Persistent Organochlorinated Pesticides and Mechanisms of Their Toxicity

Home , DDT, Diosmetin, Methoxychlor

Toxicology 307 (2013) 74–88

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Toxicology

j ournal homepage: www.elsevier.com/locate/toxicol

Persistent organochlorinated pesticides and mechanisms of their toxicity

a,b,∗ a a c

Ezra J. Mrema , Federico M. Rubino , Gabri Brambilla , Angelo Moretto ,

d a

Aristidis M. Tsatsakis , Claudio Colosio

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 Laboratory

for Analytical Toxicology and Metabolomics, Via San Vigilio 43, 20142, Milan, Italy

Department of Environmental and Occupational Health, Muhimbili University of Health and Allied Sciences, P.O. Box 65015, Dar es Salaam, Tanzania

Department of Biomedical and Clinical Sciences, Luigi Sacco of the University of Milan and International Centre for Pesticides and Health Risk Prevention (ICPS) of the University

Hospital Luigi Sacco, Via GB Grassi 74, 20157 Milan, Italy

Centre of Toxicology Sciences and Research, Division of Morphology, Medical School, University of Crete, Voutes, Heraklion, 71003 Crete, Greece

a r a

t i c l e i n f o b s t r a c t

Article history: Persistent organic pollutants comprised of organic chemicals like polychlorinated biphenyls, dibenzo-p-

Received 22 October 2012

dioxins, dibenzofurans and organochlorinated pesticides which have many characteristics in common.

Received in revised form

Once released in the environment they resist physical, biological, chemical and photochemical breakdown

24 November 2012

processes and thus persist in the environment. They are subject to long transboundary air pollution trans-

Accepted 27 November 2012

port. They accumulate in the food chain due to their lipophilicity, bioaccumulation and biomagniﬁcation

Available online 3 December 2012

properties. Human exposure occurs through inhalation of air, ingestion of food and skin contact. Because

most of them bioaccumulate and remain preferentially in fat, their long-term effects are still a matter of

Keywords:

public health concern. They are condemned for health adverse effects such as cancer, reproductive defects,

Human exposure

neurobehavioral abnormalities, endocrine and immunological toxicity. These effects can be elicited via a

Adverse health effect

Organochlorinated pesticides number of mechanisms among others include disruption of endocrine system, oxidation stress and epi-

Mechanisms of action genetic. However most of the mechanisms are not clear thus a number of studies are ongoing trying to

elucidate 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 to

some existing epidemiological data. Both genomic and nongenomic pathways are used to describe these

effects. 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),

1. Introduction

Persistent organochlorinated pesticides are a structurally het-

erogeneous class of organic compounds composed primarily of

•

Abbreviations: NO, nitrogen oxide radical; 16␣-OHE, 16␣-hydroxyestrone;

carbon, hydrogen and several chlorine atoms per molecule. They

2-OHE, 2-hydroxyestrone; 6 CpG, Cytosine Guanine base pair; DDT,

share similar properties with other persistent organic pollutants

dichlorodiphenyltrichloroethane; GABAA, gamma butyric acid type A

• (POPs) such as polychlorinated biphenyls (PCBs) and polychlori-

Receptor; HCB, hexachlorobenzene; HO , hydroxyl radical; o,p -DDD,

1,1-dichloro-2-(o-chlorophenyl)-2-(p chlorophenyl)ethane; o,p -DDE, nated dibenzo-p-dioxins/furans (PCDDs/Fs). Once released in the

1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethylene; o,p -DDT, 1,1,1- environment, they break down very slowly in air, water, soil and

•−

trichloro-2-(o-chlorophenyl)-2-(p chlorophenyl)-ethane; O2 , superoxide

in living organisms and are thus subject to long trans-boundary

radical; p,p -DDD, 1,1-dichloro-2, 2-bis (4-chlorophenyl)ethane; p,p -DDE,

air pollution (LTAP) transport where they are carried over long dis-

1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene; p,p -DDT, 1,1,1-trichloro-

tances via the atmospheric transport and human activities. Even the

2,2-bis(p-chlorophenyl) ethane; PCBs, polychlorinated biphenyls; PCDDs,

polychlorinated dibenzo-p-dioxins; PCDFs, polychlorinated dibenzofurans; PI3K, body burden of the ﬂocks of migratory birds is a sizeable contribu-

phosphatidylinositol 3-kinase; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; ␤-HCH,

tion to LTAP (WHO, 2003). As OCPs bio-magnify through the food

beta-hexachlorocyclohexane.

∗ chain, consumers of food of animal origin such as ﬁsh, meat, milk

Corresponding author at: Department of Health Sciences of the University of

and dairy products end up with high levels of exposure and due

Milan, Via San Vigilio 43, 20142 Milan, Italy. Tel.: +39 02 81843469;

to slow biodegradation, OCPs accumulate in the body: for example

fax: +39 02 49538671; mobile: +39 327 2262702.

E-mail addresses: [email protected], [email protected] (E.J. Mrema). DDT can remain in the body for 50 years, lifelong sequestrated into

E.J. Mrema et al. / Toxicology 307 (2013) 74–88 75

the soft tissue compartments (mainly the adipose tissues), from 2. Mechanisms of OCPs toxicity

which they partition to the bloodstream into plasma or serum lipids

and are actively secreted into breast milk as the main elimination 2.1. Endocrine activity

pathway in mammals.

Human exposure begins during early prenatal and continues The endocrine system controls, balances and produces hor-

during the breast-feeding neonatal periods, which are critical mones and modulates their actions in the body through a network

stages for the development and differentiation of sensitive body of activation and repression pathways at multiple levels of synthe-

organs and systems. In fact, these chemicals cross the placenta to sis, secretion, transport, binding, action or elimination of natural

the foetus and are secreted into breast milk (Perera et al., 2005). hormones. Steroid hormones are involved in cell development,

In adults the dietary exposure route accounts for more than 90% metabolism, protein synthesis, behavior, reproduction and devel-

of total organochlorine compounds (OCs) burden for the general opment. These responses are elicited through binding of hormones

population, while workers are exposed mainly through inhalation to their speciﬁc receptors such as nuclear receptors (NRs). Among

and skin contact. OCs are rapidly absorbed in the small intestine the members of NRs are estrogen receptors (ERs), androgen

and enter circulatory system where they are distributed throughout receptor (AR), progesterone receptor (PR), glucocorticoid receptor

the body and accumulate in body tissues with high lipid contents, (GR), constitutive androstane receptor (CAR), rodent pregname X

with a continuous exchange between blood and tissues. As such receptor (PXR), mineralocorticoid receptor and thyroid hormone

these chemicals have been detected in human tissues such as receptors (TRs). Aromatic hydrocarbon receptor (AhR) is a key reg-

blood (whole blood, cord blood, serum and plasma), adipose tis- ulator of the cellular response to xenobiotic exposure (Swedenborg

sues (in autopsies and living subjects), breast milk, muscles and hair et al., 2009). NRs and AhR are involved in the induction of xenobiotic

(Appenzeller and Tsatsakis, 2012; Covaci et al., 2002; Dewailly et al., metabolizing enzymes.

1999; Jakszyn et al., 2009; Mrema et al., 2011; Rappolt and Hale, In the absence of ligands NRs and AhR stay in the cytoplasm or

1968; Tsang et al., 2011; Tsatsakis and Tutudaki, 2004; Tsatsakis in the cell nucleus in a complex form (Picard, 2006). In the presence

et al., 1998, 2008; Tutudaki et al., 2003). of ligands, receptor-ligand binding occurs, followed by translo-

Exposure to these persistent chemicals has been associated with cation of NRs/AhR to the nucleus. In the nucleus, a complex of

health effects including cancer (Aronson et al., 2000; Cohn et al., aryl receptor nuclear translocator (ARNT) protein and co-activators

2007; Mathur et al., 2002; Recio-Vega et al., 2011), reproductive is formed. The complex binds to the speciﬁc AhR-DNA recogni-

defects (Nicolopoulou and Stamanti, 2001) and behavioral changes tion site and ﬁnally induces transcription of target genes. The

(Zala and Penn, 2004). These effects are believed to be related to ligand binding triggers conformational changes that lead to dissoci-

their ability to disrupt the functions of certain hormones, enzymes, ation of the repressive complex, the recruitment of transcriptional

growth factors, neurotransmitters and to induce key genes involved co-activators and receptor dimerization (Hankinson, 2005). NRs

in metabolism of steroids and xenobiotics (Gourounti et al., 2008). homodimerize or heterodimerize with retinoid X receptor and AhR

The mechanism of action involves toxicokinetic and toxicody- dimerizes with ARNT leading to gene transcription of NR or AhR

namic processes, at several levels between chemical exposure and target genes (Claessens and Gewirth, 2004; Swanson, 2002). NRs

the endpoints at individual level. Variation within humans can control transcription through binding to promoter regions of DNA.

affect the endpoint at any toxicodynamic and/or toxicokinetic steps Several chlorine atoms in the molecular structures of OCPs

(Mortensen and Euling, in press). Direct, non/genomic mechanisms impart their highly lipophilic character and fairly rigid confor-

of toxicity involve perturbation of the homeostatic redox level(s) of mation (Fig. 2). The chlorine atoms are poorly reactive towards

biological compartments (oxidative stress) and consequent cellular nucleophilic displacement and elimination reactions and thus their

death through apoptosis. Indirect, genomic mechanisms of toxic- biotransformation and biodegradation reactions are limited and are

ity involve permanent modiﬁcation of the transcription of gene mostly conﬁned, albeit to poor yield, only to anaerobic environment

elements by interference at the epigenetic level of DNA functioning. of sludges, but seldom in mammalian organisms. As a consequence,

Since little is known on the relationship among events that their interaction with biological systems is mostly limited to ago-

result in OCPs’ toxicity in humans (Mortensen and Euling, in press), nistic or antagonistic binding to the intracellular receptors for

a number of studies are currently ongoing to investigate mecha- which natural hydrophobic substances, such as steroid derivatives,

nisms responsible for OCPs’ toxicity. A better knowledge on how are the endogenous ligands. Agonistic binding leads to recruit-

these compounds inﬂuence the development and progression of ment of coactivators and thus increases transcriptional activity

diseases is a crucial step towards addressing the emerging dis- while antagonistic binding prevents coactivator recruitment and/or

eases and hence improving public health. In this review, we provide attracts corepressors, leading to decreased transcriptional activity

an overview on available information of possible mechanisms of of the receptors.

toxicity of some selected OCPs and where possible a linkage is Many POPs including OCPs are known or suspected to be

made to epidemiological data. We summarize ﬁndings of epidemi- endocrine active. When present in the body they may interfere

ological studies and both in vitro and in vivo experimental studies at several control points in the hormone signaling pathways. As

(Tables 1–3). a result, the response cascade of natural hormones can either be

The main OCPs for which we review available information inhibited or excessively enhanced, at the wrong time, in the wrong

are dichlorodiphenyl-trichloroethane (DDT) and its metabolites, tissue (Swedenborg et al., 2009). Endocrine activity of OCPs can be

hexachlorobenzene (HCB), beta-hexachlorohexacyclohexane (␤- due to direct binding with hormone receptors due to their confor-

HCH) and endosulfan. These chemicals have been employed in mational similarity with the receptor-binding portions of natural

agriculture and in public health as insecticides and biocides for hormones, mainly of the steroid and diphenylether (thyroxine)

several decades but were mostly banned between the ‘1970 and structural groups. This is the case of aromatic polychlorinated sub-

1990s’. Only a few active substances, essentially DDT is still used stances such as DDT and its structural cognates, endosulfan and

in tropical countries to control malaria vectors through indoor lindane. Other compounds indirectly alter hormonal pathway by

residual spraying (Bouwman et al., 2011) and lindane for treat- directly inhibiting enzyme activities responsible for biosynthesis

ment of lice and scabies (Engeler, 2009). So, most exposures of the precursors of steroid hormones. In particular, the DDT ana-

derive from past uses and, in certain cases, are still signiﬁcant log, mitotane, a pharmaceutical drug which is used to suppress

because of the long persistence and biomagniﬁcation of these cortisol production also causes a sharp raise in plasma cholesterol compounds. levels.

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

Table 1

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

Author Effects assessed OCP measured OCP level OCP level (␮g/g Results ptrend

(␮g/L serum) lipid serum)

Cohn et al. (2007) Breast cancer DDT Tertiles Tertiles OR, 95% CI

<8.09 <1.1 1.00 0.01

8.09–13.9 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 0.049

0.265–0.382 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 0.0024

0.265–0.382 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 0.0002

0.158–0.250 1.01 (0.75–1.36)

0.251–0.390 1.00 (0.73–1.38)

>0.390 1.71 (1.23–2.38)

2.1.1. Interference of OCPs in estrogen hormone metabolism DDT. In fact, the concentration of DDT employed in the Bradlow

−5

␤ ␮

17 -Estradiol is the main estrogen hormone, responsible for experiments is 10 M (10 M), while the physiological concen-

development of female sex characteristics such as growth of tration of steroid hormones such as estradiol is of at least four to

the breasts and suppression of body hair growth. Biotransfor- six orders of magnitude lower (low-picomolar). On the other hand

mation of 17␤-estradiol proceeds via two common metabolic DDT and its metabolites as well as many other OCPs are known

pathways yielding catechol estrogen 2-hydroxyestrone (2-OHE), phenobarbital-type cytochrome P450 inducers as they have been

a weak estrogen; and 16␣-hydroxyestrone (16␣-OHE), a fully shown to induce cytochrome P450 2B (CYP 2B) in rat liver or cul-

potent estrogen. Enzymes CYP1A1, CYP1B1, CYP1A2 and catechol- tured rat hepatocytes (Campbell et al., 1983; Li et al., 1995; Lubet

O-methyltransferase (COMT) are responsible for this metabolism. et al., 1990, 1992; Yoshioka et al., 1984). The enzyme activation

␣

16 -OHE, but not 2-OHE increases unscheduled DNA synthesis, upon exposure to these pesticides can thus lead to alterations in

uncontrolled cell division and increased anchorage-independent the endogenous levels of hormones and consequently compromise

growth. Interrupting these pathways might lead to formation of hormone signaling. In addition to xenobiotics metabolism, CYP1A1

more potent products as exempliﬁed by some xenoestrogens. has been shown to participate in the activation of dietary polyphe-

Exposure of cell cultures of MCF-7 to concentrations as low as nols (Androutsopoulos et al., 2008, 2009a) and it is inhibited by

−5

10 M of various OCPs such as DDT, lindane, chlordecone and various polyphenolics (Androutsopoulos et al., 2010, 2011).

endosulfan blocked the 2-OHE pathway and increase levels of 16␣-

OHE in estrogen positive (ER+) cells (Bradlow et al., 1995). This 2.1.2. DDT and DDT metabolites

␣

interference in estrogen biotransformation led to increasing 16 - DDT is produced as a technical mixture of 3 isomeric forms, the

OHE/2-OHE ratio relative to the ratio in the untreated control cell most prevalent being the p,p -isomer; o,o - and o,p -being present in

cultures. This increasing ratio has been associated with breast and minor and variable amounts. DDT was initially used in public health

other cancers of animals (Telang et al., 1992) (Fig. 1); possibly control of lice and malaria mosquitoes during the Second World

␣

because of 16 -OHE has a strong estrogenic activity (Bradlow et al., War and afterwards it was used as pesticide in agriculture. Globally

␣

1995). Levels of 16 -hydroxylation have been found increased by DDT production was ∼1.8 million tonnes since 1940s (ATSDR, 2002)

about 50% in human breast cancer as compared to control cases and more than 40,000 tonnes were used in agriculture annually

(Schneider et al., 1982). (Geisz et al., 2008). It is estimated 600,000 tonnes of DDT were

CYP1A1 metabolizes estradiol and environmental contami- used domestically in USA 30 years prior to its ban. Over 80% of

nants into the corresponding hydroxylated derivatives through the the quantity of the pesticide used in 1970–1972 was applied to

highly reactive intermediates of arene-oxide connectivity (Hayes cotton crops and the remainder was used on peanut and soybean

et al., 1996). The expression of CYP1A1 is regulated by the aryl crops. The global production was reported to drop to 3314 tonnes

hydrocarbon receptor (AhR), a ligand-dependent transcription in 2009; the production is meant for use in control of malaria and

factor that activates the transcription of target genes, such as leishmaniasis (UNEP, 2010a).

CYP1A1, CYP1A2, CYP1B1 and oncogenes (Androutsopoulos et al., DDT and its metabolite DDE exhibit hormonal activity in vari-

2009b). DDT and its metabolites are capable of up regulating ous tissues with mechanisms involving the steroidogenic pathway,

CYP1A activity (Van Tonder, 2011). According to Navas et al. (2004) receptor mediated changes in protein synthesis or antiandrogenic

CYP1A inducers have deﬁned structures which make them capable and estrogenic actions. Most of their endocrine effects result from

of binding AhR. DDT has two aromatic rings linked by sp3 carbon the ability to mimic 17-␤-estradiol. o,p -DDT is a well character-

and thus each of the rings can rotate along its single bond with the ized DDT isomer; its intracellular mechanism of action is mediated

C2 carbon. This allows some degree of conformational mobility through the ER pathway (Steinmetz et al., 1996) described in Sec-

and in principle the possibility to bind AhR but the rings cannot lie tion 2.1. It has been shown to bind and activate the ER, to promote

in the same plane, as in the estrogen molecules (Fig. 3). Lack of this the expression of estrogen-dependent genes and to induce prolif-

molecular recognition motif greatly limits the afﬁnity of AhR for eration of ER-competent cells such as breast cancer cells (MCF-7)

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

Table 2

Summary 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 of Endosulfan was 10 times less potent than

pesticides 17␤-estradiol. Effects observed at doses of

10 ␮M endosulfan or greater.

Soto et al. (1995) In vitro MCF-7 cell culture Relative binding afﬁnity of Endosulfan hPR receptor binding afﬁnity was

pesticides 10 less than 17␤-estradiol.

Wade et al. (1997) In vitro MCF-7 cell culture Cell proliferation potential of Endosulfan was 10 less potent as compared to

pesticides 17␤-estradiol. Effect observed at the highest

soluble dose.

Sinha et al. (1999) In vitro Rat testicular cells in culture Cell viability and lactate Endosulfan produced cytotoxic change. Effects

dehydrogenase leakage observed at 20 ␮M of endosulfan at or above.

Sinha et al. (2001b) In vitro Rat testicular cells in culture Activity of polyol pathway Increased aldose reductase activity and

enzymes decreased sorbitol dehydrogenase activity at

or >20 ␮M of endosulfan.

Grunfeld and In vitro MCF-7 BUS cells Human ER␣ and ER␤ mRNA Endosulfan decreased human ER␣ mRNA

Bonefeld-Jorgensen expression levels weakly with no effect on human EB␤

(2004) expression.

Li et al. (2006) In vitro MCF-7 cell culture Activation of MAPK, PI3-K, At 20–75 ␮M endosulfan activated P13-K and

PKC, PKA and CaMKIV MAPK. The effect observed at 10 nM of

17␤-estradiol and DES.

Sinha et al. (1995) In vivo Adult Druckrey rats Testicular enzyme activities, Increased testicular enzyme activities at all

administered with repeat sperm count, sperm doses tested; sperm count decreased in a

dose of endosulfan (0, 2.5, 5 morphology, and dose-dependent manner; decreased spermatid

or 10 mg/kg, 5 days per week intra-testicular spermatid counts and reduced daily sperm production at

for 70 days) count the two highest test doses was reported.

Sinha et al. (1997) In vivo Weanling Druckrey rats Testicular enzyme activity, No observed effects on testes weights;

administered with repeat testes weights, sperm count, increased levels of testicular enzyme activity;

dose of endosulfan (0, 2.5, 5 sperm abnormality, sperm count decreased in a dose-dependent

or 10 mg/kg, 5 days per week spermatid count and daily manner; decreased spermatid count and

for 90 days) sperm production sperm production rate.

Raizada et al. (1991) In vivo Ovariectomized Wistar rats Organs weights, glycogen No effects on weights of uterus, cervix, vagina

administered with repeat content of organs, and and pituitary; no effects on glycogen content of

dose of endosulfan histopathological changes in the organs examined; no histopathological

(1.5 mg/kg daily for 30 days organs changes reported.

and injected daily with

␮

estradiol 1 g/rat i.p.)

Shelby et al. (1996) In vivo Mice administered with Competitive binding of No competitive inhibition of estrogen receptor

repeat dose of endosulfan on endosulfan to mouse observed; no increase in uterine wet mass;

postnatal days 17–19 at a estrogen receptor and DES and estradiol signiﬁcantly affected uterine

dose of 10 mg/kg uterine growth growth.

Wade et al. (1997) In vivo 18 days rats injected with Uterine estrogen receptor Estradiol binding inhibited at high doses

3 mg/kg/day endosulfan daily binding (10 M endosulfan); no change in uterine

for 3 days weights, uterine peroxidase activity or

numbers of uterine estrogen and progesterone

receptors.

Saiyed et al. (2003) Cohort Male children lived in the SMR assessment; serum Sexual maturity delayed in the exposed

village sprayed with testosterone, LH, and FSH; population; controlled for age, only serum LH

endosulfan and children who and endosulfan residues in levels differed between the populations.

lived in 20 km away from blood

village

Ibarluzea et al. (2004) Case–control Breast cancer women (cases) Relationship between cancer Endosulfan residues were not associated with

and non-cancer women diagnosis and the adipose increased risk of breast cancer

(controls) pesticides

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

MCF-7, Michigan Cancer Foundation-7; SMR, sexual maturity rating; LH, luteinizing hormone; FSH, Follicle Stimulating Hormone; i.p., intraperitoneal injection; ER, estrogen

receptor; hPR, human progesterone; DES, diethylbestrol.

in vitro (Soto et al., 1995). At 10 ␮M concentration, o,p -DDT ago- promoter with a luciferase reporter vector revealed that androgen-

␣

nized both ER - and ER␤-mediated transcription a potency that is induced AR transcriptional activity was inhibited by p,p -DDE or by

approximately 0.1% that of estradiol (Lemaire et al., 2006). A signif- the potent antiandrogen, hydroxyﬂutamide at 0.2 ␮M (63.6 ppb)

icant dose-dependent increase in cell number in MCF-7 cell culture concentration. This level of p,p -DDE is lower than 140 ppb found

−8

was observed starting at 10 M and maximum response achieved among residents living in DDT-treated homes (Bouwman et al.,

−6

at 10 M (Steinmetz et al., 1996). Enantiomer-speciﬁc estrogenic 1991). In utero exposure to DDE has been shown to decrease

activity has been suggested (Hoekstra et al., 2001; McBlain, 1987; anogenital distance (a morphologic quantitative indicator of fem-

−5

Wang et al., 2009). At 5.26 × 10 M (S)-(−)-o,p -DDT produced half inization of male offspring) in male rat offspring, to increase

of the maximum effect (EC50) whereas (R)-(+)-o,p -DDT had negli- retention of male nipple and to cause an alteration in expression of

gible estrogen activity (Hoekstra et al., 2001). The (+)-enantiomer androgen receptor (Kelce et al., 1995) suggesting that these abnor-

exhibited estrogen-like response only at an environmentally unre- malities might be mediated at the level of the androgen receptor.

−3

alistic concentration of 10 M, and thus estrogenic activity of In addition to anti-androgen activity, p,p -DDE can activate the

o,p -DDT is due to the (−)-enantiomer. ER and induce cell proliferation (Kelce et al., 1995). 17␤-Estradiol

−8 −5 −5

Transient cotransfection assay of monkey kidney CV1 cells with (1 × 10 M), o,p -DDT (1 × 10 M) and ␤-HCH (1 × 10 M) sim-

human AR expression vector and a mouse mammary tumor virus ilar showed gene expression proﬁle of estrogen-responsive genes

78 E.J. Mrema et al. / Toxicology 307 (2013) 74–88 B at by

The of ␬ in ROS

M. B these vitro ROS

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MDA MDA cells

group bak group;

SP600125,

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␮ NAC;

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␮ ␮

and groups;

effect. ␮

were

and caspase-3 germ nuclear

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increase

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FasL

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Bax in

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and NAC B any

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50;

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NAC;

and M lactate ␬

JNK2

human 30

doses attenuated in apoptosis lindane. p38

groups; Fas,

blocked

␮

3 increased or

mg/kg h. respectively;

groups, had

% by of was 6 in

of 6 basement NF- group

all have

attenuated higher

dose 50

and increase

and all germ decreased NAC phosphorylation

LDH,

100 extract attenuated as M in the

cytosol, ␮ cells

tested

the NAC by

This

not degeneration in

␮ increased

dose

and 10,

level cytochrome

antinomycin

B; NAC;

caspase-8 to

signiﬁcantly

10 group.

time-dependent JNK1

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cells and signiﬁcantly

M. levels

JNKs

and single

enhanced NAC early

decreased

did early Fas. SB202190,

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near a

germ time-dependent apoptosis

increased cells

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as with

and

lower as a activity and

with NAC; b.wt ␬ neutralized

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and group

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kappa group;

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selective children. progressively.

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treatments, by after

FasL group,

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Fas

signiﬁcantly NF-

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region

M M M -BHC of 30 ␮ p -3

production in

mg/kg ,

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-8

␮ over

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observed. ␤ DDD

␮ ␮ death activity induced FasL

mg/kg

elevated

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elevated factor NAC.

50 LDH

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M D; NAC;

50 50

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level

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SOD

inhibited or ␮ increased

-DDD

and in M TUNEL-labeled

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unexposed

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phosphorylation

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respectively, with

50 peripheral

reduced

and ␮ 30

p65

of 30

DDE >30

and

testis cells group Nuclear the 50 >30 blood GSH-Px B group;

␬ in FasL FasL

in and 50 treated

at 30, group, number

␬

the

b.wt M in at from

declined loss M and B,

co-localization procaspase induced procapase-9 apoptotic high

and by M 30 ␮ in

DDT,

and NF-

␬ and

␮

cytochrome

Fas, Fas,

NF- attenuated

increased germ antinomycin increase dosage observed.

The ␮ in

cells

observed

Procaspase-9 following

with in

procaspase-3

30 50

than expression

not

and and NF- level higher

increased,

or dependent

50 -DDE in

mg/kg

SOD

c c neutralized or and

in in

time-dependent

p treatments FasL. FasL found mRNA exhibited

with with signiﬁcantly , groups; observed peripheral

induced

changed

level

p and weights

induced

30 in NAC;

of of

100 M induced

labeled

dose -8

and

observed; tubules tubules. levels 30

␮

manner increase

activities reduced ␮ in

FasL cells

by p65 protein

children increased in 8

groups death

death ␮

and effectively. PBMC, body 50

blood -DDT,

decrease

increased SB202190 procaspase-8;

␬ procaspase-3, procaspase-3,

colocalized M

increased increased cells p 50 or bcl-w

in TUNEL regions

, or

group;

enhanced enhanced

mRNA and cell rate

cell

␮ of or characters effects

in in p

in cytochrome cytochrome

Bcl-w

doses;

of NF-

and reduction or

of decreased of

all 30

treatment g/mL; elevated

phosphorylation

exposed increased

kinase;

␮ in in NAC

M enhanced -8 attenuated malondialdehyde;

seminiferous 30, seminiferous

-DDT, change

␮

the

p , o Viability Cytosolic Cytosolic Apoptotic No Apoptotic Observed Apoptotic elevation signiﬁcantly activation production TUNEL-positive caspase-3 peripheral caspase-3 50 viability different the after10, time-dependent observed and apoptotic immunoreactivity and time-dependent MAPK Procaspase-3 levels reduction increase number production at rate in elevation changes extracts activation with immunoreactivity manner. translocation the level level the changes; NAC; MDA,

. , B m ␬ on of NH2-terminal

on on

vivo

of cell

Fas; weight.

NF- B or B; rat in and

FasL;

NAC

assay. ␬ ␬ mRNA

of c-Jun

dismutase;

and on level, of

germ organs testis; bcl-w;

SOD

levels

and MAPKs

of body

on induction extracts and

ROS

NF- NF-

cellular

level, adult

production,

cytoplasmic testis. FasL, on Fas;

JNKs,

the rats

and

TUNEL levels

Fas

apoptosis.

p65,

(in MDA

and vitro

effect

b.wt, FasL,

levels. -DDE SB202190 B

caspase-3 ROS

levels rat

assay. MDA

proteins,

by on

procaspase-3,

levels,

in ␬

the

and bak activity,

, weights, nuclear

testis in caspase-3 leakage,

FasL

in the

translocation,

superoxide

and activation. and-9, and-9 NF- p65 rats

FasL;

c generation,

of (in

both

levels, bax, and

FasL;

B activity,

protein

TUNEL apoptosis, body LDH of -cysteine;

B species; ␬

p65 ␬ mRNA, c,

l protein cleavage;

of ␬

metabolites -DDE-induced

Fas,

GSH-Px B ROS

SOD,

by apoptosis, on on on

␬ apoptosis. NF- -8 -3

SOD , p65 effect NF- caspase-3

activities

its

-DDE-induced activity NF-

testis B on

protein

p of humans

, -HCH). rats

-8 NF- ␬ or caspase-3 caspase-3

level,

antinomycin

oxygen

lindane: methoxychlor: and and and on p

, ,

␥

-DDE -DDE -DDE

rate,

in c c

the localization of SOD of

cytochrome

N-acetyl- FasL, p p p of of

NF-

, , , caspase-8

cytoplasmic

on and

-BHC localization

in p p p DDT

and apoptosis, MAPK cytochrome reduction, ␤ NAC NAC,

MDA

Fas, of of of of the

testis Peroxidase; of investigated of of NAC, of

cell

reactive

leakage in

effects effects family,

testis,

-DDE-induced procaspase-8,

localization immunolocalization -HCH

the apoptosis

p ␤ ,

ROS, Effects Effect Effects Effects The Effect The levels Effect of Effects production, extract); cytochrome activation, caspase-3 SP600125 phosphorylation; weights, -8; p caspase-3, immunolocalization procaspase-8 on JNK/p38 testis), effect Bax the in procaspase-9 procaspase-3 activity, viability LDH p65 cytochrome localization on germ

kinases;

Glutathione to

labeling; rats

or 10

days days vitro

protein to 18 18 18

with

M 60, for used

in 50 of of of ␮ of

for

with end

methoxychlor,

vivo 20, and

treated single single

b.wt) 50 for

-BHC

GSH-Px, 30,

in a a were -DDE 0,

(80–90 (80–90

␤

-DDE

nick 30

-DDE p DDE, Prepubertal testes testes testes testes , rats p

10,

M and

for maximum p p

volunteers

incubated treated , p

cells.

␮ rats rats

with with p

with mg/kg

M Sprague-Dawley rats M

of dUTP -HCH)

the

from from from from 70 of (5 ␮ male (DDT,

␮

␤

experiments from Sprague-Dawley Sprague-Dawley ( exposure) Thus10, were were

various 70

50, 70

old male

male exposed Wistar Wistar children

mitogen-activated

to proteinases;

cells cells cells cells b.wt

-DDE

b.wt)

OCPs

DDT

cells cells male male p

30, -BHC dose.

old viability

PBMC old with lindane methoxychlor

(s), treatment

50 ␤ p

days

DDT 50

sample/material

male male

10, of of of old old transferase The The

M M administered administered found administered

the mg/kg

Sertoli Sertoli Sertoli Sertoli

mg/kg 30,

days

h ␮ ␮

subsequent

effects MAPKs Human Rat rats. 10, study. (50 Study Rat 20-Day Rat Rat Adult Adult 24 10,30, 70 (without old) old) test tolerant with 18–20 days unexposed treated rats. was rats 100 in 20 dose dose 50 days days

aspartate-speciﬁc

and apoptotic

potential;

type

deoxynucleotidyl

the

vitro vivo vitro vitro vitro vitro vivo vivo vivo

on in Study In In In In In In In In

cysteinyl

membrane terminal

studies

al.

et Caspases,

TUNEL,

: al. recent (2008) (2011)

(2009) (2010) (2011) et

al. al.

(2004)

al. al. al.

mitochondrial et et

al. 3

et et et

inhibitor; (2009) et (2010) m

Author Shi Shi Shi Song Pérez-Maldonado Song Saradha Vaithinathan Summary Table Abbreviations JNK

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-hydroxysteroid

dehydrogenase. 17␤-Estradiol metabolites affect cell proliferation and BCA development either directly via receptor-independent mechanisms involving structural/functional

alterations in DNA, or indirectly via receptor-dependent mechanisms involving phenotypic growth regulation. These mechanisms upregulate aberrant proliferation and

development of BCA.

Source: 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 - of rats with 50 or 100 mg/kg body weight p,p -DDT (repeated

−5

DDE (1 × 10 M) was a weak transcriptional inducer (Silva et al., doses for ten successive days) induced decrease in the number and

2010). motility of epididymal spermatozoa in a dose dependent manner

In adult mice p,p -DDT induced a dose-dependent effect on and DDT induction of testosterone metabolism causing reduced

estrogen receptor activity in liver, brain, thymus and prostate of a testosterone levels (Ben Rhouma et al., 2001). The same treatment

transgenic mouse strain (ERE-tkLUC). The same effect was caused led to increased thyroid hormones and to a decrease in serum

by o,p -DDT, except in the liver. This study showed that DDT isomers T4 levels and hypothyroidism (Tebourbi et al., 2010). Given the

modulate ER activity also in nonreproductive organs (Di Lorenzo very high doses used in the experiments, the results of this study

et al., 2002). At a dose known to interfere with animal fertility are hardly comparable with effects forecasted at human environ-

(50 ␮g/kg), o,p -DDT signiﬁcantly affected ER response. Treatment mental exposure levels. In fact, a cross-sectional study conducted

Fig. 2. Molecular structures of phenobarbital (a prototypic CYP2B inducer) and some OCPs reviewed.

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

and detoxiﬁcation in the human placenta, any malfunction of this

enzyme will disrupt the placental detoxiﬁcation machinery. This

may 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 a

Greek rural population, revealed a signiﬁcant association between

CYP1A1*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 ailments

such as chronic obstructive pneumonopathy (p = 0.045), peripheral

Fig. 3. The 3D molecular structure of DDT showing diphenyl rings which are not

circulatory problems (trend p = 0.042), arteritis (p = 0.022), allergies

within the same plane.

(trend p = 0.046), hemorrhoids (trend p = 0.026) and allergic der-

matitis (p = 0.0016). Thus it was suggested that CYP1A1 may play

among 781 young men did not ﬁnd association between DDE a signiﬁcant role in the incidence of several diseases among the

levels (median: 2.7 ␮g/g serum lipid; range 0.1–56.1 ␮g/g serum population with long exposure to organochlorinated pesticides.

lipid) and anogenital distance (Longnecker et al., 2007). Also a

nested case–control study by Torres-Sánchez et al. (2008) among 37 2.1.3. Endosulfan

young men showed that higher DDE levels (10th–90th percentile: Endosulfan is synthesized via Diels–Alder addition of

0.21–5.89 ␮g/g serum lipid) signiﬁcantly reduced the anogenital hexachloro-cyclopentadiene and cis-butene-1,4-diol in xylene.

distance. A study by Bornman et al. (2010) on urogenital malforma- The annual production is estimated to range between 18,000 and

tions in male newborns suggests increased rates of malformations 20,000 tonnes worldwide (UNEP, 2010b). In USA, 400 tonnes are

among those whose mothers lived in DDT-treated homes. Although estimated to be used annually for domestic purpose. The main

these studies do not provide sufﬁcient information for determi- uses include control of pests in vegetables, fruits, cereal grains and

nation of a quantitative risk to humans, biological plausibility is cotton, ornamental shrubs, trees, vines and ornamental plants. It

strengthened by animal ﬁndings (WHO, 2011). was also used to control ectoparasite on beef and lactating cattle.

Cohn et al. (2007) conducted an epidemiological study and In Africa it is commonly used in cotton production at a dose rate

showed an association, with a dose response relationship, between between 1000 and 2000 g/ha while in India it is used to control

DDT exposure and development of breast cancer when exposure is pests on cashew plantations at the dose rate of 1200 g/ha. The

prepubertal. This was the case for the younger women (<14 years estimated annual production in India is 9500 tonnes, of which

of age) in second and third tertile of exposure who had DDT expo- 4500–5000 tonnes are consumed domestically.

sure in their childhood [2nd tertile: 8.09–13.90 ␮g/L (∼1.1–1.8 ␮g/g Most studies investigating endosulfan estrogenicity in vitro

5 6

lipid), OR = 2.8, 95% CI, 1.1–6.8; 3rd tertile: >13.90 ␮g/L, OR = 5.4, show that its potency is 10 –10 times lower than that of estradiol

95% CI, 1.7–17.1]. McGlynn et al. (2006) found signiﬁcant increas- (Table 2; Arcaro et al., 1998; Soto et al., 1994, 1995; Wade et al.,

ing risk of liver cancer among individuals with values in the 1997). Briz et al. (2011) showed that endosulfan directly interacts

highest versus the lowest quintile of serum DDT concentration with ERs in primary cultures of cortical neurons (CN) and cerebel-

(geometric mean = 0.49 ␮g/g lipids; OR = 3.8; 95% CI, 1.7–8.6; p for lar granule cells (CGC). It inhibited the binding of [ H]-estradiol to

trend = 0.0024) when analysis was adjusted for age, sex, hepatitis the ER in both CN and CGC with a fairly high IC50 of 21.7 ± 4.2 and

B surface antigen status, places of residence and DDE level. The 35.0 ± 8.3 ␮M, respectively. These IC50 values are very close to that

lowest and the highest quintile of serum DDT concentrations were of ␣-endosulfan with recombinant human steroid receptors partic-

<0.265 and >0.787 ␮g/g lipids, respectively. However there was no ularly hPR (20 ␮M) (Scippo et al., 2004). Exposure of endosulfan at

evidence of signiﬁcant association between liver cancer and serum 10 ␮M for 5 h increased Akt phosphorylation and activated ERK1/2

DDE concentration (p = 0.51). McGlynn et al. (2008) showed an through a mechanism involving GABAA and glutamate receptors

association between testicular germ cell carcinomas and DDE expo- in CN. The observed alterations on ER-mediated signaling and ER

sure (upper quartile: >0.39 ␮g/g lipid, RR = 1.71; 95% CI, 1.23–2.38; levels in neurons were suggested to contribute to the neurotoxicity

Table 1). of endosulfan.

Several epidemiological data showed an association between In vitro assay showed that endosulfan, dieldrin and lindane

DDT exposure and miscarriage or preterm birth (Dewailly et al., act as antagonists of androgen receptors (Andersen et al., 2002;

1993; Gladen et al., 2003; Karmaus and Zhu, 2004). To understand Li et al., 2008; Nativelle-Serpentini et al., 2003). Endosulfan and

the involvement of the AhR/CYP1A1 pathway in the mechanism dieldrin were shown to inhibit the aromatase enzyme (CYP19); the

of action of DDT and DDE, Wójtowicz et al. (2011) used human rate-limiting enzyme of estrogen synthesis from the non-aromatic

placenta explants cultures originated from normal pregnancy and precursor androstenedione. Like other organochlorines, endosul-

cotreated them with 3.2 ng/mL TCDD and 1, 10 or 100 ng/mL fan and dieldrin alter the estradiol metabolism by inducing CYP1

(approx. 0.18, 1.81, 18.10 ␮g/g lipid) of p,p -DDT o,p -DDT, p,p -DDE enzymes (Badawi et al., 2000; Bradlow et al., 1995). Endosulfan

or o,p -DDE for 24, 48 or 72 h (these concentrations are known and dieldrin were shown to inhibit androgen response at 20 ␮M

to occur in serum of pregnant women). Immunoblot analyses and thus acted as very weak antiandrogens. At 50 ␮M endosulfan

showed that the isomers of DDT and DDE inhibited the expres- reduced the aromatase activity in human placenta microsomes to

sion of CYP1A1 most effectively at 48 and/or 72 h after treatment. 87% of the control level (i.e. 1 ␮M of 4-Androsten-4-ol-3,17-dione)

In a separate experiment to study the expression of AhR, the pla- and hence exhibited weak aromatase-inhibiting effect (Andersen

centa explants were cultured in the presence of these isomers at et al., 2002).

100 ng/mL for 1, 3, 6, 14, 24, 48 and 72 h. The level of AhR pro- In vitro assay shows that dieldrin (5 ␮M) and endosulfan (1 ␮M)

tein decreased gradually with time starting after 3 h of treatment signiﬁcantly increase cell proliferation and ER transactivation gene

in all treatment experiments, with exception of that with o,p -DDE, response in MCF-7 cells (Andersen et al., 2002). The maximum

where the decrease was delayed at 24 h after the start of the exper- response occurred at 25 ␮M for both dieldrin and endosulfan. How-

iment. The author argued that as CYP1A1 is involved in metabolism ever, only endosulfan was shown to potentiate the 17␤-estradiol

E.J. Mrema et al. / Toxicology 307 (2013) 74–88 81

induced proliferation when tested together with a concentration (Song et al., 2012). However, at the moment no ﬁrm conclusion can

of 17␤-estradiol causing a sub maximum response. Other authors be drawn on these proposed mechanisms.

have also shown the ability of endosulfan to promote MCF-7

cell proliferation in vitro (Grunfeld and Bonefeld-Jorgensen, 2004; 2.1.4. Hexachlorobenzene

Ibarluzea et al., 2004; Soto et al., 1994). However, there are no epi- Hexachlorobenzene, a fully chlorinated aromatic hydrocarbon,

demiological studies linking exposure to endosulfan and increase was widely used as fungicide. Its half life in human is estimated

of cancer risk in humans. In vitro studies examining the effects of to be in the range of 6 years (To-Figueras et al., 1997). The most

endosulfan on androgen-responsive systems showed that endosul- sensitive target organs are the liver, the ovary and the central

fan exhibited cytotoxic effects on testicular cells at a concentration nervous system (ATSDR, 2002). HCB is well known to induce por-

of 20 ␮M or greater (Sinha et al., 1999, 2001a). These doses are phyria through a free-radical generation mechanism (Mazzetti

environmentally irrelevant. In vivo assays have not demonstrated et al., 2004), due to its rather unique chemical characteristics

estrogenic effects (Gellert, 1978; Raizada et al., 1991; Shelby et al., of lipophilicity and possibly to conversion into the redox-active

1996; Wade et al., 1997). Endosulfan caused alterations in testicular tetrachloro-1,4-benzoquinone. HCB is a weak agonist of the AhR

functions at very high doses. At doses of 2.5, 5 and 10 mg/kg admin- protein (Hahn et al., 1989). Unlike estrogen, HCB does not have

istered orally to adult Druckrey rats for 5 days per week for 70 days, a signiﬁcant afﬁnity for the ER thus suggesting other intracellular

endosulfan was shown to increase testicular enzyme activities at all pathways than the one taken by endogenous estrogen in mediating

tested doses. Sperm count decreased in a dose-dependent manner these actions.

(Sinha et al., 1995). Similar study conducted for 90 days using wean- HCB can elicit tumorigenic activity through AhR-dependent and

ing Druckrey rats yielded similar results where spermatid count independent pathways. In vivo administration of HCB has shown

and sperm production rate decreased (Sinha et al., 1997). Repeated to induce alterations in insulin-like growth factors (IGFs) signaling

dose of 3 mg/kg/day of endosulfan administered to immature rats pathway in mammary gland and mammary tumors in rat only when

for 3 days caused no change in uterine weights, uterine peroxidase co-administered with N-nitroso-N-methylurea (Randi et al., 2006).

activity, or numbers of uterine estrogen and progesterone recep- Since the latter compound is a strong mutagen, HCB plays the role of

tors. Only at a high dose (10 ␮M) endosulfan inhibited estradiol a tumor cocarcinogen in rat mammary gland, as an inducer of cell

binding (Wade et al., 1997) as was in the case for in vitro studies proliferation and of c-Src kinase activity in MCF-7 breast cancer

discussed above. cells.

Exposure of adult rats to 2.5, 5 or 10 mg/kg endosulfan for 10 Garcia et al. (2010) used ER␣ (+) MCF-7 and ER␣ (−) MDA-

weeks (5 day per week) or 3, 6 or 1000 mg/kg b.wt/day endosul- MB-231 cell lines to investigate ability of HCB to promote cell

fan have shown to cause reduction in intratesticular spermatid proliferation and alter Insulin/IGF-I signaling pathway. At 0.005 and

counts, sperm abnormalities, and changes in the marker enzymes of 0.05 ␮M, HCB signiﬁcantly increased the proliferation of only MCF-

testicular activities, such as lactate dehydrogenase, sorbitol dehy- 7 cell lines, with no effect at 0.5 and 5 ␮M. In estrogen-depleted

drogenase, g-glutamyl transpeptidase, and glucose-6-phosphate medium, HCB stimulated proliferation of MCF-7 cells only at the

dehydrogenase (Khan and Sinha, 1996; Sinha et al., 1995). More- lower concentrations showing that the effect of HCB on cell prolifer-

over exposure of pregnant rats to endosulfan at 1 mg/kg/day from ation was not dose responsive. Thus HCB stimulates proliferation in

day 12 through parturition has shown to decrease spermatogene- estrogen-sensitive cells in an ER␣-dependent manner. At 0.005 and

sis in offspring (Sinha et al., 2001a). It should be noted that these 0.05 ␮M, HCB increased the level of IGF-IR and IR in ER␣ (+) MCF-7

doses are orders of magnitude higher than those to which humans cell line. However at 0.5 and 5 ␮M, HCB did not alter the level of

are usually exposed in the living environment and at the workplace. these receptors but increased CYP1A1 gene expression and induced

Limited human data exist on the effect of endosulfan. A cohort apoptosis in MCF-7 cell lines, suggesting HCB effect on apoptosis is

study conducted by Saiyed et al. (2003) among male school children AhR-dependent. At 0.005 and 0.05 ␮M of HCB did not induce the

(10–19 years old) showed that endosulfan exposure was associ- expression of CYP1A1 mRNA. On the other hand 0.005 ␮M of HCB

ated with delayed male sexual maturity and interfered with the led to c-Src activation signifying this effect is not AhR-dependent.

male sex-hormone synthesis. Sexual maturity rating (SMR) scoring At 0.005, 0.05, 0.5 and 5 ␮M, HCB increased IRS-1 phosphoryla-

for development of pubic hair, testes, penis, and serum testos- tion with no effect in IRS-1 protein levels. IGF-I receptor is known

terone level was positively related to age and negatively related to regulate proliferation, survival and differentiation in mammary

to aerial exposure to endosulfan (AEE; p < 0.01). Serum LH levels gland. Insulin-like growth factor-insulin receptor (IGF-IR) path-

were signiﬁcantly positively related to AEE after controlling for way is involved in development of breast cancer and IR content

age (p < 0.01). Serum endosulfan levels were signiﬁcantly higher is known to increase in human breast cancer whereas IRS-1 is the

(p < 0.001) in the exposed population (mean 7.47 ± 1.19 ppb) than main intracellular substrate activated by IGF-I and insulin in human

controls (1.37 ± 0.40 ppb). Moreover, a big proportion of exposed breast cancer cells. c-Src can promote growth of tumor cells, par-

group (78%) had detectable serum endosulfan as compared to 29% ticipating in or augmenting mitogenic signaling pathways that are

of the controls. Although these ﬁndings match those expected on initiated by extracellular growth factors or intracellular oncogenes

the basis of experimental evidences, nevertheless, it was not clear (Biscardi et al., 2000). As per authors’ point of view, these results

if endosulfan was the only chemical that could be linked to the give a clue to the molecular mechanism of HCB in breast cancer

observed associations. development.

Several cellular and molecular mechanisms of endosulfan toxic- To investigate the effects of HCB, Pontillo et al. (2011) showed

ity have been proposed. These include mitochondrial dysfunction, that at 0.05 ␮M HCB produced an early increase of c-Src, human

induction of oxidative stress, modulation of activities of stress- epidermal growth factor receptor (HER1) activation, signal trans-

responsive signal transduction pathways, activating protein-1 ducers and activators of transcription (STAT) 5b, ERK1/2, but not

(AP-1) and antioxidant response element (ARE)-mediated tran- Akt, in ER␣ (−) MDA-MB-231 cell lines in a dose-dependent manner

scription. Recently a model has been proposed in which endosulfan (0.005, 0.05, 0.5, and 5 ␮M).

is shown to increase extracellular signal regulated kinases (ERK) Young rats (65 days of age) were administered 100 mg/kg b.wt of

1/2 and p38 activities and, in turn, these proteins activate HCB three times a week (total dose of 1800 mg/kg b.wt over 45 day

mitogen-activated protein kinases (MAPKs) and increase c-Jun period) (Pena˜ et al., 2012). When the test animals reached oestrous

phosphorylation. Phosphorylated c-Jun, in turn, increases AP-1 phase they were sacriﬁced. In mammary glands, HCB increased c-

activity, which results in activation of ARE-mediated transcription Src and HER1 activation, c-Src/HER1 association; and STAT5b and

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

ERK1/2 phosphorylation. Moreover HCB enhanced ER␣ phosphory- This is one reason why this isomer shows a higher bioaccumula-

␣

lation and ER /c-Src physical interaction. In tumors, HCB induced tion (half-life in human body of about 7.2 years; Jung et al., 1997)

c-Src and HER1 activation, c-Src/HER1 association, as well as Akt and a higher bioaccumulation factor of 5274 (WHO, 1992). Since

and STAT5b phosphorylation. Additionally, HCB increased ER␣ pro- this isomer is eliminated very slowly from human body, it makes a

tein content and decreased p-ER␣ levels and ER␣/c-Src association. signiﬁcant contribution to the total HCH body burden. ␤-HCH was

␤

HCB increased serum 17- estradiol and prolactin contents and widely used on cotton plants during 1960s and 1970s.

decreased progesterone, FSH and LH levels in rats without tumors. ␤-HCH (1 ␮M) has mitogenic activity in MCF-7 cell lines in vitro

This might be a consequence of a negative feedback on the pituitary (Coosen and van Velsen, 1989) but not in ER␣-MDA-MB231 cell

hormones secretion, by E2. The opposite effect was observed in rats lines which express a non-functional ER (Steinmetz et al., 1996).

with tumors. These results indicate that HCB induces an estrogenic This result indicates that a functional ER is necessary to elicit the

effect in mammary gland, increasing c-Src/HER1 and ER␣ signaling mitogenic activity.

−8

pathways. HCB stimulates c-Src/HER1 pathway, but decreases ER␣ In vivo assay with mouse xenograft showed that 10 M ␤-HCH

−5

activity in tumors, appearing to shift them towards a higher malig- signiﬁcantly increased MCF-7 cell number and at 10 M maximal

nancy phenotype. The enhanced c-Src and HER1 activation could be responses were achieved (Steinmetz et al., 1996). Moreover ␤-HCH

associated to the increase in the hyperplasia found in the mammary increased pS2 gene mRNA level. Like HCB, ␤-HCH does not com-

gland and the higher malignancy observed in tumors (Pena˜ et al., pete with estradiol for binding to the ER (Coosen and van Velsen,

2012). The synergism between c-Src and HER1 serves to upregu- 1989; Hatakeyama et al., 2002; Steinmetz et al., 1996). Its chemical

late the mitogenic activity of HER1 downstream effectors involved structure characteristics may not allow binding the classical ligand

in tumorigenesis (Biscardi et al., 2000). However, the very high binding domain and thus fail to activate ER in the nucleus, but it

doses tested signiﬁcantly affect the relevance of this information shows afﬁnity to the alternative pocket and consequently triggers

for human environmental and occupational risk assessment. the Src/Ras/ERK pathway (Silva et al., 2010). Although unable to

bind the ER, ␤-HCH is capable of activating ER target genes and the

proliferation of estrogen-responsive cell lines (Silva et al., 2007;

2.1.5. ˇ-Hexachlorocyclohexane

Steinmetz et al., 1996) by other chemical and biochemical mecha-

Complex and variable mixtures of hexachlorocyclohexane

nisms.

(HCH) products are produced by photochemical chlorination of

−5

Treatment of MCF-7 cells with 1 × 10 M ␤-HCH resulted in

benzene. HCH exists in 9 stereoisomeric forms, which include (+)-

strong activation of the Src kinase, ERK1 and ERK2. Thus ␤-HCH

−

and ( )-alpha- (␣), beta- (␤), gamma- (␥), delta- (␦), epsilon- (␧),

has been shown to induce rapid and sustained Src/ERK signaling

zeta- (␨), eta- (␩) and theta- (␪) isomers. Technical-grade HCH is

in MCF-7 cell lines lasting for at least 30 min, while this path-

␣ comprised of 60–70% -HCH, 5–12% ␤-HCH, 10–15% ␥-HCH, 6–10%

way was not signiﬁcantly affected by p,p -DDE (Silva et al., 2010).

␦-HCH, and 3–4% ␧-HCH. Puriﬁed HCH isomers, as well as technical-

Hatakeyama et al. (2002) observed similar activation of ERK1/ERK2

grade HCH are used either as fungicides or in the synthesis of other

chemicals. by ␤-HCH.

␥ In epidemiological study Mussalo-Rauhamaa et al. (1990) found

Lindane ( -isomer) was used as seed treatment for barley, corn,

that ␤-HCH frequently occurs in breast fat of breast cancer patients

oats, rye, sorghum and wheat to protect seeds for sowing from

with mean concentration of 0.13 ± 0.06 mg/kg fat. Controlling for

molds and ants during storage before sowing and in the ground

age and parity, ␤-HCH remained a signiﬁcant risk factor (OR, 10.51;

before sprouting. The estimation of global production between

95% CI, 2.00–55.26). The cutoff point for the residue level in breast

1950 and 2000 was 600,000 tonnes and the majority was used in

adipose tissue was >0.1 mg/kg fat.

agriculture. In the past, ␥-HCH was also used in veterinary products

to control mites, lice and other pests, but recent data suggest that

2.2. Oxidative stress, apoptosis and OCPs’ toxicity

no products are currently registered, at least in the United States

for this use. It was also used as an insecticide to treat fruit, vegeta-

Apoptosis is the mechanism whereby damaged or unnecessary

bles, forest crops, animals and animal premises. It is also currently

cells are naturally eliminated without causing damage to the sur-

used in treatment of scabies and lice in humans in form of lotion,

rounding tissue (Ameisen, 1996). This effect is accomplished by a

cream or shampoo, especially in tropical countries.

partial, yet exhaustive digestion of cell structures (DNA, structural

Another important HCH isomer is the beta-isomer (␤-HCH). This

proteins, enzymes and lipids) prior to rupture of the cell membrane

isomer has all chlorine atoms in an equatorial position, and thus (a)

and release of products in the extracellular medium for clear-

while it lacks aromatic character, its molecular shape is more sim-

ance. When apoptosis is excessive or inappropriate, incomplete or

ilar to that of strictly planar hexachlorobenzene than those of all

insufﬁcient, defections trigger pathological-related condition such

other isomers and (b) it lacks any axial chlorine atom(s) which can

as immunodeﬁciency, autoimmunity diseases, cancer (Alison and

be the site for 1,2-elimination by chemical mechanisms (Fig. 4).

Sarraf, 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 towards

a more oxidative (more negative) value of the electrochemi-

cal potential with respect to that of the relevant cell phase, is

one main trigger in the induction of apoptosis (Kannan et al.,

2000; Pérez-Maldonado et al., 2005). Many pesticides share as

a possible mechanism of toxicity the ability to trigger apopto-

sis through alterations in redox homeostasis generated by a

decrease of antioxidant defenses and by accumulation of reac-

tive oxygen species (ROS). An over-production of ROS leads

to processes such as oxidative modiﬁcations of redox signaling

protein, oxidative DNA damage, endoplasmic reticulum stress

and alterations in mitochondrial function which in turn trig-

Fig. 4. 3D molecular shapes of: (A) beta-hexachlorocyclohexane (␤-HCH or

ger the activation of speciﬁc signaling cascades. Activation of

1,2,3,4,5,6-HCH [1␣,2␤,3␣,4␤,5␣,6␤]); (B) hexachlorobenzene (HCB) (Plots by ACD-

Labs/3D, Advance Chemistry Development, 2007). stress-activated protein kinases (SAPKs) such as c-Jun N-terminal

E.J. Mrema et al. / Toxicology 307 (2013) 74–88 83

kinases (JNK) and of transcription-dependent p53 signaling percentage of apoptotic cells and the blood levels of only DDE of

cascades act as important sensors of electrophilic (and of oxidized) exposed children recruited in 2003 (p = 0.010) and those recruited

substances among which xenobiotic metabolites and induce apop- in 2004 (p = 0.040). The association between DDT or DDE exposure

totic cell death. Pesticides also induce the activation of survival and DNA damage was signiﬁcant (p = 0.004 and p = 0.005 respec-

responses such as DNA repair mechanisms, mitogen-activated pro- tively), whereas the association between DDT or DDE and oxidative

tein kinase/phosphatidylinositol 3-kinase (MAPK/PI3K) signaling DNA damage and that of oxidative damage and apoptosis were not

cascades and up-regulation of antioxidant defenses in an attempt signiﬁcant.

to cope with and counteract the deleterious effects of higher lev- An isomer of DDD, o,p -DDD, marketed under the name Mitotane

els of intracellular reactive species which in turn trigger cell death is used as an antineoplastic medication in the treatment of adreno-

pathways. In most cases apoptotic and survival signaling cascades cortical carcinoma. Mitotane alters steroid peripheral metabolism,

are activated simultaneously in response to exposure to pesticides. directly suppresses the adrenal cortex through the controlled

Table 3 summarizes some studies on apoptotic effects of some destruction of adrenal tissue, which leads to a decrease in cortisol

OCPs. production (Maher et al., 1992; Wu et al., 2006).

DDT derivatives have been shown to induce neural cell death Exposure of Wistar rats to a single dose of lindane (␥-HCH;

by apoptosis through the activation of mitogen-activated protein 5 mg/kg b.wt) induced testicular apoptosis through the involve-

kinases (MAPKs) which play signiﬁcant roles in controlling cell ment of Fas-FasL and mitochondria-dependent pathways (Saradha

survival, proliferation, differentiation and cellular responses to et al., 2009). Shi et al. (2011) showed that concentrations >30 ␮M

␤

various harmful signals (Shinomiya and Shinomiya, 2003). ␤-HCH, of -HCH induced apoptotic cell death in rat Sertoli cells associated

•−

p,p -DDE and other OCPs can induce elevation of ROS such as O2 , with increased expression of FasL levels which could lead to the

• •

HO and NO (Samanta and Chainy, 1997; Song et al., 2008; Sujatha Fas activation. These two genes are known to induce apoptosis.

et al., 2001). Moreover ␤-HCH treatment induced an increase of nuclear factor

Shi et al. (2009) hypothesized the signaling pathways involved kappa B (NF-␬B) p65. The latter can directly stimulate the expres-

in p,p -DDE-induced apoptosis: in his model ROS generation plays sion of these genes. Oxidative stress has been reported to enhance

␬

critical role in the initiation Sertoli cells apoptosis through two NF- B activation (Nakamura and Omaye, 2008). ␤-HCH has been

mechanisms. The ﬁrst one is mitochondria-mediated pathway shown to induce activation of caspase-8, that plays the role in

involving elevation of ROS, decrease in mitochondrial trans- transduction of death signal (Said et al., 2004) and caspase-3, that

membrane potential along with the cytochrome c release from initiates cell apoptosis (Khan et al., 2000).

mitochondria into the cytosol and activation of the caspase-9 and Moreover, Shi et al. (2011) showed that ␤-HCH induces increase

-3. Other mechanism involves elevation of ROS, which resulted in apoptotic rate of Sertoli cells by possible mechanisms of

in activation of NF-B, expression of FasL and triggered FasL- ROS/JNK/FasL pathway. In vitro exposure to ␤-HCH in rat Sertoli

dependent pathway (i.e. FasL/caspase-8/-3 signaling module). In cells can enhance ROS and oxidative stress, and then induce activa-

their investigation, Song et al. (2008) found at levels above 30 ␮M, tion of JNKs and NF-␬B and expression of FasL. Signiﬁcant increase

p,p -DDE induced apoptotic cell death of cultured rat Sertoli cells of FasL protein expression was observed with 30–50 ␮M ␤-HCH

in a pro-oxidant and mitochondria dependent manner by acti- treatment. This increase could lead to activate the Fas system. Upon

vating the intrinsic programmed cell death pathway. Moreover engagement of FasL to Fas, an intrinsic program of apoptotic death

p,p -DDE elevated the apoptotic rate of Sertoli cells in vitro (at is stimulated in a target cell leading to the activation of caspase-8.

>30 ␮M) and germinal cells in vivo (at >20 mg/kg b.wt) by mech- Finally, apoptosis of Sertoli cells is mediated by caspase-3, thereby

anism suspected to involve FasL-dependent pathway (Shi et al., disturbing the spermatogenic process (Shi et al., 2011; Fig. 5). These

2009, 2010). results led the authors hypothesize that ROS generation may play

Exposure to p,p -DDE can enhance ROS production and oxida- a critical role in the initiation of ␤-HCH-induced apoptosis by acti-

tive stress, and then induce activation of NF-␬B and expression vation of the JNKs, translocation of NF-␬B, expression of FasL and

of Fas-FasL. As a result, an intrinsic program of apoptotic death further activation of caspase cascade.

is stimulated in a target cell resulting to the activation of cas- Yu et al. (2008) studied the individual effect of p,p -DDE and

␤

pase 8. Ultimately, apoptosis of Sertoli cells and germinal cells is -HCH as well as the effect of their mixture on JNK and MAPK

mediated by caspase 3, thereby disturbing the spermatogenesis pathway in rat Sertoli cells. The latter were exposed to these OCPs

(Shi et al., 2010). Song et al. (2011) reported the induction of Ser- at the ﬁnal concentration of 10, 30 and 50 ␮M/L for 24 h in each

toli cell apoptosis by p,p -DDE at above 30 ␮M through oxidative case. In both treatments, the expression of JNK and c-jun was ele-

stress-mediated p38 MAPK and mitochondria-related pathway. vated in a dose dependent manner after 24 h of exposure. Liang

Methoxychlor, which was intended to replace DDT, has been shown et al. (2008) showed that at these levels p,p -DDE, ␤-HCH and their

to induce testicular apoptosis in rats following oral exposure to sin- mixture could induce apoptosis in rat Sertoli cells which was associ-

gle doses of 50 mg/kg b.wt where apoptosis was induced through ated with activation of caspase-3 mediated by cleavage of caspase-8

involvement of Fas-FasL and mitochondria-dependent pathways and caspase-9. As stated for the case of p,p -DDE above, these lev-

(Vaithinathan et al., 2010). It is necessary to remark that the doses els are also unrealistically high if compared even with the highest

tested are unrealistically high if compared to those typical of occu- occupational exposures.

pational and environmental exposures. As for speciﬁc mechanisms, OCPs, which are themselves fairly

Pérez-Maldonado et al. (2004) showed that o,p -DDT, p,p -DDT, un-reactive from the chemical point of view, are able to cause

p,p -DDE and p,p -DDD can induce apoptosis of human periph- or trigger oxidative stress. However, little is known or hypothe-

eral blood mononuclear cells in vitro at 20 ␮g/mL. This level is sized at the molecular level. Only the porphyrinogenic pesticide

400-fold higher than the mean blood total DDT level (0.05 ␮g/mL) hexachlorobenzene is known for its ability to directly gener-

observed in children in their pilot study (Pérez-Maldonado et al., ate oxidative stress due to its unique property to act as an

2004). The signiﬁcant level of ROS, as detected by ﬂow cytometry, electron sink either as such or, more likely, through biotransforma-

was induced at 60 and 80 ␮g/mL (Pérez-Maldonado et al., 2005). tion products such as tetrachloro-1,4-benzoquinone (van Ommen

A marginal inﬂuence of DDT and DDE on percentage of apoptosis and van Bladeren, 1989). That HCB is able to trigger apoptosis

was observed (p = 0.06) conﬁrmed by a weak positive association through the mitochondrial pathway has been demonstrated in the

between apoptosis and DDT exposure. In the follow-up study Pérez- liver (Giribaldi et al., 2011) and in the thyroid (Chiappini et al.,

Maldonado et al. (2006) found signiﬁcant association between the 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 apoptosis

of 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-3

signaling module). N-Acetyl-cystein (NAC) can inhibit triggers by protecting the sensor thiol residue of the signalling proteins.

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

2.3. Epigenetics and OCPs’ toxicity methylation pattern in the hypothalamus of young rats (3 weeks of

age) exposed at a dosage of 0.06 mg/kg/day. The 6 CpG islands were

Epigenetics is an emerging ﬁeld of study of heritable changes considerably hypomethylated as compared with controls. Thus the

that inﬂuence chromosomal stability and gene expression while authors speculated that under low level of oxidation stress, the DNA

not directly affecting changes in DNA sequence, such as by point methylation machinery malfunctions and this leads to incomplete

mutations or chromosome disruption and re-assembly (Rodenhiser methylation of speciﬁc gene regions. Following pyrosequencing

and Mann, 2006). One main mechanism of epigenetic modulation methylation analysis of the rat livers from the rats exposed to

of genetic expression is methylation at 5-position of cytosines in high dose of mixture of twelve OCPs (1.9 mg/kg/day) showed no

repeated CpG sequences, a modiﬁcation which suppresses expres- decrease in the methylation of CpG sites which is contrary to other

sion of downstream DNA sequences. Thus suppression of speciﬁc tested chemical mixtures (Desaulniers et al., 2009).

genes or re-activation of silenced genes determines the destiny of To our knowledge only two epidemiological studies have been

individual cells towards quiescence or proliferation, thus opening conducted to investigate exposure to POPs and DNA methyla-

the way to uncontrolled cell proliferation and cancer. Moreover, tion levels in a human population. The ﬁrst study was carried on

when epigenetic changes occur during certain stages of develop- Greenlandic Inuit and suggested a signiﬁcant inverse linear rela-

ment, they become permanent and can be inherited by offsprings tionship between DNA methylation and plasma concentrations of

and disturbance of epigenetic modulation is thus thought to be an DDT, DDE, ␤-HCH, oxychlordane, ␣-chlordane and mirex (Rusiecki

important mechanism in many diseases (Ozanne and Constancia, et al., 2008). This relationship was replicated by Kim et al. (2010)

2007). when they investigated the relationship between POPs exposure

Epigenetic effects have been hypothesized as a possible mecha- and DNA hypomethylation among healthy Koreans. These two

nism of POPs toxicity (Porta, 2006). For example, pregnant rats were studies demonstrated epigenetic changes related to environmen-

treated with methoxychlor at the fairly high dose of 50–150 mg/kg tal exposure although the two populations differ in magnitude of

during the sex-determining time window of pregnancy (days exposure, Greenland Inuit people being more exposed than Korean

E7–E10). Gonads were collected from embryos in gestating moth- population.

ers at E16 and from postnatal males showed a reduced germ

cell to testis area at developmental age and an almost doubled 3. Conclusions

fraction of apoptotic germ cells, although males were fertile and

produced normal offspring (Cupp et al., 2003). In a further experi- The general population is exposed to OCPs through several ways,

ment (Anway et al., 2005) exposition of pregnant rats to 100 mg/kg food being the major route of exposure. A few groups of the human

produced male offsprings with reduced sperm capacity and fer- population may show much higher levels of exposure than others

tility and the compromised fertility trait was passed through the due to the fact that they live in areas with a historical uncon-

adult male germ line for four generations. Altered patterns of DNA trolled use of these pesticides in agriculture (typical examples being

methylation was demonstrated to occur in the germ cells of gener- some areas of Central Asia) or that their diet mainly consists of

ations two and three. bio-accumulating organisms, such as large marine mammals (the

In vitro and animal studies have suggested that exposure to typical example is that of Arctic Inuits). In both cases the hazard

endocrine active compounds, such as POPs, may adversely affect is transmitted trans-generationally starting with breastfeeding of

DNA methylation patterns (Anway and Skinner, 2006; Anway et al., newborns and consequently the impairment of fertility can deter-

2006). It has been proved that chemicals can alter the epigenetic mine a demographic decline of some populations.

marks and that epigenetic marks can be found in patients with A number of adverse health effects such as endometriosis, infer-

the disease of concern or in diseased tissue (Baccarelli and Bollati, tility, immunotoxicity, neurotoxicity and spontaneous abortions,

2009). breast cancer, prostate cancer and neurodegenerative disorders

DDT has been shown to affect DNA methylation in experimental have been suggested as a result of this exposure. OCPs may exert

animals as shown by Shutoh et al. (2009). DDT was found to alter the these effects through various mechanisms. Animal and in vitro

E.J. Mrema et al. / Toxicology 307 (2013) 74–88 85

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