, B., Skinner, M.K., 2003. Effect oftransient embryonic in vivo exposure to the endocrine disruptor methoxychloron embryonic and postnatal testis development. J. Androl. 24, 736–745.

esaulniers, D., Xiao, G.H., Lian, H., Feng, Y.L., Zhu, J., Nakai, J., Bowers, W.J.,2009. Effects of mixtures of polychlorinated biphenyls, methylmercury, andorganochlorine pesticides on hepatic DNA methylation in prepubertal femaleSprague-Dawley rats. Int. J. Toxicol. 4, 294–307.

avis, D.L., Axelrod, D., Bailey, L., Gaynor, M., Sasco, A.J., 1998. Rethinking breast can-cer risk and the environment: the case for the precautionary principle. Environ.Health Perspect. 106, 523–529.

ewailly, É., Mulvad, G., Pedersen, H.S., Ayotte, P., Demers, A., Weber, J., Hansen,J.C., 1999. Concentration of organochlorines in human brain, liver, and adi-pose tissue autopsy samples from Greenland. Environ. Health Perspect. 107,823–828.

ewailly, É., Ayotte, P., Bruneau, S., Laliberte, C., Muir, D.C., Norstrom, R.J., 1993. Inuitexposure to organochlorines through the aquatic food chain in arctic Quebec.Environ. Health Perspect. 101, 618–620.

i Lorenzo, D., Villa, R., Biasiotto, G., Belloli, S., Ruggeri, G., Albertini, A., Apos-toli, P., Raviscioni, M., Ciana, P., Maggi, A., 2002. Isomer-specific activity ofdichlorodyphenyltrichloroethane with estrogen receptor in adult and sucklingestrogen reporter mice. Endocrinology 143, 4544–4551.

ngeler, E., 2009. UN: Treaty Expanded by 9 more Dangerous Chemicals, Available:http://www.guardian.co.uk/world/feedarticle/8498685 (accessed 18.12.11).

arcia, M.A., Pena, D., Alvarez, L., Cocca, C., Pontillo, C., Bergoc, R., de Pisarev, D.K.,Randi, A., 2010. Hexachlorobenzene induces cell proliferation and IGF-I signalingpathway in an estrogen receptor alpha-dependent manner in MCF-7 breastcancer cell line. Toxicol. Lett. 192, 195–205.

eisz, H.N., Dickhut, R.M., Cochran, M.A., Fraser, W.R., Ducklow, H.W., 2008. Meltingglaciers: a probable source of DDT to the Antarctic marine ecosystem. Environ.Sci. Technol. 42, 3958–3962.

ellert, R.J., 1978. Kepone, mirex, dieldrin, and aldrin: estrogenic activity and theinduction of persistent vaginal estrus and anovulation in rats following neonataltreatment. Environ. Res. 16, 131–138.

iribaldi, L., Chiappini, F., Pontillo, C., Randi, A.S., Kleiman de Pisarev, D.L., Alvarez,L., 2011. Hexachlorobenzene induces deregulation of cellular growth in rat liver.Toxicology 289, 19–27.

laden, B.C., Shkiryak-Nyzhnyk, Z.A., Chyslovska, N., Zadorozhnaja, T.D., Little, R.E.,2003. Persistent organochlorine compounds and birth weight. Ann. Epidemiol.13, 151–157.

ougeon, M.L., Lecoeur, H., Dulioust, A., 1996. Programmed cell death in peripherallympocytes from HIV-infected persons. J. Immunol. 156, 3509–3520.

ourounti, K., Lykeridou, K., Protopapa, E., Lazaris, A., 2008. Mechanisms of actionsand health effects of organochlorine substances. A review. HSJ 2, 89–98.

runfeld, H.T., Bonefeld-Jorgensen, E.C., 2004. Effect of in vitro estrogenic pesticideson human oestrogen receptor alpha and beta mRNA levels. Toxicol. Lett. 151,467–480.

ahn, M.E., Goldstein, J.A., Linko, P., Gasiewicz, T., 1989. Interaction of hexachloro-benzene with the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro andin vivo. Arch. Biochem. Biophys. 270, 344–355.

ankinson, O., 2005. Role of coactivators in transcriptional activation by the arylhydrocarbon receptor. Arch. Biochem. Biophys. 433, 379–386.

atakeyama, M., Zou, E., Matsumura, F., 2002. Comparison of the characteristic ofestrogenic action patterns of beta-HCH and heregulin beta1 in MCF-7 humanbreast cancer cells. J. Biochem. Mol. Toxicol. 16, 209–219.

ayes, C.L., Spink, D.C., Spink, B.C., Cao, J.Q., Walker, N.J., Sutter, T.R., 1996. 17 Beta-estradiol hydroxylation catalyzed by human cytochrome P450 1B1. Proc. Natl.Acad. Sci. U.S.A. 93, 9776–9781.

oekstra, P.F., Burnison, B.K., Neheli, T., Muir, D.C., 2001. Enantiomer-specific activityof o,p′-DDT with the human estrogen receptor. Toxicol. Lett. 125, 75–81.

barluzea, J.J., Fernandez, M.F., Santa-Marina, L., Olea-Serrano, M.F., Rivas, A.M., Aur-rekoetxea, J.J., Exposito, J., Lorenzo, M., Torne, P., Villalobos, M., Pedraza, V., Sasco,A.J., Olea, N., 2004. Breast cancer risk and the combined effect of environmentalestrogens. Cancer Causes Control 15, 591–600.

akszyn, P., Goni, F., Etxeandia, A., Vives, A., Millan, E., Lopez, R., Amiano, P., Ardanaz,E., Barricarte, A., Chirlaque, M.D., Dorronsoro, M., Larranaga, N., Martinez, C.,Navarro, C., Rodriguez, L., Sanchez, M.J., Tormo, M.J., Gonzalez, C.A., Agudo, A.,2009. Serum levels of organochlorine pesticides in healthy adults from fiveregions of Spain. Chemosphere 76, 1518–1524.

ung, D., Becher, H., Edler, L., Flesch-Janys, D., Gurn, P., Konietzko, J., Manz, A., Papke,O., 1997. Elimination of beta-hexachlorocyclohexane in occupationally exposedpersons. J. Toxicol. Environ. Health 51, 23–34.

annan, K., Holcombe, R.F., Jain, S.K., Alvarez-Hernandez, X., Chervenak, R., Wolf,R.E., Glass, J., 2000. Evidence for the induction of apoptosis by endosulfan in ahuman T-cell leukemic line. Mol. Cell Biochem. 205, 53–66.

armaus, W., Zhu, X., 2004. Maternal concentration of polychlorinated biphenylsand dichlorodiphenyldichlorethylene and birth weight in Michigan fish eaters,a cohort study. Environ. Health 3, 1–9.

elce, W.R., Stone, C.R., Laws, S.C., Gray, L.E., Kemppainen, J.A., Wilson, E.M., 1995.

Persistent DDT metabolite p,p′-DDE is a potent androgen receptor antagonist.Nature 375, 581–585.

han, P.K., Sinha, S.P., 1996. Ameliorating effect of vitamin C on murine sperm tox-icity induced by three pesticides (endosulfan, phosphamidon and mancozeb).Mutagenesis 11, 33–36.

y 307 (2013) 74– 88

Khan, S.M., Dauffenbach, L.M., Yeh, J., 2000. Mitochondria and caspases in inducedapoptosis in human luteinized granulosa cells. Biochem. Biophys. Res. Commun.269, 542–545.

Kim, K.Y., Kim, D.S., Lee, S.K., Lee, I.K., Kang, J.H., Chang, Y.S., Jacobs, D.R., Steffes,M., Lee, D.H., 2010. Association of low-dose exposure to persistent organic pol-lutants with global DNA hypomethylation in healthy Koreans. Environ. HealthPerspect. 118, 370–374.

Lemaire, G., Mnif, W., Mauvais, P., Balaguer, P., Rahmani, R., 2006. Activation of �-and �-estrogen receptors by persistent pesticides in reporter cell lines. Life Sci.79, 1160–1169.

Li, H.C., Dehal, S.S., Kupfer, D., 1995. Induction of the hepatic CYP2B and CYP3Aenzymes by the proestrogenic pesticide methoxychlor and by DDT in the rat.Effects on methoxychlor metabolism. J. Biochem. Toxicol. 10, 51–61.

Li, J., Li, N., Ma, M., Giesy, J.P., Wang, Z., 2008. In vitro profiling of the endocrinedisrupting potency of organochlorine pesticides. Toxicol. Lett. 183, 65–71.

Li, X., Zhang, S., Safe, S., 2006. Activation of kinase pathways in MCF-7 cells by 17beta-estradiol and structurally diverse estrogenic compounds. J. Steroid Biochem.Mol. Biol. 98, 122–132.

Liang, X.M., Hu, Y.F., Yu, H.G., Yang, K.D., 2008. Effects of p,p′-DDE and beta-BHCon the apoptosis of Sertoli cells in vitro. Zhonghua Yu Fang Yi Xue Za Zhi 42,648–652.

Longnecker, M.P., Gladen, B.C., Cupul-Uicab, L.A., Romano-Riquer, S.P., Weber, J.P.,Chapin, R.E., Hernández-Ávila, M., 2007. In utero exposure to the antiandrogen1,1-dichloro-2,2-bis(pchlorophenyl)ethylene (DDE) in relation to anogenitaldistance in male newborns from Chiapas, México. Am. J. Epidemiol. 165,1015–1022.

Lubet, R.A., Dragnev, K.H., Chauhan, D.P., Nims, R.W., Diwan, B.A., Ward, J.M., Jones,C.R., Rice, J.M., Miller, M.S., 1992. A pleiotropic response to phenobarbital-typeenzyme inducers in the F344/NCr rat. Effects of chemicals of varied structure.Biochem. Pharmacol. 43, 1067–1078.

Lubet, R.A., Guengerich, F.P., Nims, R.W., 1990. The induction of alkoxyresorufinmetabolism: a potential indicator of environmental contamination. Arch. Envi-ron. Contam. Toxicol. 19, 157–163.

Maher, V.M., Trainer, P.J., Scoppola, A., Anderson, J.V., Thompson, G.R., Besser, G.M.,1992. Possible mechanism and treatment of o,p′-DDD-induced hypercholestero-laemia. Q. J. Med. 84, 671–679.

Mathur, V., Bhatnagar, P., Sharma, R.G., Acharya, V., Sexana, R., 2002. Breast cancerincidence and exposure to pesticides among women originating from Jaipur.Environ. Int. 28, 331–336.

Mazzetti, M.B., Taira, M.C., Lelli, S.M., Dascal, E., Basabe, J.C., de Viale, L.C., 2004.Hexachlorobenzene impairs glucose metabolism in a rat model of porphyriacutanea tarda: a mechanistic approach. Arch. Toxicol. 78, 25–33.

McBlain, W.A., 1987. The levo enantiomer of o,p’-DDT inhibits the biding of 17�-Estradiol to the estrogen receptor. Life Sci. 40, 215–221.

McGlynn, K.A., Abnet, C.C., Zhang, M., Sun, X.D., Fan, J.H., O’Brien, T.R., Wei,W.Q., Ortiz-Conde, B.A., Dawsey, S.M., Weber, J.P., Taylor, P.R., Katki, H.,Mark, S.D., Qiao, Y.L., 2006. Serum concentrations of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene(DDE) and risk of primary liver cancer. J. Natl. Cancer Inst. 98, 1005–1010.

McGlynn, K.A., Quraishi, S.M., Graubard, B.I., Weber, J.P., Rubertone, M.V., Erickson,R.L., 2008. Persistent organochlorine pesticides and risk of testicular germ celltumors. J. Natl. Cancer Inst. 100, 663–671.

Mortensen, H.M., Euling, S.Y. Integrating mechanistic and polymorphism datato characterize human genetic susceptibility for environmental chem-ical risk assessment in the 21st century. Toxicol. Appl. Pharmacol.,http://dx.doi.org/10.1016/j.taap.2011.01.015, in press.

Mrema, E.J., Turci, R., Rubino, F., Fugnoli, L., Pitton, M., Mandic-Rajcevic, S., Colo-sio, C., Minoia, C., 2011. Serum levels of polychlorinated biphenyls (PCBs) andorganochorinated pesticides (OCPs) among individuals of general population inthree Italian geographic regions. Toxicol. Lett. 205S, S60–S179.

Mussalo-Rauhamaa, H., Hasanen, E., Pyysalo, H., Antervo, K., Kauppila, R., Pantzar, P.,1990. Occurrence of �-hexachlorocyclohexane in breast cancer patients. Cancer66, 2124–2128.

Nakamura, Y.K., Omaye, S.T., 2008. Alpha-tocopherol modulates human umbilicalvein endothelial cell expression of Cu/Zn superoxide dismutase and catalaseand lipid peroxidation. Nutr. Res. 28, 671–680.

Nativelle-Serpentini, C., Richard, S., Seralini, G.E., Sourdaine, P., 2003. Aromataseactivity modulation by lindane and bisphenol-A in human placental JEG-3 andtransfected kidney E293 cells. Toxicol. In Vitro 17, 413–422.

Navas, J.M., Chana, A., Herradón, B., Segner, H., 2004. Induction of cytochromeP4501A (CYP1A) by clotrimazole, a non-planar aromatic compound. Compu-tational studies on structural features of clotrimazole and related imidazolederivatives. Life Sci. 76, 699–714.

Nicolopoulou, P., Stamanti, P., 2001. The impact of endocrine disrupters on thefemale reproductive system. Hum. Reprod. Update 7, 323–330.

Ozanne, S.E., Constancia, M., 2007. Mechanisms of disease: the developmental ori-gins of disease and the role of the epigenotype. Nat. Clin. Pract. Endocrinol.Metab. 3, 539–546.

Pena, D., Pontillo, C., García, M.A., Cocca, C., Alvarez, L., Chiappini, F., Bourguignon,N., Frahm, I., Bergoc, R., Kleiman de Pisarev, D., Randi, A., 2012. Alterations inc-Src/HER1 and estrogen receptor � signaling pathways in mammary gland and

tumors of hexachlorobenzene-treated rats. Toxicology 293, 68–77.

Perera, F.P., Rauh, V., Whyatt, R.M., Tang, D., Tsai, W.Y., Bernert, J.T., Tu, Y.H., Andrews,H., Barr, D.B., Camann, D.E., Diaz, D., Dietrich, J., Reyes, A., Kinney, P.L., 2005. Asummary of recent findings on birth outcomes and developmental effects ofprenatal ETS, PAH, and pesticide exposures. Neurotoxicology 26, 573–587.

xicolog

P

P

P

P

P

P

R

R

R

R

R

R

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

E.J. Mrema et al. / To

érez-Maldonado, I.N., Athanasiadou, M., Yánez, L., González-Amaro, R., Bergman,A., Diaz-Barriga, F., 2006. DDE-induced apoptosis in children exposed to the DDTmetabolite. Sci. Total Environ. 370, 343–351.

érez-Maldonado, I.N., Diaz-Barriga, F., De la Fuente, H., González-Amaro, R.,Calderón, J., Yánez, L., 2004. DDT induces apoptosis in human mononuclear cellsin vitro and is associated with increased apoptosis in exposed children. Environ.Res. 94, 38–46.

érez-Maldonado, I.N., Herrera, C., Batres, L.E., González-Amaro, R., Díaz-Barriga, F.,Yánez, L., 2005. DDT-induced oxidative damage in human blood mononuclearcells. Environ. Res. 98, 177–184.

icard, D., 2006. Chaperoning steroid hormone action. Trends Endocrinol. Metab. 17,229–235.

ontillo, C.A., García, M.A., Pena, D., Cocca, C., Chiappini, F., Alvarez, L., Kleiman dePisarev, D., Randi, A.S., 2011. Activation of c-Src/HER1/STAT5b and HER1/ERK1/2signaling pathways and cell migration by hexachlorobenzene in MDA-MB-231human breast cancer cell line. Toxicol. Sci. 120, 284–296.

orta, M., 2006. Persistent organic pollutants and the burden of diabetes. Lancet 368,558–559.

aizada, R.B., Srivastava, M.K., Dikshith, T.S.S., 1991. Lack of estrogenic effectsof endosulfan: an organochlorine insecticide in rat. Natl. Acad. Sci. Lett. 14,103–107.

andi, A.S., Cocca, C., Carbone, V., Nunez, M., Croci, M., Gutierrez, A., Bergoc, R.,Kleiman de Pisarev, D.L., 2006. Hexachlorobenzene is a tumor co-carcinogenand induces alterations in insulin growth factors signaling pathway in the ratmammary gland. Toxicol. Sci. 89, 83–92.

appolt, R.T., Hale, W.E., 1968. p,p′-DDE and p,p′-DDT residues in human placentas,cords and adipose tissue. Clin. Toxicol. 1, 57–61.

ecio-Vega, R., Velazco-Rodriguez, V., Ocampo-Gómez, G., Hernandez-Gonzalez,S., Ruiz-Flores, P., Lopez-Marquez, F., 2011. Serum levels of polychlorinatedbiphenyls in Mexican women and breast cancer risk. J. Appl. Toxicol. 31,270–278.

odenhiser, D., Mann, M., 2006. Epigenetics and human disease: translating basicbiology into clinical applications. CMAJ 174, 341–348.

usiecki, J.A., Baccarelli, A., Bollati, V., Tarantini, L., Moore, L.E., Bonefeld-Jorgensen,E.C., 2008. Global DNA hypomethylation is associated with high serum-persistent organic pollutants in Greenlandic Inuit. Environ. Health Perspect. 116,1547–1552.

aid, T.M., Paasch, U., Glander, H.J., Agarwal, A., 2004. Role of caspases in maleinfertility. Hum. Reprod. Update 10, 39–51.

aiyed, H., Dewan, A., Bhatnagar, V., Shenoy, U., Shenoy, R., Rajmohan, H., Patel, K.,Kashyap, R., Kulkarni, P., Rajan, B., Lakkad, B., 2003. Effect of endosulfan on malereproductive development. Environ. Health Perspect. 111, 1958–1962.

amanta, L., Chainy, G.B., 1997. Comparison of hexachlorocyclohexane-inducedoxidative stress in the testis of immature and adult rats. Comp. Biochem. Physiol.C: Pharmacol. Toxicol. Endocrinol. 118, 319–327.

aradha, B., Vaithinathan, S., Mathur, P.P., 2009. Lindane induces testicular apoptosisin adult Wistar rats through the involvement of Fas-FasL and mitochondria-dependent pathways. Toxicology 255, 131–139.

chneider, J., Kinne, D., Fracchia, A., Pierce, V., Anderson, K.E., Bradlow, H.L., Fish-man, J., 1982. Abnormal oxidative metabolism of estradiol in women with breastcancer. Proc. Natl. Acad. Sci. U.S.A. 79, 3047–3051.

cippo, M.L., Argiris, C., Van De, W.C., Muller, M., Willemsen, P., Martial, J., Maghuin-Rogister, G., 2004. Recombinant human estrogen, androgen and progesteronereceptors for detection of potential endocrine disruptors. Anal. Bioanal. Chem.378, 664–669.

helby, M.D., Newbold, R.R., Tully, D.B., Chae, K., Davis, V.L., 1996. Assessing environ-mental chemicals for estrogenicity using a combination of in vitro and in vivoassays. Environ. Health Perspect. 104, 1296–1300.

hi, Y., Song, Y., Wang, Y., Liang, X., Hu, Y., Guan, X., Cheng, J., Yang, K., 2009. p,p′-DDEinduces apoptosis of rat Sertoli cells via a FasL-dependent pathway. J. Biomed.Biotechnol. 2009, 181282.

hi, Y., Song, Y., Wang, Y., Wang, Y., Liang, X., Hu, Y., Yu, H., Guan, X., Cheng, J., Yang,K., 2011. �-Benzene hexachloride induces apoptosis of rat Sertoli cells throughgeneration of reactive oxygen species and activation of JNKs and FasL. Environ.Toxicol. 26, 124–135.

hi, Y.Q., Wang, Y.P., Song, Y., Li, H.W., Liu, C.J., Wu, Z.G., Yang, K.D., 2010. p,p′-DDE induces testicular apoptosis in prepubertal rats via the Fas/FasL pathway.Toxicol. Lett. 193, 79–85.

hinomiya, N., Shinomiya, M., 2003. Dichlorodiphenyltrichloroethane suppressesneurite outgrowth and induces apoptosis in PC12 pheochromocytoma cells.Toxicol. Lett. 137, 175–183.

hutoh, Y., Takeda, M., Ohtsuka, R., Haishima, A., Yamaguchi, S., Fujie, H., Komatsu, Y.,Maita, K., Harada, T., 2009. Low dose effects of dichlorodiphenyltrichloroethane(DDT) on gene transcription and DNA methylation in the hypothalamus ofyoung male rats: implication of hormesis-like effects. J. Toxicol. Sci. 34,469–482.

ilva, E., Kabil, A., Kortenkamp, A., 2010. Cross-talk between non-genomic andgenomic signalling pathways–distinct effect profiles of environmental estro-gens. Toxicol. Appl. Pharmacol. 245, 160–170.

ilva, E., Scholze, M., Kortenkamp, A., 2007. Activity of xenoestrogens at nanomolarconcentrations in the E-Screen assay. Environ. Health Perspect. 115, 91–97.

inha, N., Adhikari, D., Saxena, D.K., 2001a. Effect of endosulfan on the enzymes ofpolyol pathway in rat Sertoli-germ cell coculture. Bull. Environ. Contam. Toxicol.67, 821–827.

inha, N., Adhikari, N., Narayan, R., Saxena, D.K., 1999. Cytotoxic effect of endosulfanon rat Sertoli-germ cell coculture. Reprod. Toxicol. 13, 291–294.

y 307 (2013) 74– 88 87

Sinha, N., Adhikari, N., Saxena, D.K., 2001b. Effect of endosulfan during fetal gonadaldifferentiation on spermatogenesis in rats. Environ. Toxicol. Pharmacol. 10,29–32.

Sinha, N., Narayan, R., Saxena, D.K., 1997. Effect of endosulfan on the testis of growingrats. Bull. Environ. Contam. Toxicol. 58, 79–86.

Sinha, N., Narayan, R., Shanker, R., Saxena, D.K., 1995. Endosulfan induced biochem-ical changes in the testis of rats. Vet. Hum. Toxicol. 37, 547–549.

Song, M.O., Lee, C.H., Yang, H.O., Freedman, J.H., 2012. Endosulfan upregulates AP-1binding and ARE-mediated transcription via ERK1/2 and p38 activation in HepG2cells. Toxicology 292, 23–32.

Song, Y., Liang, X.M., Hu, Y.F., Wang, Y.N., Yu, H.G., Yang, K.D., 2008. p,p′-DDE inducesmitochondria-mediated apoptosis of cultured rat Sertoli cells. Toxicology 253,53–61.

Song, Y., Shi, Y., Yu, H., Hu, Y., Wang, Y., Yang, K., 2011. p,p′-Dichlorodiphenoxydichloroethylene induced apoptosis of Sertoli cellsthrough oxidative stress-mediated p38 MAPK and mitochondrial pathway.Toxicol. Lett. 202, 55–60.

Soto, A.M., Chung, K.L., Sonnenschein, C., 1994. The pesticides endosulfan,toxaphene, and dieldrin have estrogenic effects on human estrogen-sensitivecells. Environ. Health Perspect. 102, 380–383.

Soto, A.M., Sonnenschein, C., Chung, K.L., Fernandez, M.F., Olea, N., Olea-Serrano, F.,1995. The E-SCREEN assay as a tool to identify estrogens: an update on estrogenicenvironmental pollutants. Environ. Health Perspect. 103, 113–122.

Steinmetz, R., Young, P.C., Caperell-Grant, A., Gize, E.A., Madhukar, B.V., Ben-Jonathan, N., Bigsby, R.M., 1996. Novel estrogenic action of the pesticide residuebeta-hexachlorocyclohexane in human breast cancer cells. Cancer Res. 56,5403–5409.

Sujatha, R., Chitra, K.C., Latchoumycandane, C., Mathur, P.P., 2001. Effect of lindaneon testicular antioxidant system and steroidogenic enzymes in adult rats. AsianJ. Androl. 3, 135–138.

Swanson, H.I., 2002. DNA binding and protein interactions of the AHR/ARNT het-erodimer that facilitate gene activation. Chem. Biol. Interact. 141, 63–76.

Swedenborg, E., Ruegg, J., Makela, S., Pongratz, I., 2009. Endocrine disruptive chem-icals: mechanisms of action and involvement in metabolic disorders. J. Mol.Endocrinol. 43, 1–10.

Tebourbi, O., Hallegue, D., Yacoubi, M.T., Sakly, M., Rhouma, K.B., 2010. Subacutetoxicity of p,p′-DDT on rat thyroid: hormonal and histopathological changes.Environ. Toxicol. Pharmacol. 29, 271–279.

Telang, N.T., Suto, A., Wong, N.Y., Bradlow, H.L., Osborne, M.P., 1992. Induction bythe estrogen metabolite, 16�-hydroxyestrone, of genotoxic damage and aber-rant proliferation in mouse mammary epithelial cells. J. Natl. Cancer Inst. 84,634–638.

To-Figueras, J., Sala, M., Otero, R., Barrot, C., Santiago-Silva, M., Rodamilans, M.,Herrero, C., Grimalt, J., Sunyer, J., 1997. Metabolism of hexachlorobenzene inhumans: association between serum levels and urinary metabolites in a highlyexposed population. Environ. Health Perspect. 105, 78–83.

Torres-Sánchez, L., Zepeda, M., Cebrián, M.E., Belkind-Gerson, J., Garcia-Hernandez, R.M., Belkind-Valdovinos, U., López-Carrillo, L., 2008. Dichloro-diphenyldichloroethylene exposure during the first trimester of pregnancyalters the anal position in male infants. Ann. N. Y. Acad. Sci. 1140, 155–162.

Tsang, H.L., Wu, S., Leung, C.K.M., Tao, S., Wong, M.H., 2011. Body burden of POPs ofHong Kong residents, based on human milk, maternal and cord serum. Environ.Int. 37, 142–151.

Tsatsakis, A., Tutudaki, M., 2004. Progress in pesticide and POPs hair analysis for theassessment of exposure. Forensic Sci. Int. 145, 195–199.

Tsatsakis, A.M., Tutudaki, M., Tzatzarakis, M., Psaroudakis, K., Dolapsakis, G.P.,Michalodimitrakis, M.N., 1998. Pesticide disposition in hair: preliminary resultsof a model study of methomyl incorporation into rabbit hair. Vet. Hum. Toxicol.40, 200–204.

Tsatsakis, A.M., Tzatzarakis, M.N., Tutudaki, M., 2008. Pesticide levels in head hairsamples of Cretan population as an indicator of present and past exposure.Forensic Sci. Int. 176, 67–71.

Tsatsakis, A.M., Zafiropoulos, A., Tzatzarakis, M.N., Tzanakakis, G.N., Kafatos, A., 2009.Relation of PON1 and CYP1A1 genetic polymorphisms to clinical findings ina cross sectional study of a Greek rural population professionally exposed topesticides. Toxicol. Lett. 186, 66–72.

Tutudaki, M., Tsakalof, A.K., Tsatsakis, A.M., 2003. Hair analysis used to assess chronicexposure to the organophosphate diazinon: a model study with rabbits. HumanExp. Toxicol. 22, 159–164.

UNEP, 2010a. Report of the Third Expert Group Meeting on the assessmentof the production and use of DDT and its Alternatives for Disease Vec-tor Control. Stockholm Convention on Persistent Organic Pollutants, Geneva,Switzerland.

UNEP, 2010b. Report of the Sixth Expert Meeting on Endosulfan Risk ManagementEvaluation. Stockholm Convention on Persistent Organic Pollutants, Geneva,Switzerland.

Vaithinathan, S., Saradha, B., Mathur, P.P., 2010. Methoxychlor induces apoptosisvia mitochondria- and FasL-mediated pathways in adult rat testis. Chem. Biol.Interact. 185, 110–118.

van Ommen, B., van Bladeren, P.J., 1989. Possible reactive intermediates in the oxida-tive biotransformation of hexachlorobenzene. Drug Metab. Drug Interact. 7,

213–243.

Van Tonder, J.J., 2011. Development of in vitro mechanistic toxicityscreening model using cultured hepatocytes. PhD Thesis. Univer-sity of Pretoria, Pretoria, viewed 2012 September, 27. Available:http://upetd.up.ac.za/thesis/available/etd-04262012-112100/

8 icolog

W

W

WW

WW

Yu, H.G., Liang, X.M., Hu, Y.F., Wang, Y.N., Yang, K.D., 2008. Action mechanism of

8 E.J. Mrema et al. / Tox

ade, M.G., Desaulniers, D., Leingartner, K., Foster, W.G., 1997. Interactions betweenendosulfan and dieldrin on estrogen-mediated processes in vitro and in vivo.Reprod. Toxicol. 11, 791–798.

ang, L., Zhou, S., Lin, K., Zhao, M., Liu, W., Gan, J., 2009. Enantioselective estro-genicity of o,p′-dichlorodiphenyltrichloroethane in the MCF-7 human breastcarcinoma cell line. Environ. Toxicol. Chem. 28, 1–8.

HO, 1992. Alpha- and Beta Hexachlorocyclohexane. EHC 123.HO, 2003. Joint WHO/Convention Task Force on the Health Aspects of Air Pollution.

Health Risks of Persistent Organic Pollutants from Long Range TransboundaryAir Pollution. Copenhagen, Denmark.

HO, 2011. DDT in Indoor Residual Spraying: Human Health Aspects. EHC 241.ójtowicz, A.K., Honkisz, E., Zieba-Przybylska, D., Milewicz, T., Kajta, M., 2011.

Effects of two isomers of DDT and their metabolite DDE on CYP1A1

y 307 (2013) 74– 88

and AhR function in human placental cells. Pharmacol. Rep. 63, 1460–1468.

Wu, Y., Foster, W.G., Younglai, E.V., 2006. Rapid effects of pesticides on humangranulosa-lutein cells. Reproduction 131, 299–310.

Yoshioka, H., Miyata, T., Omura, T., 1984. Induction of a phenobarbital-inducibleform of cytochrome P-450 in rat liver microsomes by 1,1-di(p-chlorophenyl)-2,2-dichloroethylene. J. Biochem. 95, 937–947.

p,p′-DDE and/or beta-BHC on JNK and MAPK signal transduction pathways inrat Sertoli cells. Zhonghua Nan Ke Xue 14, 311–316.

Zala, S., Penn, D.J., 2004. Abnormal behaviours induced by chemical pollution: areview of the evidence and new challenges. Anim. Behav. 68, 649–664.