Endocrine-Disrupting Chemicals: Associated Disorders and Mechanisms of Action

The incidence and/or prevalence of health problems associated with endocrine-disruption have increased. Many chemicals have endocrine-disrupting properties, including bisphenol A, some organochlorines, polybrominated flame retardants, perfluorinated substances, alkylphenols, phthalates, pesticides, polycyclic aromatic hydrocarbons, alkylphenols, solvents, and some household products including some cleaning products, air fresheners, hair dyes, cosmetics, and sunscreens. Even some metals were shown to have endocrine-disrupting properties. Many observations suggesting that endocrine disruptors do contribute to cancer, diabetes, obesity, the metabolic syndrome, and infertility are listed in this paper. An overview is presented of mechanisms contributing to endocrine disruption. Endocrine disruptors can act through classical nuclear receptors, but also through estrogen-related receptors, membrane-bound estrogen-receptors, and interaction with targets in the cytosol resulting in activation of the Src/Ras/Erk pathway or modulation of nitric oxide. In addition, changes in metabolism of endogenous hormones, cross-talk between genomic and nongenomic pathways, cross talk with estrogen receptors after binding on other receptors, interference with feedback regulation and neuroendocrine cells, changes in DNA methylation or histone modifications, and genomic instability by interference with the spindle figure can play a role. Also it was found that effects of receptor activation can differ in function of the ligand.


Introduction
The objectives of the present paper include to give an overview of the wide spectrum of substances having endocrinedisrupting properties; to list a series of observations suggesting that endocrine disruptors contribute to human health problems, however, without attempting to bring an allinclusive review for each individual substance accompanied by a final conclusion; to give an overview of the many mechanisms on which endocrine disruption rests.

Incidence and Prevalence of Health Problems associated with Endocrine Disruption Have Increased
Epidemiological data show increases in incidence and prevalence of diseases associated with endocrine-disrupting chemicals, such as breast, prostate, and testis cancer, diabetes, obesity, and decreased fertility over the last 50 years. A short overview of supporting data is presented below. These increases might partly reflect an increase in the likelihood of diagnosis and certainly do not constitute proof of the impact of endocrine-disrupting chemicals. Time trends and ecological studies are not well suited to study a possible association between exposure to endocrine disrupting chemicals and risk of disease, as assessment of exposure is extremely difficult. The data on time trends are, however, consistent with such an impact, as are data on the incidence or prevalence of some diseases in migrants and on differences in function of geographical area. Huge geographical differences in cancer incidence are well documented in the report "Cancer incidence in five continents, volume IX" of the International Agency for Research on Cancer [1], often showing higher risks of hormone-related cancers in more industrialized Year of diagnosis Rate per 100,000 population countries. In France, cancer mortality among migrants is positively associated with the Human Development Index of the country-of-birth [2]. In migrants to California, time since immigration is associated with convergence of the odds for mortality from cancer and from some other diseases to that of the native population [3]. In the Netherlands (for cancer mortality) [4], in British Columbia (for cancer incidence) [5], in the San Francisco Bay area (for risk of breast cancer) [6], and in Hawai (for cancer incidence among Japanese) [7], convergence towards the rates of the native population was reported. A systematic review and meta-analysis revealed that prevalence of obesity is higher among migrant Asian Indians than among Indians living in India [8]. In India, migration from rural to urban area's is associated with an increase in the prevalence of diabetes and obesity [9].

Cancer.
In Great Britain, from 1978 to 2007, the overall age-standardized incidence rate of cancer has increased by 25%, an increase of 14% in men and a 32% increase in women [10] (see Figure 1).
According to Great Britain statistics, breast and prostate cancer incidence have a higher relative increase in incidence. Prostate cancer incidence has almost tripled from 33 per 100,000 in 1975 to 97 per 100,000 in 2007, while the incidence of breast cancer for women increased by 57%, from 77 per 100,000 in 1978 to 120 per 100,000 in 2007 (see Figures  2 and 3) [10]. These trends are being observed worldwide [11,12]. Also the incidence of testis cancer has increased worldwide [13]. The increase in cancer incidence can partly be explained by the introduction of screening, which will result in a transient increase in incidence and a stage shift to earlier stages, microinvasive, and in situ cancers in the steady state situation [14]. It is, however very unlikely that early screening explains the entire rise in cancer incidence, because of the progressive nature of the cancer process, which leads almost always to clinically evident disease. Also, in the United States Incidence rates for all childhood   1975  1977  1979  1981  1983  1985  1987  1989  1991  1993  1995  1997  1999  2001  2003  2005  2007 Year of diagnosis/death cancers combined increased 0.6% per year from 1975 to 2002 [15], while cancer is a disease which, since many decades, is diagnosed more easily in children compared to elderly because of the particular impact of the disease on children.

Diabetes.
The United States Centre for Disease Control and Prevention (CDC) reports percentages of diabetes incidence of 0.93% in 1958 against 6.29% in 2008, which is a more than 6-fold increase, as is shown in Figure 4 [16].

Decreased Fertility.
In men fertility has been decreasing in the last decades, at least in some countries. Observations by Comhaire and colleagues [75] on candidate sperm donors show that this has been the case in Flanders (see Figure 6).
Similar observations were made in Denmark [76], in France [77], and in the United Kingdom [78]. From a thorough analysis of 101 studies published 1934-1996 Swan et al. [79] concluded that sperm quality declined in the United States, Australia, and several countries in Europe. It seems likely that a decrease in semen quality plays a role in the recent decline in fertility rates [80]. Travison et al. [81] have recently  reported declining levels of testosterone in US men of 1% per year, the same rate of decline seen for sperm concentrations.
However, sperm quality is only one of several parameters determining human fertility. In terms of fertility of couples, in Sweden a transient increase in subfertility was seen in the early 1990s, but generally fecundability increased in Sweden between 1983 and 2002 [82]. As to ectopic pregnancies, in a Norwegian county population-based study age-adjusted ectopic pregnancy incidence rates increased from 4.3 to 16.0 per 10,000 women-years over the period 1970-1974 to 1990-1994 and declined to 8.4 per 10,000 women-years in 2000-2004 [83]. Joffe [84] found that couple fertility had increased in Britain, except for slight dips during 1976-80 and 1986-90.
Sallmén et al. [85] consider that, with the exception of rare settings in which the factors affecting reproductive choices have not changed, it is probably impossible to identify biologic changes in fertility of humans over recent decades.
The sex ratio (the ratio of the number of males born and the number of females) has decreased in many countries and most recently in the United States and Japanese populations [86].

Xenoandrogens.
Xenoandrogenic activity is less frequently reported than xenoestrogenic activity. There are, however, several authors describing xenoandrogenic activity (Table 1). For instance, Delor 103, a commercial mixture of PCB congeners, was reported to have androgenic activity in a bioluminescent yeast strain [51]. Using a Yeast androgen assay expressing the human androgen receptor, Kunz and Fent [55] tested 18 UV filters for androgenic activity and found that benzophenone-2 and homosalate produced full dose-response curves, while octyl-methoxycinnamate, octyl salicylate, octocrylene, and isopentyl-4-methoxycinnamate displayed a partly agonistic behaviour indicated by submaximal dose-response curves.

10
Journal of Environmental and Public Health sedimentation dust from subway stations (in yeast) [116]; extracts of motorcycle exhaust particulate (in MCF-7 human breast cancer cells and immature female rats) [117]; PAH mixtures in by-products of manufactured gas plant (MGP) residues [118]. Interestingly, some mixtures of polychlorinated biphenyls had antiestrogenic activity in MCF-7-BUS cells, whereas other mixtures, containing some of the same PCB congeners, had not [52]. The structural basis for estrogenic or rather antiestrogenic activities of polybrominated diphenyl ethers was studied by Yang et al. [119]. They showed, with docking studies on the human estrogen receptor alpha, that some of the PBDE compounds with antiestrogenic activity extended into the channel of the estrogen receptor (ER), which is usually occupied by the alkylamine side chain of the ER antagonists raloxifene and 4-hydroxytamoxifen, while most PBDE compounds without antiestrogenic activity adopted binding modes similar to that of ER agonist 17beta-estradiol (E2), located in the binding cavity and which did not protrude into the channel.
The omnipresence of antiestrogenic pollutants was demonstrated by Sanfilippo et al. [120] who, based on the yeast estrogen screen (YES) and chemical analysis (GC/MS), found antiestrogenic activity due to the presence of bis(2ethylhexyl) phthalate (DEHP) in ultrapure water for laboratory use.
Some phytoestrogen, such as flavones and isoflavones, have antiestrogenic effects through inhibition of the aromatase enzyme converting testosterone to estradiol [122]. Conceivably, some man-made substances or pollutants might also have antiestrogenic effects through inhibition of the aromatase [123].

Antiandrogens.
Many substances binding on androgen receptors have antiandrogenic effects rather than androgenic effects. Table 1 shows some data on substances for which antiandrogenic effects were observed. Other data are mentioned below.
The polychlorinated biphenyl PCB#138 showed an antiandrogenic effect on androgen receptor activity in transiently cotransfected Chinese Hamster Ovary cells [54]. Using the stable prostatic cell line PALM, which contains a human androgen receptor (hAR) expression vector and the reporter MMTV-luciferase, Lemaire et al. [64] found several organochlorine pesticides to have antiandrogenic activity (Table 1). Of 18 UV filters tested, 16 substances (see Table 1) displayed antiandrogenic activity and showed full doseresponse curves with complete inhibition of dihydrotestosterone activity in a Yeast androgen assay expressing the human androgen receptor [55]. The antiandrogenic activities of phenyl-and benzyl salicylate, benzophenone-1 and -2, and of 4-hydroxybenzophenone were higher than that of flutamide, a known hAR antagonist. For the DDT metabolite Dichlorodiphenyldichloroethylene (p,p -DDE) and for 23 pesticides intensively used in Europe in recent years, antiandrogenic activity (Table 1) was reported [66]. The antiandrogenic activity of these pesticides was observed in vitro on MDA-kb2 cells, human breast cancer cells stably transfected with a firefly luciferase reporter gene that is driven by an androgen-response element-containing promoter [66]. Of 200 pesticides tested for antagonism to a human androgen receptor (hAR) by highly sensitive transactivation assays using Chinese hamster ovary cells, 66 of 200 pesticides exhibited antiandrogenic activity [63]. In particular, the antiandrogenic activities of two diphenyl ether herbicides, chlornitrofen and chlomethoxyfen, were higher than those of vinclozolin and p,p -dichlorodiphenyl dichloroethylene, known AR antagonists [63]. The antiandrogenic activity of the organophosphorus pesticide fenthion was observed to be similar in magnitude to that of the antiandrogenic drug flutamide in an androgen-responsive element luciferase-reporter-responsive assay using NIH3T3 cells [71]. Whereas the insecticide permethrin might have estrogen-like effects on female rats, it showed antiandrogenlike effects on males [69]. Polycyclic musks were found to have antiandrogenic effects (on human U2OS cells with an androgen receptor linked reporter gene) [60]. Some methoxylated brominated diphenyl ethers (primarily of natural origin in the marine environment) were shown to be antiandrogenic in reporter gene assays [26].
Also complex mixtures of pollutants occurring in the environment were shown to have antiandrogenic activity. This was observed for some soils collected near highways (on MVLN cells) [114]; gaseous and particulate fractions of ambient air (on MVLN cells) [115]; diesel exhaust particles (in vivo in animals) [104].

Disruption of Corticoid Function and Other Metabolic
Effects. Endocrine disruption can also affect corticoid hormonal function, as observed for hexachlorobenzene in Wistar rats [138]. Hexachlorobenzene was also observed to induce oxidative stress, disruption of arachidonic acid metabolism, and porphyria [139,140].

Activation of the Aryl Hydrocarbon
Receptor. The aryl hydrocarbon receptor (AhR) regulates enzymes important to the metabolism of both endogenous substances (e.g., hormones) and exogenous substances, involved in both the detoxification and bioactivation of xenobiotics [143]. However, the AhR has been shown to regulate a much wider spectrum of cellular processes including cell proliferation, differentiation, apoptosis, and intercellular communication [144,145]. TCDD, a prototype ligand to the AhR, is one of the most potent carcinogens [146] and designated by IARC as a human carcinogen. Activation of the AhR receptor probably plays an important role in tumorpromotion [144,147]. Dioxins and other lipophylic dioxin-like substances constitute an important risk to human health through their presence in food items [148]. Also, activation of the AhR by polycyclic aromatic hydrocarbons might be a major toxic mode of action of air pollution particulate matter. Andrysik et al. [149] found that polycyclic aromatic hydrocarbons and their polar derivatives present in the organic fraction of air pollution were potent inducers of a range of AhR-mediated responses, including induction of the AhR-mediated transcription, such as cytochrome P450 1A1/1B1 expression, and the AhR-dependent cell proliferation. Importantly, these toxic events were observed at doses one order of magnitude lower than DNA damage. The AhR-mediated activity of the neutral fraction was linked to PAHs and their derivatives, as polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls, were only minor contributors to the overall AhRmediated activity.

Internal Exposure to Endocrine Disrupting Chemicals Is
Not Negligible. Andersen et al. [150] observed that occupational exposure to estrogen-like pesticides can result in detectable impacts on hormonal activity in the blood. Also, it has been shown that, in human placentas, xenoestrogenic activity amounted to 9.27% of the endogenous estrogenic activity in terms of the geometric mean, and to 9.84% in terms of the median value according to the MCF-7 breast cancer cell-based E-Screen [151]. Furthermore, Bonefeld-Jorgensen et al. [152] found that serum levels of the polychlorobiphenyl CB-153 and p,p -DDE alone were not sufficient to predict xenoestrogenic serum activity, and that other xenoestrogens must have been present. In 2006, a bisphenol A (BPA) Expert Panel Consensus Statement was released by NIEHS/EPA, stating that most humans are exposed to BPA (95% of human urines are positive in the assay of the USA Center for Disease Control (CDC)). Unconjugated BPA in human serum is in the 0.3 to 0.5 ng/mL range, and the concentration of BPA in fetal serum, umbilical cord blood, amniotic fluid, and placenta indicate that the developing fetus is chronically exposed to BPA in the 0.7-9.2 ng/mL range (unconjugated BPA) [36].  [154]; the organophosphorus pesticides malathion and parathion [155]; PCBs [156,157]; bisphenol A [158,159]; cadmium [160][161][162]; butyl benzyl phthalate [163]; organochlorine pesticides (p,p -DDD plus p,p -DDE plus o,p -DDE plus aldrin plus dieldrin) [164]; cosmetics benzyl salicylate, benzyl benzoate and butylphenylmethylpropional (Lilial) [165]; nitrite [166]. The antiandrogenic pesticide vinclozolin induced, through epigenetic changes, a number of disease states or tissue abnormalities, including cancer of the breast, in adult rats from the F1 generation and all subsequent generations examined (F1-F4) [167].

Observations Indicating That Endocrine
Animal experiments showed that in utero exposure to dioxins induced alterations in breast development and increased susceptibility for mammary cancer development [168]. Mice [169] and rats [170] exposed perinatally to BPA showed preneoplastic lesions (intraductal hyperplasias) in mammary tissue and such rats developed carcinoma in situ. Rats exposed perinatally [171] or through lactation [172] to BPA showed an increased susceptibility to neoplastic development. Perinatal exposure to environmentally relevant levels of BPA induced an increase in the number of terminal end buds in the Mouse mammary gland and by 6 months of age, the mammary glands showed a dramatic expansion of the ductal network with a significant increase in terminal ducts and alveolar structures relative to the control [173]. Terminal end buds are the structures in which mammary cancer originates in both rodents and humans [174]. Table 2 resumes the main data from a number of epidemiological studies. Below some other data and additional details of some of these studies are mentioned.
López-Carrillo et al. [89] reported that exposure to diethyl phthalate may be associated with increased risk of breast cancer, whereas exposure to the parent phthalates of monobenzyl phthalate and mono (3-carboxypropyl) phthalate might be negatively associated.
In a prospective study on young women, from Oakland, California, who provided blood samples in the period 1959-1967, high levels of serum p,p -DDT predicted a statistically Significant effect only in subgroup of leaner women.
[90]  No effect for other DDT-related compounds [98] significant 5-fold increased risk of breast cancer among women who were born after 1931 [98]. These women were under 14 years of age in 1945, when DDT came into widespread use, and mostly less than 20 years. DDT use peaked. These findings suggest that exposure to p,p -DDT early in life may increase breast cancer risk [98]. In a systematic review and meta-analysis of studies concerning cyclodiene insecticides and breast cancer, Khanjani et al. [175] found a significant association between heptachlor and breast cancer, but no significant association with other cyclodiene insecticides. Mills and Yang [176] performed a registrybased case-control study of breast cancer in farm labor union members in California. Controlling for covariates, adjusted ORs (and 95% CIs) for breast cancer in quartiles of pesticide use were 1.00, 1.30 (0.73-2.30), 1.23 (0.67-2.27), and 1.41 (0.66-3.02). Chlordane, malathion, and 2,4-D were associated with increased risk. Risk associated with chemical use was stronger in younger women, those with early-onset breast cancer, and those diagnosed earlier [176].

Experimental Studies.
As noted by Gail Prins [177], who contributed importantly to the study of prostate carcinogenesis, there is substantial evidence from animal models that specific endocrine-disrupting compounds may influence the development or progression of prostate cancer. In large part, these effects appear to be linked to interference with estrogen signaling, either through interacting with ERs or by influencing steroid metabolism and altering estrogen levels within the body. Studies in animal models show augmentation of prostate carcinogenesis with several environmental endocrine disruptors including diethylstilboestrol, PCBs, cadmium, ultra violet filters, Bisphenol A, and arsenic. Importantly, there appears to be heightened sensitivity of the prostate to these endocrine disruptors during the critical developmental windows including in utero and neonatal time points as well as during puberty. Thus infants and children may be considered a highly susceptible population for endocrine-disrupting exposures and increased risk of prostate cancers with aging [177].
Bisphenol A, a plastic component that can be considered a model agent for endocrine disruption, was shown to induce changes in differentiation patterns, cell proliferation, and size in the prostate, changes that are probably associated with an increase in cancer risk [178].
Exposure to diethylstilbestrol (DES), especially prenatal and early in life, has been associated with prostate abnormalities, including prostatic squamous neoplasia [177].
Cadmium is a known ER ligand. In vitro work has shown proliferative action of cadmium in human prostate cells, and in rats prostatic tumors have been induced by oral cadmium exposure or by injection [177,182,183].
According to a review by Benbrahim and Waalkes, arsenic can induce malignant transformation of human prostate epithelial cells and also appears to impact prostate cancer cell progression by precipitating events leading to androgen independence in vitro [184].
Vinclozolin, a fungicide, has known antiandrogenic properties. On the one hand prostate gland growth in rats was reduced by vinclozolin, but on the other hand, prenatal exposure leads to aging-associated prostatitis in the next four generations of offspring. This suggests a role in prostate cancer induction [177]. Table 3 resumes the main data from a number of epidemiological studies. Below some other data and additional details of some of these studies are mentioned.
The Agricultural Health study in Iowa and North Carolina showed that Farmers and commercial pesticide applicators have a significantly increased risk of prostate cancer (http://www.aghealth.org/).
Among  [110]. Still in the same study, the thiocarbamate herbicide butylate, the organophosphorothioate insecticides chlorpyrifos and coumaphos, the organophosphorodithioate pesticide fonofos, the pyrethroid insecticide permethrin, and the organophosphorodithioate insecticide phorate showed a significantly increased risk of prostate cancer among study subjects with a family history of prostate cancer but not among those with no family history [110,186].

Testis Cancer.
In the recent past, there have been several new epidemiological papers (reviewed by Aitken et al. [187] that point towards widespread declines in sperm quality and increases in testis cancer incidence and suggesting that these likely have an endocrine etiology.
Skakkebaek et al. [188] hypothesized that the testicular dysgenesis syndrome originates from conception and can result in a cascade of defects in Sertoli and Leydig cells that ultimately affect maldescent of the testes, cryptorchidism, fertility, and the probability of testicular cancer. Jorgensen et al. [189] observed that the increasing incidence of testis  Limited info on specific chemicals [112] 18 Journal of Environmental and Public Health cancer in Finnish birth cohorts was associated with a substantial decrease in semen quality and concluded that these simultaneous and rapidly occurring adverse trends suggested that the underlying causes are environmental and, as such, preventable.

Non-Hodgkin Lymphoma and Other Hematopoietic
Cancers. Table 4 resumes the main data from epidemiological studies. Below some other data and additional details of some of these studies are mentioned.
Merhi et al. [190] performed a meta-analysis of 13 case control studies that examined the occurrence of hematopoietic cancers in pesticide-related occupations. The overall meta-odds ratio obtained after pooling 44 ORs from 13 studies was 1.3 (95% CI: 1.3-1.5). In particular, a significant increased risk of NHL was found (OR = 1.35; 95% CI = 1.2-1.5).

Diabetes. According to Harrison's Principles of Internal
Medicine (16th edition), diabetes is a metabolic disease caused by an attenuated production of insulin by pancreas Bcells (type I diabetes) or by development of resistance against insulin action resulting in a relative insulin-shortage (type II diabetes).

Experimental Data.
According to a review by Alonso-Magdalena et al. [203], widespread EDCs, such as dioxins, pesticides and bisphenol A, cause insulin resistance and alter β-cell function in animal models. Many of them act as estrogens in insulin-sensitive tissues and in β cells, generating a pregnancy-like metabolic state characterized by insulin resistance and hyperinsulinemia.
Adult male Sprague-Dawley rats exposed to crude salmon oil, containing persistent organic pollutants (POPs), developed insulin resistance, abdominal obesity, and hepatosteatosis. The contribution of POPs to insulin resistance was confirmed in cultured adipocytes where POPs, especially organochlorine pesticides, led to robust inhibition of insulin action [204].
Parathion and other organophosphate pesticides induce a prediabetic state in Sprague-Dawley rats in a sex-selective manner [205].
Long-term exposure to the endocrine disrupting herbicide atrazine induces morphological and functional changes in mitochondria, a decrease in metabolic rate, insulin resistance and obesity in Sprague-Dawley rats [206].
The xenoestrogen Bisphenol A disrupted the action of the endocrine pancreas and blood sugar homeostasis in mice: a postprandial hyperinsulinemia and insulin resistance were induced after an injection of 100 μg/kg per day during 4 days in mice [207].
In Wistar rats, prenatal exposure to diisobutyl phthalate reduced plasma leptin and insulin in male and female fetuses [208].
Arsenic inhibits, in 3T3-L1 adipocytes in vitro, insulin signaling by inhibiting the PDK-1/PKB/Akt signal transduction pathway, which might explain its diabetogenic effects [209].  [196], the U.S. Department of Veterans Affairs added type 2 diabetes to the list of presumptive diseases associated with the exposure to dioxin-containing Agent Orange in Vietnam.

Epidemiological Studies.
Inhabitants of Seveso who were exposed to very high concentrations of dioxin during the disaster in 1976 showed significantly more diabetes than nonexposed inhabitants [191].
Longnecker and Daniels [210] identified 11 reports on the relation of TCDD with type 2 diabetes, 5 of which showed an unequivocally positive association with 3 others showing an equivocally positive association, but all these studies showed notable weaknesses.
After adjustment for age and other covariates, serum total toxic equivalent activity (sum of PCDD/Fs and coplanar PCBs) was 62% (P = 0.0005) higher in diabetic patients, than in controls [192].

Other POPs (Mainly Organochlorine).
After adjustment for age and other covariates, concentration of 12 marker PCBs was 39% (P = 0.0067) higher in diabetic patients than in controls [192].
In a cross-sectional study of 2,245 pregnant women, of whom 44 had diabetes (primarily type 1), the adjusted mean serum level of PCBs among the subjects with diabetes was 30% higher than in the control subjects (P = 0.0002), and the relationship of PCB level to adjusted odds of diabetes was linear [193].
According to the observations of Lee et al. [197], the dioxin-like PCBs and the organochlorine pesticides showed the strongest associations with diabetes.
In a nested case-control within the Coronary Artery Risk Development in Young Adults (CARDIA) cohort, Lee et al. [197]  POPs showed nonlinear associations with diabetes risk. The highest risk was observed in the second quartiles of trans-nonachlor, oxychlordane, mirex, highly chlorinated PCBs, and PBB153, a finding that suggests low-dose effects. Sextiles of a summary measure for all 31 pops measured were associated with following odds ratios after adjustment       To calculate the cumulative measure, the category number of each POP (0 assigned to the nondetectable category, and 1 through 5 assigned to successively increasing categories) was added to make the sum of POP levels (SUMPOPs), producing a value of 0-30, which was itself categorized at its 25th, 50th, 75th, and 90th percentiles, making five groups.
c To calculate the cumulative measure of three PCDDs, the ranks of the three PCDDs were summed, then divided into quartiles. Cumulative measures were calculated in the same way subclasses similarly for the three PCDFs, the four dioxin-like PCBs, the five nondioxin-like PCBs, and the four organochlorine pesticides. Lee et al. [196] reported strong and highly significant associations, among participants in the NAHNES study, between serum concentrations of persistent organic pollutants and the prevalence of diabetes, with high Odds ratio's (Table 5) after correction for age, sex, race and ethnicity, poverty, income, BMI, and waist circumference. In nondiabetic adults participating in the same NAHNES study, successive quartiles of a parameter for sum of serum concentrations of organochlorine pesticides (Oxychlordane, Trans-nonachlor, p,p-dichlorodiphenyltrichloroethane and -Hexachlorocyclohexane) were associated with HOMA-IR insuline resistance values of 3.27 ± 0.39, 3.36 ± 0.33, 3.48 ± 0.35, and 3.85 ± 0.45 (P trend < 0.01) [198], after adjustment for age, sex, race, poverty income ratio, BMI, waist circumference, cigarette smoking, serum cotinine concentration, alcohol consumption, and exercise. This association increased in power in function of increasing HOMA-IR values: correlated odds-ratio for the comparison of the highest and the lowest quartile of serum concentration of organochlorine pesticides were 1.8 for an insulin resistance value equal to or higher than the 50th percentile, 4.4 for the 75th percentile and 7.5 for the 90th percentile. This means that, when comparing high and low exposed individuals, the odds-ratio for a strong abnormal insulin resistance is clearly higher than for a weakly increased insulin resistance [198].
The associations found between some PCBs and organochlorine pesticides with insulin resistance among subjects without diabetes [198] are consistent with the notion that these substances contribute to the induction of diabetes. When all five subclasses of POPs were included in one model, only OC pesticides were significantly associated with insulin resistance [198].
The rather high correlation between serum levels of different organochlorine pollutants can lead to noncausal associations between some organochlorines and diabetes, simply because these organochlorines are themselves correlated with other organochlorines, which do have a causal association with diabetes.
Interestingly, in the National Health and Nutrition Examination Survey, obesity did not increase the prevalence of diabetes among subjects with nondetectable levels of POPs, even though there were sufficient numbers of study subjects at risk in each BMI category [196].
Everett et al. [211] argue that the probability of a causal association between PCBs and diabetes is weakened by the fact that many observations fail to show a linear dose-effect relationship. The existence of nonlinear and nonmonotonic effects is, however, to be expected for receptor-binding substances (see Section 5.2).
Stahlhut et al. [212] studied 622 (only 327 for MEHHP and MEOHP) adult US male participants in the NHANES 1999-2002 to evaluate six phthalate metabolites with prevalent exposure and known or suspected antiandrogenic activity as predictors of insulin resistance measured through the log-transformed homeostatic model assessment (HOMA, a measure of insulin resistance). Using multiple linear regression, adjusted for age, race/ethnicity, fat and total caloric consumption, physical activity level, serum cotinine, and urine creatinine, urinary concentrations of three metabolites (log transformed to normalize the data) were associated (regression coefficient (SE), P), with increased insulin resistance: MBP (0.064 (0.024), P = 0.011), MBzP (0.079 (0.023), P = 0.002), and MEP (0.056 (0.020), P = 0.008). Converting regression coefficients to clinically interpretable measures, an increase in phthalate metabolites from the 10th to the 90th percentile corresponded to an increase in HOMA (at the 2.50 median) with 1.3-1.4 (52-57% of median) in association with three metabolites: MBP (1.

4.2.2.5.
Arsenic. Epidemiologic evidence points to an increased chance for the development of type 2 diabetes in populations exposed to arsenic. These associations were, however, only found at relatively high, occupational exposures, or in areas with very high drinking water concentrations of arsenic, such as parts of Bangladesh and Taiwan [210]. According to Longnecker [213], more studies are needed on the association of diabetes with arsenic. Higher cadmium, arsenic, and lead levels have also been found in diabetic mothers compared to controls [214].

Tentative Conclusion.
The associations between exposure to xenobiotics and diabetes in epidemiological studies are quite complex, and it is still difficult to establish a causal relation between a specific chemical and diabetes in humans, amongst others because clear dose-effect relationships are often lacking. However, taking into account the results of animal experiments, the consistency between epidemiological findings as to diabetes, insulin resistance, and the metabolic syndrome (see Section 4.4) and the mechanistic data suggesting that nonlinear and nonmonotonic exposure-effect relations are to be expected (see Section 5.2), it is likely that some organochlorines (especially some pesticides), some brominated chemicals, phthalates, and high levels of exposure to arsenic contribute to the risk of diabetes.

Obesity.
Fat soluble xenobiotics can accumulate in adipose tissue, which then functions as a reservoir from which these substances can return to the blood, where they are detected in concentrations that show, for some xenobiotics-a positive correlation to the body mass index [215]. Irigaray et al. [216] observed that benzo(a)pyrene can induce obesity in mice by inhibition of beta-adrenergic stimulation of lipolysis in adipose tissue. However, negative correlations are often observed between body mass index and serum levels of organochlorine pollutants, probably due to a dilution effect. In Flemish adolescents, we found a significant negative association between internal organochlorine exposure (especially marker PCBs and hexachlorobenzene) and body mass index [217], negative association that might in part be explained by a dilution effect, but that might also, at least partly, result from an endocrine disruption effect suggesting that some organochlorines might, at least in adolescents, have a limiting effect on weight gain.
Several studies also suggest a role in obesity for the endocrine disrupting organotins tributyltin (TBT) and triphenyltin (TPT). TBT and TPT are potent agonists of the nuclear hormone receptors peroxisome proliferatoractivated receptor gamma and retinoid X receptor, which serve as metabolic sensors for lipophilic hormones, dietary fatty acids and their metabolites, and consequently influence lipid biosynthesis and storage [218,219].
Polybrominated diphenyl esters (PBDE's) are potential obesogens as well. In a rat study PBDE's were found to lower thyroxin levels and affect lipolysis and insulin stimulated glucose oxidation in isolated adipocytes [221]. Low-dose exposure to PBDE's during gestation and lactation has also been observed to cause long-term changes in thyroid gland morphology [218].
Dithiocarbamates (present in cosmetics and pesticides) are suggested obesogens, through interference with glucocorticoid receptor signaling [219].
Heindel and vom Saal [223] have also reviewed effects of perinatal exposure to several endocrine disruptive chemicals on homeostatic control systems required to maintain normal body weight troughout life. These chemicals include cadmium, organotins, and bisphenol-A. Table 6 resumes the main data from a number of epidemiological studies. Below some other data and additional details of some of these studies are mentioned.

Metabolic Syndrome.
The metabolic syndrome is characterized by a high blood pressure, hyperglycemia and hypertriglyceridemia, low HDL concentrations and too much fat in the waist area.

Organochlorines.
Lee et al. [224] found a correlation between serum concentrations of persistent organic pollutants and the prevalence of the metabolic syndrome in nondiabetic adults. Organochlorine pesticides correlated most strongly, with corrected odds ratios of 1.0, 1.5, 2.3, and 5.3 for the OC pesticide quartiles. Dioxin-like PCB's also showed a positive correlation with ORs 1.0, 1.1, 2.2, and 2.1. Non-dioxin-like PCBs showed an inverted-U association, with odds ratio's of 1.0, 1.3, 1.8, and 1.0 (P for quadratic term <0.01). OC pesticides showed ORs >2 for four of the five components of metabolic syndrome: waist circumference, elevated triacylglycerol, low HDL-cholesterol, and high fasting glucose. PCBs were linearly or quadratically associated with three of the five components: waist circumference, elevated triacylglycerol, and high fasting glucose. PCDDs and PCDFs were not associated with 4 of the 5 metabolic syndrome parameters but were positively and significantly associated with high blood pressure.
In a cross-sectional study on 1,374 subjects, not occupationally exposed to dioxins and related compounds, representative of the general population in Japan, the toxic equivalents (TEQs) of PCDDs, PCDFs, and DL-PCBs and total TEQs had significant adjusted associations with metabolic syndrome. The DL-PCB TEQs and total TEQs were associated with all components of the syndrome, and the odds ratios (ORs) in the highest quartile of DL-PCB TEQs in four of the five components were higher than those for PCDDs or PCDFs. Also congener-specific associations with metabolic syndrome were observed; in particular, the 28 Journal of Environmental and Public Health

Perfluorinated Compounds.
Data from NHANES and from the C8 Health Project, mentioned in a recent review on perfluorooctanoic acid [226], suggest that perfluorooctanoic acid (PFOA) contributes to higher blood lipid concentrations.

In Animals.
Serious problems of reproductive health in wildlife caused by pesticides were reported in the important book "Silent Spring" [227]. Later work on the reproductive and developmental anomalies in Great Lakes gulls [228] and in alligators [229] and advances in understanding of the mechanism of action of diethylstilbestrol and other xenoestrogens [230] contributed to the formulation of the endocrine disruptor hypothesis [231]. Recent studies have documented disturbing reproductive effects of ambient levels of the pesticide atrazine on frogs, characterised by feminization of males that had been exposed to concentrations that can be encountered through permitted uses in the United States [232]. There are increasing data from wildlife studies and laboratory studies with rodents, ungulates, and nonhuman primates that support a role of EDCs in the pathogenesis of several female reproductive disorders, including polycystic ovarian syndrome, aneuploidy, premature ovarian failure (POF), reproductive tract anomalies, uterine fibroids, endometriosis, and ectopic gestation [153]. In sheep and rhesus monkeys, prenatal androgenic stimulation gives rise to the polycystic ovary syndrome. In rats, exposure to 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in utero and through the end of reproductive life results in a dose-dependent onset of premature reproductive senescence, likely due to direct effects on ovarian function [233]. Perinatal exposure of rats to Bisphenol A affects the fertility of male offspring [234].

Changes in Sex Ratio. In a small First Nation
Chippewa community surrounded by chemical manufacturing plants in Sarnia, Ontario, Canada, a marked decline in the sex ratio was observed, inferring that almost 40% of the boys had been lost [235]. In Seveso, Italy, a 2,4,5, trichlorophenol explosion in 1976 resulted in an immediate loss of males with subsequent recovery [236]. Van Larebeke et al. [237] proposed that sex ratio changes might constitute sentinel health events of endocrine disruption.

Fertility of Women.
Daughters of women exposed to Diethylstilboestrol showed a reduced fertility [238].
According to the endocrine society scientific statement, bisphenol A and other endocrine disrupting chemicals might contribute to the risk of polycystic ovary syndrome (PCOS) in women [153]. PCOS is a debilitating disorder in women, occurring in 6.6% of the reproductive age population; it is a leading cause of subfertility and is associated with increased lifetime risks of cardiovascular disease and type II diabetes [153].
Adult exposure in women to cigarette smoke results in decreased fecundity, decreased success rates of in vitro fertilization (IVF), decreased ovarian reserve, earlier menopause by 1-4 years, and an increased miscarriage rate [239,240]. Maternal smoking during pregnancy appears to be a threat to the future fecundity of the daughter [241].
Higher PCB serum levels were reported to be associated with increasing menstrual cycle length and a trend towards irregular cycles [242].
Among 50 Southeast Asian immigrant women, after correction for confounding, mean luteal phase length was shorter by approximately 1.5 days at the highest quartile of serum DDT (95% CI = −2.6 to −0. 30) or DDE (−2.6 to −0.20). Progesterone metabolite levels during the luteal phase were consistently decreased with higher DDE concentration [243]. Cohn et al. [244] investigated time to pregnancy (fecundability), a sensitive indicator of environmental effects on reproduction, among 289 women exposed to p,p -DDT and p,p -DDE in utero. Maternal p,p -DDE was associated with a raised probability of pregnancy in their daughters, and p,p -DDT was associated with a reduced probability of pregnancy in daughters.
Among 1240 women from the Danish National Birth Cohort recruited from 1996 to 2002, longer "time to pregnancy" (TTP) was associated with higher maternal levels of PFOA and PFOS (P = 0.001) [245]. Compared with women in the lowest exposure quartile, the adjusted odds of infertility (defined as TTP > 12 months) increased by 70-134% and 60-154% among women in the higher three quartiles of PFOS and PFOA, respectively. Fecundity odds ratios (FOR) were virtually identical for women in the three highest exposure groups of PFOS (FOR (1/4) 0.70, 0.67, and 0.74, resp.) compared with the lowest quartile. A linear-like trend was observed for PFOA (FOR 0.72, 0.73, and 0.60 for three highest quartiles versus lowest quartile) [245].
Endometriosis, occurring in 6 to 10% of women, is associated with infertility. In utero exposure of humans to diethylstilboestrol results in an increase in relative risk of endometriosis (RR = 1.9, 95% confidence interval, 1.2-2.8) [246].

Fertility of Men (i) Maternal Smoking.
Maternal smoking during pregnancy appears to be a threat to the future fecundity of the son [249,250].
Exposure to dioxin during the Seveso incident led to a decreased sperm quality in men exposed prepubertally, was positively associated with sperm quality in men exposed between ages 10-17, and did not affect sperm quality in men exposed at ages 18-26 [253].
(iii) PCBs. Between groups of 34 men with poor semen quality and 31 men with normal sperm quality, Dallinga et al. [254] found no significant differences in organochlorine blood levels. However, among the men with normal semen quality, sperm count and sperm progressive motility were inversely related to the concentration of PCB metabolites [254].
Among 305 young Swedish men 18-21 years old from the general population, Richthoff et al. [255] found weak but statistically significant, negative correlations between PCB-153 levels and both the testosterone : SHBG ratio (r = −0.25, P < 0.001)-a measure of the biologically active free testosterone fraction-and sperm motility (r = −0.13, P = 0.02).
Among 212 male partners of subfertile couples who presented to the Massachusetts General Hospital Andrology Laboratory, there were dose-response relationships among PCB-138 and sperm motility (odds ratio per tertile, adjusted for age, abstinence, and smoking, and P value for trend were, resp., 1.00, 1.68, 2.35, P value = 0.03) and morphology (1.00, 1.36, 2.53, P value = 0.04). The lack of a consistent relationship among semen parameters and other individual PCB congeners and groupings of congeners may indicate a difference in spermatotoxicity between congeners [256]. Among 195 Swedish fishermen, aged 24-65 years, the subjects in the quintile with the highest PCB 153 concentration (>328 ng/g lipid) tended to have decreased sperm motility compared with the subjects in the lowest quintile (<113 ng/g lipid). The age-adjusted mean difference was 9.9% (95% confidence interval −1.0to21% P = 0.08) [257]. No significant associations between p,p -DDE and semen characteristics or reproductive hormones were found [257].
(v) Perfluorinated Substances. Sperm quality was also reported to be diminished in association with more intensive exposure to the perfluorinated substances perfluorooctanoic acid and perfluorooctane sulfonate [260]. Among 105 Danish men from the general population (median age 19 years), men in the highest quartile of combined levels of PFOS and PFOA had a median of 6.2 million normal spermatozoa in their ejaculate in contrast to 15.5 million among men in the lowest quartile (P = 0.030) [260].
(vi) Heavy Metals. Sperm quality was also reported to be diminished in association with exposure to heavy metals. Among 149 healthy male industrial workers 20-43 years of age residing in Zagreb (Croatia), (98 subjects with slight to moderate occupational exposure to Pb and 51 reference subjects), a Pb-related decrease in sperm density, in counts of total, motile, and viable sperm, in the percentage and count of progressively motile sperm, in parameters of prostate secretory function, and an increase in abnormal sperm head morphology was found. These associations were confirmed by results of multiple regression, which also showed significant (P < 0.05) influence of blood cadmium, serum zinc, serum copper, smoking habits, alcohol consumption, or age on certain reproductive parameters. Blood cadmium contributed to a decrease in sperm motility and an increase in abnormal sperm morphology [261].
(vii) Trihalomethanes. Higher trihalomethane levels in drinking water might be associated with a decrease in the fecundity of men [262].
(viii) Air Pollution. Selevan et al. [263] studied semen quality of young men (18 years of age) living in Teplice, a highly industrialized district with seasonally elevated levels of air pollution, or in Prachatice, a rural district with relatively clean air. Periods of elevated air pollution in Teplice were significantly associated with decrements in several semen measures including proportionately fewer motile sperm, proportionately fewer sperm with normal morphology or normal head shape, and proportionately more sperm with abnormal chromatin [263]. In later research, 36 young men from Teplice were sampled up to seven times over 2 years allowing evaluation of semen quality after periods of exposure to both low and high air pollution. A significant association was found between exposure to periods of high air pollution (at or above the upper limit of US air quality standards) and the percentage of sperm with DNA fragmentation according to sperm chromatin structure assay (SCSA). Other semen measures were not associated with air pollution [264].
DEHP metabolites were measured in spot urine of 463 male partners of subfertile couples who presented for semen analysis to the Massachusetts General Hospital and of men with all semen parameters above WHO reference values. Dose-response relationships were observed for MBP with low sperm concentration (odds ratio per quartile adjusted for age, abstinence time, and smoking status = 1.00, 3.1, 2.5, 3.3; P for trend = 0.04) and motility (1.0, 1.5, 1.5, 1.8; P for trend = 0.04) [266].
Among 379 men from an infertility clinic, sperm DNA damage was associated with MEP and with MEHP after adjusting for DEHP oxidative metabolites, which may serve as phenotypic markers of DEHP metabolism to "less toxic" metabolites [267].
Swan et al. [268] found that mothers with higher levels of phthalates in their blood during pregnancy had a significantly greater chance of bearing male infants with reduced anogenital distance, a marker for testicular dysgenesis in rodents.

(x) Anabolic Steroids Used in Food Production.
Residues of anabolic steroids and other xenobiotics used in food production may pose long-term risks for developmental processes in males. For example, in a large study of sperm concentration and fertility in American men by Swan et al. [269], there was a negative association with the number of servings of beef their mothers ate per week while pregnant. The findings of Swan et al. [268,269] are consistent [270] with the developmental origins of human health and disease (DoHAD) hypothesis [271] and with the testicular dysgenesis syndrome identified by Skakkebaek et al. [188].
(xi) Endocrine Disruption, Reproductive, and Overall Male Health. That endocrine disruption has an important impact on the reproductive organs in men is also suggested by considerable evidence indicating that endocrine disruptors contribute to the risk of testicular cancer and prostate cancer (see above) and male urinary tract malformations (see below). Poor semen quality and increased incidence of testis cancer might find their origin in the testicular dysgenesis syndrome [187,188].
Jensen et al. [272] found significant associations between self-rated health and semen quality and testicular size. Interestingly, good semen quality was associated with a higher life expectancy [273]. The decrease in mortality among men with good semen quality was due to a decrease in a wide range of diseases and was found among men both with and without children; therefore, the decrease in mortality could not be attributed solely to lifestyle and/or social factors. Semen quality may therefore be a fundamental biomarker of overall male health [273]. [274][275][276]. Hypospadias is associated with a slight increase in Follicle Stimulating hormone concentrations indicative of primary testicular dysfunction [275]. Recorded temporal trends, geographical differences, and observations made in wildlife after environmental accidents are compatible with a role of endocrine disrupters in the causation of male urinary tract malformations [274][275][276]. That endocrine disruption is involved in cryptorchydism and hypospadias is also suggested by the association of cryptorchydism and hypospadias with reduced anogenital distance [277].

High Impact of Exposures during Early Life and Certain
Time Windows. In utero and during early postnatal life mammalian organisms are much more sensitive than during adult life, not only to mutagenic agents [278], but also to endocrine disruption. Exposures during these periods can entail a high impact, not only on development but also on the risk of disease much later in life. This might be the case for cancer [279][280][281][282][283], obesity [284][285][286], the metabolic syndrome [286], diabetes [286], cardiovascular disease [286], neurodegenerative disease such as Alzheimer and Parkinson [287][288][289][290][291][292][293][294], and mental retardation [295]. Prenatal exposures might even contribute to psychoses [296,297]. Especially during certain time windows, when critical proliferation, differentiation, or migration processes take place, a highly increased sensitivity to disrupting agents must be taken into account, especially when these agents are receptor binding [271]. This insight led to the new paradigm on "developmental origins of human health and disease" [271].
Early life exposure (late embryonic and/or early postnatal) to low doses of PCBs [298][299][300] or soy [301] significantly and adversely affected mating behaviors in female rats. Early postnatal treatment with coumestrol (a phytoestrogen) diminished masculine and feminine sexual behaviors [302,303]. These results are consistent with an impact of EDCs on the neuroendocrine hypothalamus (see also Section 5.1.10).
Internal prenatal exposure to perfluorooctanoic acid (PFOA) was inversely associated with birth weight in some studies, but not in others [226].

Males Often More Sensitive Than Females.
In mammalians, the ontogenetic evolution towards the female phenotype appears in some way to be the default process and occurs to a large extent without sex hormone influence [304]. The ontogenetic evolution towards the male phenotype on the contrary occurs under the influence of sex hormones produced by the testis (both testosterone and estradiol are necessary for normal sexual differentiation of the male brain) [304] and so probably is more vulnerable to endocrine disruption. Also, sexual differentiation of the female and male brain differs due in part to alfa-fetoprotein, which protects the brain from effects of maternal estrogens. Alfa-fetoprotein knockout mouse females are masculinised and defeminised in brain and behaviour, providing further support for a role of estrogens in the masculinisation of the brain. Additional differences in brain development between the sexes may result from even subtle differences in the timing of hormone exposures, as the mammalian brain is exquisitely sensitive to hormones in late embryonic and early postnatal time periods, and even small differences may exert large effects [304]. There has been considerable and consistent research that shows that PCBs, phytoestrogens, fungicides, pesticides, and other xenobiotics can disrupt brain sexual differentiation [304].
Even adult males and females do not necessarily react in the same way to exogenous substances. In the Flemish biomonitoring, changes in gene expression associated with internal exposure to DDE, hexachlorobenzene, marker PCBs and dioxin-like activity were predominantly in opposite direction for men and women [305].

Mechanistic Considerations
Endocrine-disrupting agents can act through many different mechanisms. This is best documented for agents interfering with sex hormones, especially xeno-estrogens, and most of the evidence below stems from this field.

Activation of the Classical Nuclear Receptors.
In the classic view, estrogens control gene networks and modulate target cell activities through activation of the ERα and ERβ nuclear receptors, which bind estrogen responsive elements (EREs) in the promoters of target genes and regulate target gene expression [306].
Initially, due to the often limited effect of interaction with ERs (e.g., BPA is, in this respect, 1000-2000 fold less potent than 17β-estradiol), it was thought that the significance of xenoestrogens was low [307,308]. However, the last decade(s), a number of other mechanisms were discovered, often triggered at low concentrations of xenoestrogen. This will be further discussed in this section.
Endocrine disruption of the thyroid system too can occur through interference with thyroid hormone receptordependent transactivation [126].

Effects of Activation of Receptors Differs in Function of the Ligand.
After activation by binding to a ligand resulting in a conformation change, receptors can act as transcription factors, interacting with coactivators and corepressors and with DNA sequences [309,310]. However, the change in conformation differs in function of the ligand [311][312][313][314]. Differences in conformation can be expected to be associated with differences in function and thus differences in regulatory activity on gene expression. That is what has been observed: receptors bound to xenobiotic ligands do not have exactly the same influence on gene expression as receptors bound by endogenous ligands [315,316]. This has been studied for several xenoestrogens, such as octylphenol, nonylphenol, endosulfan, and kepone by Wu et al. [317], who demonstrated the highly structure-dependent induction of luciferase activity in MCF-7 and MDA-MB-231 breast cancer cells transfected with a construct linked to 34 Journal of Environmental and Public Health ERα and luciferase. 17Beta-estradiol and the phytoestrogen coumestrol even had opposite effects on the regulation of estrogen receptor beta mRNA in the brain of rats [318].

Activation of Membrane-Bound Estrogen Receptors
: mERα, mERβ, and GPR30. Until recently, most studies have focused on the slow, genomic phase of steroid responses based on activation of nuclear receptors and acting through modifications of transcription and protein synthesis. However, steroid hormones can also induce rapid (seconds to minutes) nongenomic responses based on plasma membrane receptors and acting through second messenger-triggered signal cascades [319].
In the past, the basis for calling an estrogen "weak" or "strong" has been entirely dependent upon the nuclear transcription signalling mechanism [320]. It is now becoming clear that "weak" activity via one pathway does not necessarily predict the potency of a hormone or mimetic acting via another signalling pathway. Though the activities of most environmental estrogens have been called "weak" for many years because of their inability to initiate nuclear retention and transcriptional effects, we now see that they are quite potent initiators of signal cascades emanating from the membrane [319].
Two types of membrane estrogen receptors, mERα and mERβ, are likely the same proteins as the nuclear receptors ERα and ERβ, transported to the plasma membrane by yet undetermined mechanisms [306]. As to nongenomic actions of activated mERs, one example is Ca ++ release that can lead to changes in cell motility, intra-and extracellular signalling processes, and rapid hormone secretion (including prolactin) trough exocytosis. Changes in prolactin (PRL) secretion are associated with hormonal regulation of lactation, cell proliferation, the cellular immune response, and parental/maternal behaviour [321]. Xenoestrogens such as dieldrin, endosulfan, o,p -DDE, nonylphenol, bisphenol A, coumestrol, and diethylstilbestrol are known to affect Ca ++ influx and PRL release. Interestingly, Wozniak et al. [321] found these xenoestrogens to affect one or more of the above pathways in a specific way. Differences between xenoestrogens included the concentration range (pM or nM) in which they were active, as well as in the temporal pattern in which they induced a specific mechanism, for example, early-or late-phase activation or an early and sustained activation. This illustrates the complexity of xenoestrogen induced endocrine disruption.
The rapid "nongenomic" mechanisms have been shown to interact with cytoplasm signal transduction molecules such as cAMP and adenylate cyclase, calcium, PI3K, PKB, Src (and consequent activation of kinases Erk1 and Erk2 in the Src/Ras/Erk-cascade) and G-proteins or directly with secondary transcription factors such as AP-1 (activator protein 1), STATS (signal transducer and activator of transcription), NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells), and Sp1 (specificity protein 1) [323,324].

Cytoplasmic Interactions.
Besides with nuclear-and plasmamembrane-associated ER receptors, estrogens can also interact with targets within the cytosol. One of the best studied examples is the activation of Src/Ras/ERK (MAPK) pathway, in a mechanism linked to the proliferative effects of estrogens. Binding of estrogens to cytosolic ER receptors determines the interaction of ERs with Src, changing the conformation of the kinase to an active state and leading to the activation of the Src/Ras/ERK signaling cascade [325]. Another example is the modulation of nitric oxide (NO). Estrogen activates endothelial NO synthase (eNOS, the enzyme responsible for NO production). This activation in endothelial cells by ERα involves phosphatidylinositol 3-kinase (PI3K) and an ERα-eNOS signalling complex in the endothelial caveolae. Binding of estrogen to the ERα will, via a direct physical interaction with PI3K, activate serine/threonine protein kinase B (Akt), which in turn will phosphorylate and activate NOS [325].

Cross-Talk between Genomic and Nongenomic Pathways.
As can be deduced from previous chapters, xenoestrogens can influence several mechanisms simultaneously. Therefore, Silva et al. [323] investigated the differential action of xenoestrogens, acting on both nuclear (genomic) as well as on extranuclear (non-genomic) pathways, and possible cross-talk between these two mechanisms triggered by the xenoestrogenic chemicals o,p -DDT, p,p -DDE, and β-HCH in MCF-7 cells. Therefore, expression of estrogen responsive genes and phosphorylation of Src, Erk1, and Erk2 were measured after exposure to these estrogenic compounds. The researchers found strong similarity between E2, o,p -DDT, and β-HCH in gene expression and phosphorylation patterns, as well as in cell proliferation. This is despite of the lack of affinity of β-HCH for the ER binding domain. p,p -DDE, however, influenced estrogen-related gene expression, but did not phosphorylate Src and Erk1/Erk2. Other authors have also found diversity of effects between different xenoestrogens, such as bisphenol A (unable to induce Erk activation) and endosulfan and p-nonylphenol (rapidly inducing Erk1/Erk2 phosphorylation) [326].

Activation of Estrogen-Related Receptors.
Estrogen related receptors (ERR) are a subfamily of orphan nuclear receptors closely related to ERα and ERβ. Three of these ERR are known: ERRα, ERRβ, and ERRγ. ER and ERR show a considerable amount of similarity in aminoacid sequence, but E2 does not bind ERRs. However, ERR can bind to estrogen response elements, which suggests a possible overlap between ER and ERR action [328].
ERRs show spontaneous transcriptional activity, which is known to be repressed by a few chemicals. For example, DES represses the molecular activities of ERRs, however, to a considerably lesser extent than its action as an ER activator. Another inverse agonist of ERRγ is 4-hydroxytamoxifen (4-OHT). On the other hand, bisphenol-A has been observed to have a distinct antagonist action to the inverse agonist activity of 4-OHT, thus preserving ERR-activity in the presence of 4-OHT [328].

Cross-Talk with Estrogen Receptors after Binding on
Other Receptors. Although their antiestrogenic actions are well described, dioxins can also induce endometriosis and estrogen-dependent tumours, implying possible oestrogenic effects. A heterodimer of the dioxin receptor (AhR) and Arnt, which are basic helix-loop-helix/PAS-family transcription factors, mediates most of the toxic effects of dioxins. Ohtake et al. [329] showed that the agonist-activated AhR/Arnt heterodimer directly associates with estrogen receptors ERalpha and ER-beta. This association resulted in the recruitment of unliganded ER and the coactivator p300 to estrogenresponsive gene promoters, leading to activation of transcription and estrogenic effects. The function of liganded ER was attenuated. This mechanism is compatible with as well oestrogenic as antiestrogenic effects. Ohura et al. [330] demonstrated that AhR-induced activation of ER is dependent on ligand structure and does not necessarily occur for every AhR ligand.

Changes in DNA Methylation or Histone-Modifications.
Epigenetic changes, such as DNA methylation and histone modifications, have been shown to be involved in the mechanisms related to endocrine disruption. Developmental exposure to estradiol and bisphenol A increased susceptibility to prostate carcinogenesis and regulated phosphodiesterase type 4 variant 4 expression epigenetically, through changes in DNA methylation [331]. Transient exposure of a gestating female rat during the period of gonadal sex determination to the endocrine disruptors vinclozolin (an antiandrogenic compound) or methoxychlor (an estrogenic compound) induced an adult phenotype in the F1 generation of decreased spermatogenic capacity (cell number and viability) and increased incidence of male infertility. These effects were transferred through the male germ line to nearly all males of all subsequent generations examined (i.e., F1 to F4). The effects on reproduction correlate with altered DNA methylation patterns in the germ line [332,333]. Early life exposures to EDCs may alter gene expression in hypothalamic nuclei via nongenomic, epigenetic mechanisms, including DNA methylation and histone acetylation [304]. Figure. Reports show that estrogens, including E2, E3, and bisphenol A, induce micronuclei in MCF-7 cells, indicating that the (xeno) hormones have the ability to cause genomic instability [334]. Kabil et al. [335] observed that ER antagonists (interfering with the transcriptional activity of the ER and blocking promotion of ER-dependent gene expression) did not prevent micronucleus formation by these estrogens. On the other hand, coadministration of estrogens and kinase inhibitors, interfering with the extracellular signal triggered Scr/Raf/Erk signalling pathway (see Section 5.1.3), led to significantly less micronucleus formation. Kabil et al. [335] suggest that estrogens induce micronuclei formation by enhanced Scr/Raf/Erk stimulation, disturbing the localisation of Aurora B kinase to kinetochores and leading to improper chromosome segregation.

Interference with Hormonal Feedback Regulation and
Neuroendocrine Cells. Along with the direct influence of EDCs on estrogen or androgen actions, they can affect endogenous steroid production through negative and positive feedback, effects that may differ depending on developmental stage [153]. Neuroendocrine systems function as links between the brain and peripheral endocrine systems and are responsible for the control of homeostatic processes including reproduction, growth, metabolism, energy balance, and stress response. As stated by Gore [336], disruption of neuroendocrine homeostasis by endocrinedisrupting chemicals can lead to a series of perturbations.
GnRH (Gonadotropin-releasing hormone) neurons in the hypothalamus control reproductive function in vertebrates. GnRH binds to its receptors on cells named gonadotropes, which synthesise and release luteinizing hormone (LH) and follicle stimulating hormone (FSH). These hormones then bind to receptors on ovary and testes to cause steroidogenesis and gametogenesis. Disturbance of GnRH levels can be induced by amongst others PCBs, o,p -DDT, bisphenol A and is dependent on the chemical and on timing of treatment [336].
Evidence exists for neuroendocrine disruption of the hypothalamic-pituitary-thyroid system, with implications on (amongst others) metabolism and energy balance. For example, PCBs reduce the thyroxin and TSH (thyroid stimulating hormone) response to TRH (thyrotropin releasing hormone), which indicates hypothalamic and/or pituitary deregulation. Thyroid disruption also has consequences for neural development [336]. Disturbance of neuropsychic development in association with internal exposure to PCBs has indeed been observed in biomonitoring studies on adolescents [337] as well as on 36-month-old children, in this last instance in association with PCB levels measured in cord blood (unpublished results from the Flemish biomonitoring, Vermeir et al. in preparation).
Bisphenol A is another endocrine disruptor identified as a developmental thyroid toxicant [336]. Furthermore, the obesogenicity of DES has been supposed to involve action on the developing hypothalamic circuits, which are important for the energy balance.
Ceccarelli et al. [338] have shown in rats that exposure during early puberty to the estrogenic chemicals 17ethinylestradiol (EE) and bisphenol-A (BPA) has a distinctive effect on ER-α-expressing neurons, in key brain areas involved in reproductive behavior. The number of ER-α containing cells and the testosterone and estradiol serum levels were modified, and the changes were both sexdependent and different in the short and long term.
Also, exposure to hormonally active substances such as exogenous endocrine-disrupting chemicals (EDCs), may result in improper hypothalamic programming, thereby decreasing reproductive success in adulthood [304]. Furthermore, transmission of neuroendocrine effects to future generations has also been observed for vinclozolin with significant alterations in brain gene expression and behavior in F3 descendants [339].
Atrazine was observed to stimulate aromatase activity in some cell types, which leads to a higher synthesis of estradiol [340]. On the other hand, DDT and several metabolites, lindane, MEHP (phthalate metabolite), and several organotins were reported to inhibit aromatase activity in some cell types [123].
Hydroxylated metabolites of polyhalogenated aromatic hydrocarbons showed potent inhibition of estrogen sulfotransferase [341], an enzyme active in the excretion of estrogens, which elevates bioavailable estrogens in target organs.
Disruption of thyroid function occurs mainly through prereceptor regulation of ligand concentration [136,137]. Organohalogens are among the chemicals that can induce a decrease in circulating thyroid hormone levels [137]. This is through at least three distinct mechanisms. First, the chemical can directly affect the thyroid gland to decrease the synthesis of TH [342]. Second, reduced TH levels can be caused by enhanced biliary excretion of T4 due to the induction of UDP-glucuronyltransferases [343]. Third, the chemical may displace binding of the natural ligand, T4 to TTR, which could result in deiodinase inactivation and biliary excretion of the hormone [344,345]. Although all three mechanisms might be responsible for the decreases in circulating TH by organohalogens (parent compound and metabolites), high affinity binding of organohalogens to TTR might be the key factor in eliciting the biological effects [137].

Effects on Oxidative
Metabolism through Activation of the Pregnane X Receptor. The expression of many genes involved in xenobiotic/drug metabolism and transport is regulated by at least three nuclear receptors or xenosensors: aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR). These receptors establish crosstalk with other nuclear receptors or transcription factors controlling signalling pathways that are important to the homeostasis [346]. The pregnane X receptor (PXR) is activated by many drugs and environmental pollutants. A significant proportion (54%) of compounds with estrogenic activity or those able to bind ER were found to be hPXR activators. This was the case for classical estrogens such as estradiol and ethynylestradiol, for some antiestrogens such as 4-hydroxytamoxifen, for some mycoestrogens, for bis (2-ethylhexyl) phthalate, dibutylphthalate, and benzyl butyl phthalate, for several alkylphenols, for some UV-screens, and (but only weakly) for the fragrance galaxolide [31]. Also perfluorooctanoic acid (PFOA) was shown to activate the constitutive androstane receptor and pregnane X receptor [226]. The constitutive androstane receptor could also play a similar role but binds much less chemicals than the PXR receptor [347]. Differences in the pregnane X receptor are thought to underlie differences in xenobiotic metabolism between species [31].
The pregnane X receptor, which regulates several cytochrome P450 enzymes crucial in the oxidative metabolism of a wide range of chemicals, can thus play a role as well in the inactivation of toxic or carcinogenic chemicals as in the activation of procarcinogens and could also act as a protector of the endocrine system from chemical perturbation [31].  [54,[349][350][351][352].

Low-Dose Effects and
Furthermore, using an ordinary differential equationbased computational model, Li et al. [353] demonstrated that nonmonotonic dose-responses in gene expression can arise for exogenous ligands of steroid hormone receptors in an endogenous hormonal background.
In the case of some endocrine disruptors such as bisphenol A, exhibiting other modes of endocrine disruption in addition to binding to nuclear estrogen receptors, such as alterations in the synthesis or the metabolism of endogenous hormone and binding to plasma membrane estrogen receptors and also to androgen and thyroid hormone receptors, nontraditional dose response curves such as inverted U or U-shaped curves were observed [36]. As stated by the Chapel Hill bisphenol A expert panel [36], below the concentration range in which "pharmaceutical" effects are detected by classical toxicology, some substances, such as BPA, show biological effects due to disruption of cellular signalling mechanisms. For these substances, the safe level determined by classical toxicology does not protect against effects in the cell signal disrupting range [36].
While BPA was initially considered to be a "weak" estrogen based on a lower affinity for estrogen receptor alpha relative to estradiol [354,355], research shows that BPA is equipotent with estradiol in its ability to activate responses via recently discovered estrogen receptors associated with the cell membrane [321,[356][357][358]. It is through these receptors that BPA stimulates rapid physiological responses at low picogram per ml (parts per trillion) concentrations. Also, Lemmen et al. [354] found BPA to be more potent in activating embryonic ERs than would be expected on the basis of its in vitro activity.
That dose-effect relationships can be very complex for endocrine disruptors is evident from following examples: BPA has been found to have a greater effect on prostate tumor cell proliferation at 1 nanomole concentrations than it does at 100 nanomoles [359]. The plastic softener DEHP has been found to upregulate aromatase expression in neonatal male rats at doses above the LOAEL, but downregulate it at doses far below the LOAEL [360]. In an extreme example of different high-dose and low-dose effects, arsenic causes multiple organ failure at high doses, suppresses glucocorticoid hormone induction at low levels, yet enhances the same process at lower levels still [361].

Low-Dose Effects in Humans.
For more than two decades, the hypothesis of endocrine disruption has become a contested area of science. Some scientists have argued that isolated incidents of high levels of chemical contamination have affected health and reproduction of organisms, but that generally the levels of contamination of human beings have been far too low to have had effects. In the late 1990s, the U.S. National Academy of Sciences [362] set up a committee to examine the evidence on endocrine disruptors. The Academy considered that xeno-estrogens were at least a thousand times less powerful than endogenous hormones and that the levels encountered by humans were low and so were unlikely to yield any serious effects on human health. However, several of the observations mentioned under 4.5.2. and under 4.6. indicate that environmental exposures to very low doses do indeed have effects on humans. The observations made in the Flemish biomonitoring show that very low dose internal exposures to cadmium and to organochlorine pollutants (exposures under the median level of exposure in Flemish adolescents) are associated with detectable and sometimes pronounced differences in serum sex hormone levels [363], in sexual maturation [364], in gynaecomastia in boys [364], in height and in body mass index [217]. For several parameters, the exposure-effect relation was more pronounced per unit of dose at internal exposures under the median than above the median [217,363]. Premature thelarche has been reported in girls exposed to phthalates [365]. In the Flemish biomonitoring campaign 2007-2011, we observed that internal exposures actually occurring in adolescents aged 14-15, having no occupational exposure, to arsenic, polycyclic aromatic hydrocarbons, phthalates, organophosphorus insecticides, perfluorooctanoic acid, and the musk galaxolide, showed associations with serum hormone levels, with sexual maturation or with damage to DNA in peripheral blood cells (unpublished results, see http://www.milieu-en-gezondheid.be/resultaten/2007-2011/ studiedag%2021-12-2011/abstract%20NVL.pdf).

Effects of Combined
Exposures. The large number of endocrine-disrupting compounds present in the environment raises questions about the effects of simultaneous exposure to multiple compounds. However, relatively few studies have discussed combination effects.
Recently, Correia et al. [366] have tested combination effects of the (xeno)estrogens E 2 , EE 2 , and bisphenol A in fish, using vitellogenin induction as an endpoint. Effects of combined exposure to several (xeno)estrogens at equipotent concentrations were in high agreement with the predictions made with the model of concentration addition (based on the assumption that chemicals act via a similar mechanism to elicit an effect, such that one chemical acts as a dilution of the other and can be substituted at a constant proportion for the other), which points to additive action of these compounds. Zhang et al. [367] have also assessed the effect combinations of (xeno)estrogens (E2, EE2, bisphenol A and 4-tert-octylphenol) and observed that the concentration addition model agreed best with the observed combination effects, confirming additive effects of these substances on vitellogenin induction.
Other endpoints, however, show far more complex outcomes after combined exposure. Kochukov et al. [368] have previously demonstrated peaks of ERK-phosphorylation after E2, ethylphenol (EP), octylphenol (OP), propylphenol (PP), nonylphenol (NP), and bisphenol-A (BPA) exposure in pituitary cells. These peaks occurred at 2.5-5 minutes, 10-30 minutes, and 60 minutes for E1, E2, and BPA (3-peak oscillation), while for the alkylphenols and E3 a two-peak oscillation was observed with oscillations at 2.5-5 minutes and at 60 minutes (missing the intermediate peak). Later Jeng and Watson [369] studied the effects of combined exposure of these xenoestrogens with E1, E2, or E3. For cotreatment with alkylphenols, the first peak of the physiological estrogens (2.5-5 minutes) was abolished or blunted, while the second peak (10-30 minutes) was augmented. The 3rd peak, however, was mostly declined, often far below the response of the individual (xeno)estrogens. Thus, a temporal pattern with both synergistic (second peak) and antagonistic effects (3rd peak) was observed, which resulted in a transformation of the response from a three-peak oscillation to a single intermediate peak. The strength of the individual xenoestrogen tended to be predictive for their ability to inhibit actions of the natural estrogens when combined. BPA had an even more complex response, highly dependent on the dose. At very low concentrations (10 −14 M), combinations of physiological estrogens with bisphenol-A caused a response similar to alkylphenols (synergistic effect at the second peak, antagonistic effect at the third peak). However, at 10 −9 M concentrations, a synergistic effect was observed at both the second and third peaks [369]. Jeng and Watson [369] also studied dose dependency of effects at the 5-minute time peak. Most alkylphenols had a higher effect on ERK activation at higher doses, except for OP which showed more activity at lower doses. In combination, alkylphenols generally enhanced the effect of physiological estrogens at lower concentration but severely disrupted them at higher concentrations. BPA alone showed a non-monotonous response, with higher effects at low (10 −15 M) and high (10 −7 M) concentrations, and lower effects at intermediate concentrations. Remarkably, in combination, BPA was most disruptive of the physiological estrogen at the low (10 −15 M) and high (10 −7 M) concentrations, while physiological estrogen effects were unaffected or enhanced at intermediate concentrations. In other words, in concentration ranges where BPA had highest estrogenic activity on its own, it also had the highest inhibitory effect on physiological estrogens when combined [369].
Zsarnovszky et al. [358] have made similar observations with BPA and E2 in cerebellar neurons, with BPA induced increases of pERK positive cells at low (10 −10 -10 −12 M) and high (10 −7 -10 −6 M) concentrations, but BPA did not affect basal ERK signaling at intermediate concentrations, while coadministration of E2 (10 −10 M) and BPA (10 −12 -10 −10 M) inhibited ERK activation. Thus, BPA can both mimic or block some actions of E2. This behavior could be explained by the existence of an additional high affinity BPA-binding site with inhibitory activity, which becomes available only upon ligand binding at the high-affinity site of the rapid ERK-stimulating receptor [358].
The observation that pharmaceutical (fadrozole) and environmental aromatase inhibitors (tributyltin) do not block the effect of the xenoestrogen dichlorodiphenyltrichloroethane (o,p-DDT) suggests that in the environment, exposure to seemingly antagonistic EDCs does not necessarily lessen the harmful impacts of these compounds [370].
These observations illustrate the complexity of combined exposures to multiple endocrine disrupting compounds (and physiological estrogens), as well as the risks of endocrine disruption, even at very low doses of exposure. Furthermore, it is important to remember that a lot of different parameters, such as the coexistence of multiple ER isoforms, influence of homo-and heterodimers, posttranslational modifications of the receptors, localization at the membrane, and the changeable nature of the receptor signaling complex all likely contribute to the complex properties of (xeno)estrogens [358].

Conclusion
There is substantial evidence indicating that endocrine disruptors contribute to the risk of cancer, developmental problems, diabetes, and possibly also obesity and the metabolic syndrome. Also, it seems highly likely that endocrine disruptors can contribute to infertility and subfertility. That is why both the Endocrine Society [153] and the American Chemical Society [371] (with 161.000 chemical scientists and engineers as members, the world's largest scientific society) recently issued scientific statements on endocrine disruption. In their statements, these scientific societies recommend increased efforts at identifying and studying endocrine disruptors, expansion of education throughout the formal education structures in universities and schools, and better information to the public in general and to health care professionals and to chemists in particular. The Endocrine Society stresses the importance of the precautionary principle in the absence of direct information regarding cause and effect and considers the principle to be critical to enhancing reproductive and endocrine health. The American Chemical Society recommends more Green Chemistry research aimed at identifying and developing functional alternatives that do not have endocrine-disrupting activity. It remains, however, very difficult to determine which substances, at which point in time and at which concentrations, actually increase risk. Implementing the physical-chemical hygiene is in this context certainly indicated. Propylphenol p,p -DDE: 1, 1 -dichloro-2, 2 -bis(p-chlorophenyl) ethylene p,p -DDT: 1,1,1-trichloro-2,2-bis(pchlorophenyl) ethane PRL: