Currently, the potential effects of EDCs on human health and the proven effects of EDCs on wildlife are a major focus among the scientific community. It has been suggested that in utero exposure to environmental estrogens, antiandrogens, or chemicals like phthalates or 2,3,7,8-TCDD could be responsible for the reported 50 percent decline in sperm counts in some areas and the apparent increase in cryptorchid testes, testicular cancer, and hypospadias.
Phthalate exposures have been associated with reduced AGD in boys and lower testosterone levels in men. In females, exposure to EDCs during development could contribute to earlier age of puberty and to increased incidences of endometriosis and breast cancer. Besides pesticides and other toxic substances in the environment, many compounds that are phytosterols, estrogens, antibiotics, beta-blockers, antiepileptics, and lipid-regulating agents have significant endocrine-disrupting activity and are capable of inducing reproductive toxicity.
In the area of wildlife toxicology and ecosystem health, it is apparent that clear-cut cause and effect relationships exist between exposure to EDCs and adverse effects in several vertebrate classes from fish to mammals.
Known Effects of EDCs in Humans and Animals
The list of chemicals that are known to affect humans, domestic animals, and/or wildlife via functional developmental toxicity or endocrine mechanisms includes 2,3,7,8-TCDD, PCBs and polychlorinated dibenzofurans (PCDFs), methylmercury, ethinylestradiol, alkylphenols, plant sterols, fungal estrogens, androgens, chlordecone, DBCP, dichlorodiphenyltrichloroethane (DDT), and other organochlorine compounds. In addition to these xenobiotics, over 30 different drugs taken during pregnancy have been found to alter human development as a consequence of endocrine disruption. These drugs are not limited to estrogens, like diethylstilbestrol (DES). EDCs are known to alter human development via several mechanisms besides the estrogen receptor (ER), including binding to retinoic acid (RAR and RXR) receptors, and inhibiting synthesis of steroidogenic enzymes or thyroid hormones. Findings on the effects of background levels of PCBs on the neurobehavioral development of the child have contributed to the concerns about the effects of EDCs on human health via alteration of hormone function.
Human Sexual Differentiation
Exposure to hormonally active chemicals during sex differentiation can produce pseudohermaphroditism. Androgenic drugs like danazol and methyltestosterone can masculinize human females (i.e., “female pseudohermaphroditism”). The drug aminoglutethimide, which alters steroid hormone synthesis in a manner identical to many fungicides, also masculinizes human females following in utero exposure.
Transplacental exposure of the developing fetus to DES causes clear cell adenocarcinoma of the vagina, as well as gross structural abnormalities of the cervix, uterus, and fallopian tube. These DES-exposed women are more likely to have an adverse pregnancy outcome, including spontaneous abortions, ectopic pregnancies, and premature delivery. Some of the pathological effects that develop in males following fetal DES exposure appear to result from an inhibition of androgen action or synthesis (underdevelopment or absence of the vas deferens, epididymis, and seminal vesicles) and anti-Müllerian duct factor (persistence of the Müllerian ducts).
Known Effects of Plant and Fungal Products in Animals and Humans
Although most naturally occurring environmental estrogens are relatively inactive, the phytoestrogen miroestrol is almost as potent as estradiol in vitro and even more potent than estradiol when administered orally. In addition, many plant estrogens occur in such high concentrations that they induce reproductive alterations in domestic animals. “Clover disease,” which is characterized by dystocia, prolapse of the uterus, and infertility, is observed in sheep that graze on highly estrogenic clover pastures. Permanent infertility can be produced in ewes by much lower amounts of estrogen over a longer time period than are needed to produce “clover disease.”
Known Effects of Organochlorine Compounds in Humans
Several pesticides and toxic substances have been shown to alter human reproductive function. An accidental high-dose in utero exposure to PCBs and PCDFs has been associated with reproductive alterations in boys, increased stillbirths, low birth weights, malformations, and IQ and behavioral deficits. In addition to the effects associated with this inadvertent exposure, subtle adverse effects were seen in infants and children exposed to relatively low levels of PCBs and PCDFs.
One metabolite of DDT (mitotane, o,p′-DDD) was found to alter adrenal function with sufficient potency to be used as a drug to treat adrenal steroid hypersecretion associated with adrenal tumors. In addition, lower doses of mitotane restored menstruation in women with spanomenorrhea associated with hypertrichosis.
Occupational exposure to pesticides and other toxic substances (i.e., chlordecone and DBCP) in the workplace has been associated with reduced fertility, lowered sperm counts, and/or endocrine alterations in male workers. Workers exposed to high levels of chlordecone, an estrogenic and neurotoxic organochlorine pesticide, displayed intoxication, severe neurotoxicity, and abnormal testicular function. Male workers involved in the manufacture of 4,4′-diaminostilbene-2,2′-disulfonic acid (DAS), a key ingredient in the synthesis of dyes and fluorescent whitening agents, had lower serum testosterone levels and reduced libido as compared with control workers. Thus, it is surprising that occupational exposures to potential EDCs at effective concentrations have not been entirely eliminated from the workplace.
Androgenic activity has been detected in several complex environmental mixtures. Pulp and paper mill effluents (PME) include a chemical mixture that binds androgen receptors (AR) and induces androgen-dependent gene expression in vitro. This mode of action is consistent with the masculinized female mosquitofish (Gambusia holbrooki) collected from contaminated sites. Male-biased sex ratios of fish embryos have been reported in broods of eelpout (Zoarces viviparus) in the vicinity of a large kraft pulp mill on the Swedish Baltic coast, suggesting that masculinizing compounds in the effluent were affecting gonadal differentiation and skewing sex ratios. Effluents from beef-cattle concentrated animal feeding operations have been shown to display androgenicity.
Vinclozolin and procymidone are two members of the dicarboximide fungicide class that act as AR antagonists. These pesticides, or their metabolites, competitively inhibit the binding of androgens to AR, leading to an inhibition of androgen-dependent gene expression.
Administration of vinclozolin during sexual differentiation demasculinizes and feminizes the male rat offspring such that treated males display female-like AGD at birth, retained nipples, hypospadias, suprainguinal ectopic testes, a blind vaginal pouch, and small to absent sex accessory glands.
Procymidone induces shortening of the AGD in male pups, and older males display retained nipples, hypospadias, cryptorchidism, cleft phallus, a vaginal pouch, and reduced sex accessory gland size. Fibrosis, cellular infiltration, and epithelial hyperplasia are noted in the dorsolateral and ventral prostatic and seminal vesicular tissues in adult offspring.
This herbicide binds rat and human AR and inhibits DHT–hAR-induced gene expression in vitro. In utero linuron exposure produces male rats displaying epididymal and testicular abnormalities. Also, fetal testosterone production is significantly reduced in linuron-treated fetal males.
In utero, some phthalate esters alter the development of the male rat reproductive tract at relatively low dosages. Prenatal exposures to DBP, benzyl-butyl phthalate (BBP), di-isononyl phthalate (DINP), and diethylhexylphthalate (DEHP) cause a syndrome of effects, including underdevelopment and agenesis of the epididymis and other androgen-dependent tissues and testicular abnormalities. The phthalates are unique in their ability to induce agenesis of the gubernacular cords, a tissue whose development is dependent on the peptide hormone insulin-like peptide 3.
Methoxychlor is an estrogenic pesticide that produces estrogen-like effects. The active metabolites activate estrogen-dependent gene expression in vitro and in vivo in the female rats, thereby stimulating a uterotropic response, accelerating VO and inducing constant estrus, and reducing infertility. In the ovariectomized female rats, methoxychlor also induces estrogen-dependent reproductive and nonreproductive behaviors, including female sex behaviors, running wheel activity, and food consumption.
When given to the dam during pregnancy and lactation, both male and female offspring are affected. Females display irregular estrous cycles and reduced fecundity. Male fertility is unaffected at doses up to 200 mg/kg per day.
Ethinylestradiol is a synthetic derivative of estradiol that is in almost all modern formulations of combined oral contraceptive pills. This drug is found in many aquatic systems contaminated by sewage effluents, originating principally from human excretion. Thus, ethinylestradiol plays a major role in causing widespread endocrine disruption in wild populations of fish species and other lower vertebrate species.
The Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) proposed a tiered screening (tier 1) and testing (tier 2) strategy for EDCs. The recommended screening battery was designed to detect alterations of HPG function; estrogen, androgen, and thyroid hormone synthesis; and AR- and ER-mediated effects in mammals and other taxa.
EDSTAC recommended the laboratory rat as the species of choice for the endocrine screening and testing assays. The EDSTAC proposed three short-term in vivo mammalian assays for the tier 1 screening battery: the uterotropic, Hershberger, and pubertal female rat assays.
Estrogen agonists and antagonists are detected in a 3-day uterotropic assay using subcutaneous administration of the test compound. The selected uterotropic assays for estrogens and antiestrogens use either the intact juvenile or the castrated ovariectomized adult/juvenile female rat.
The second in vivo assay in tier 1, the Hersh-berger assay, detects antiandrogenic activity simply by weighing androgen-dependent tissues in the castrated male rat. In this assay, weights of the ventral prostate, Cowper's glands, seminal vesicle (with coagulating glands and fluids), glans penis, and levator ani/bulbocavernosus muscles are measured after 10 days of oral treatment with the test compound. This assay is very sensitive for detection of androgens and antiandrogens.
Pubertal Female Rat Assay
The third in vivo mammalian/rat assay in the screening battery is the pubertal female rat assay. Weanling female rats are dosed daily by gavage for 21 days while the age at VO (puberty) is monitored. The females are necropsied at about 42 days of age. This assay detects alterations in thyroid hormone status, HPG function, inhibition of steroidogenesis, estrogens, and antiestrogens, and has been found to be highly reproducible and very sensitive to certain endocrine activities including estrogenicity, inhibition of steroidogenesis, and antithyroid activity.