**1.5.3 Dioxins**

Tetrachlodibenzo-*p*-dioxin (TCDD) is a carcinogen, demonstrated to target the endocrine system in experimental animals. Humans are exposed to dioxins through pulp and paper industry emissions, use of contaminated herbicides (now reduced in industrialized countries), and waste incineration emissions. Dioxins are lipophilic, slowly metabolized, and thus are not easily eliminated leading to bioaccumulation. Secondary dioxin exposures include dietary uptake via contaminated breast milk, meat, fish, and other dairy. Dioxins along with polycyclic aromatic hydrocarbons (PAH) and polyhalogenated biphenyls bind to the aryl hydrocarbon receptor (AhR). AhR ligands induce cell proliferation, differentiation, and apoptosis, although the mechanisms of these stimulations are not fully understood. It is known that human sperm possess AhR and may therefore be directly susceptible to dioxin (Khorram et al., 2004). A range of endocrine effects are reported in experimental animals following dioxin exposure. These include disruption of the HPT axis feedback mechanisms leading to alterations in serum levels of testosterone, dihydrotestosterone (DHT), E2, and LH, as well as modifications of the metabolism processes/events of estrogens and androgens (Birnbaum & Tuomisto, 2000). There are few published studies reporting human exposure to dioxins. Agent Orange, which contains TCDD as its contaminant, was used during the Vietnam War and exposure was documented in veterans of Operation Ranch Hand (the unit responsible for aerial herbicide spraying in Vietnam from 1962 to 1971; (Stone, 2007). Detectable TCDD levels in serum and seminal plasma were evident in U.S. veterans two to three decades following their Vietnam military service. Reproductive parameters including serum testosterone, FSH, LH, and testicular abnormalities were not associated with serum TCDD levels in the exposed men. It may be possible that the effects of dioxin on reproductive parameters were no longer evident at the time of the study, several decades after the use of Agent Orange. As semen quality can only be evaluated by follow-up study, the acute effects of exposure to Agent Orange on male reproductive health are unknown. Another study of 101 men from the general population in Belgium assessed TCDD exposure and semen quality. TCDD exposure was measured as dioxin-like activity in serum. Increases in serum dioxin-like activity were associated with decreased seminal volume resulting in elevated sperm concentrations. Total testosterone levels were significantly reduced in men with high serum dioxin-like activity. However, there was no significant association with LH, inhibin B, FSH, total sperm numbers, or sperm morphology (Dhooge et al., 2006). There are few epidemiological studies evaluating reproductive outcomes and particularly semen quality following TCDD and dioxin exposure. However, it is more relevant to consider the effects of dioxin at environmental concentrations on male reproductive health. A possible mechanism for infertility may be mediated by dioxin interacting with AhR on human sperm with implications for capacitation, acrosome reaction, sperm–egg binding, and fertilization. More studies are required to examine the effects of dioxin on semen quality.

#### **1.5.4 Phthalates esters**

Phthalate esters are abundant industrial chemicals used in the production of plastics and are present in many personal care products including cosmetics. Phthalates are a family of

Further, more extensive correlation studies on the serum levels of DDT and metabolites in mothers of infertile men are required. These studies would provide insight into the effects of

Tetrachlodibenzo-*p*-dioxin (TCDD) is a carcinogen, demonstrated to target the endocrine system in experimental animals. Humans are exposed to dioxins through pulp and paper industry emissions, use of contaminated herbicides (now reduced in industrialized countries), and waste incineration emissions. Dioxins are lipophilic, slowly metabolized, and thus are not easily eliminated leading to bioaccumulation. Secondary dioxin exposures include dietary uptake via contaminated breast milk, meat, fish, and other dairy. Dioxins along with polycyclic aromatic hydrocarbons (PAH) and polyhalogenated biphenyls bind to the aryl hydrocarbon receptor (AhR). AhR ligands induce cell proliferation, differentiation, and apoptosis, although the mechanisms of these stimulations are not fully understood. It is known that human sperm possess AhR and may therefore be directly susceptible to dioxin (Khorram et al., 2004). A range of endocrine effects are reported in experimental animals following dioxin exposure. These include disruption of the HPT axis feedback mechanisms leading to alterations in serum levels of testosterone, dihydrotestosterone (DHT), E2, and LH, as well as modifications of the metabolism processes/events of estrogens and androgens (Birnbaum & Tuomisto, 2000). There are few published studies reporting human exposure to dioxins. Agent Orange, which contains TCDD as its contaminant, was used during the Vietnam War and exposure was documented in veterans of Operation Ranch Hand (the unit responsible for aerial herbicide spraying in Vietnam from 1962 to 1971; (Stone, 2007). Detectable TCDD levels in serum and seminal plasma were evident in U.S. veterans two to three decades following their Vietnam military service. Reproductive parameters including serum testosterone, FSH, LH, and testicular abnormalities were not associated with serum TCDD levels in the exposed men. It may be possible that the effects of dioxin on reproductive parameters were no longer evident at the time of the study, several decades after the use of Agent Orange. As semen quality can only be evaluated by follow-up study, the acute effects of exposure to Agent Orange on male reproductive health are unknown. Another study of 101 men from the general population in Belgium assessed TCDD exposure and semen quality. TCDD exposure was measured as dioxin-like activity in serum. Increases in serum dioxin-like activity were associated with decreased seminal volume resulting in elevated sperm concentrations. Total testosterone levels were significantly reduced in men with high serum dioxin-like activity. However, there was no significant association with LH, inhibin B, FSH, total sperm numbers, or sperm morphology (Dhooge et al., 2006). There are few epidemiological studies evaluating reproductive outcomes and particularly semen quality following TCDD and dioxin exposure. However, it is more relevant to consider the effects of dioxin at environmental concentrations on male reproductive health. A possible mechanism for infertility may be mediated by dioxin interacting with AhR on human sperm with implications for capacitation, acrosome reaction, sperm–egg binding, and fertilization. More studies are required to examine the

Phthalate esters are abundant industrial chemicals used in the production of plastics and are present in many personal care products including cosmetics. Phthalates are a family of

in utero exposure to DDT on semen quality of adult males.

**1.5.3 Dioxins** 

effects of dioxin on semen quality.

**1.5.4 Phthalates esters** 

compounds and only a few induce male reproductive tract abnormalities. Gray et al., *(*2000) compared the ability of six phthalate esters (diethylhexyl phthalate, DEHP; benzylbutyl phthalate, BBP; diisononyl phthalate, DINP; dimethyl phthalate, DMP; diethyl phthalate, DEP; dioctyl terephthalate, DOTP; all administered at 750 mg/kg body weight from GD14 to postnatal day (PND) 3) to induce malformations of the reproductive tract. This study assessed changes in many androgenic endpoints and found that only DEHP, BBP and to a lesser degree DINP induced alterations in all aspects of androgen-regulated male reproductive endpoints. Exposure to diethyl hexyl and dibutyl phthalates is associated with adverse effects on sperm motility (Fredricsson et al., 1993). Animal studies consistently demonstrated that phthalate esters are male reproductive toxicants (Park et al., 2002), with exposure associated with testicular atrophy, spermatogenetic cell loss, and damage to the Sertoli cell population. Phthalate monoesters target Sertoli cell functions in supporting the spermatogenesis process. This may be due to the effect of phthalates in reducing the ability of Sertoli cells to respond to FSH (Hauser et al., 2005). Initial reports on the effects of phthalates on male reproductive tract development focussed on the gross changes such as reduced anogenital distance, hypospadias, malformed epididymis and, in later studies, nipple retention (Mylchreest et al., 2000). Only a few studies give a more detailed account of the histological changes observed in the testis after in utero phthalate exposure and demonstrate that the fetal testis is directly affected by phthalates during fetal and neonatal testis differentiation (Parks et al., 2000; Fisher et al., 2003). Some of these alterations are permanent and affect the function of the testis in adult life and are similar to the histological changes which are now being shown in patients with testicur dysgenesis syndrome (TDS; Skakkebaek et al., 2003). The production of testosterone is critical for the normal masculinization of the male reproductive tract, as already discussed. It has been shown that DBP and DEHP are both capable of inhibiting the production of testosterone by the fetal testis (Parks et al., 2000; Fisher et al., 2003). Testosterone synthesis by the fetal testis is first detectable by GD15, reaches a peak at around GD18/19, and remains high until birth. However, phthalate treatment induces a 60–85% reduction in testosterone synthesis during this critical developmental window, reducing testosterone levels to a similar level to those found in females (Parks et al., 2000). This reduction in testosterone is a factor in the occurrence of hypospadias and cryptorchidism observed after phthalate treatment. This is not to suggest that phthalate exposure causes TDS in humans, merely that the administration of very high doses of DBP to pregnant rats induces a TDS-like syndrome in the male offspring that shows many analogous features to human TDS. It is plausible, given how highly conserved the pathways of fetal development are, that phthalate administration may disrupt some common mechanistic pathways which if altered in humans could be helpful in determining the pathogenesis of human TDS. In both the human syndrome and the rodent model, abnormal testicular development or dysgenesis is evident by the abnormal organisation of these tissues. In humans, histological evidence of testicular dysgenesis (immature seminiferous tubules with undifferentiated Sertoli cells, microcalcifications and Sertoli cell only (SCO) tubules, Leydig cell hyperplasia, morphologically distorted tubules and the presence of carcinoma *in situ* (CIS) cells) have been found in biopsies of the contralateral testes of testicular germ cell cancer patients and in biopsies from patients with infertility, hypospadias and cryptorchidism (Skakkebaek et al., 2003). These studies support the hypothesis that all of these disorders (low sperm counts,

Environmental Toxicants Induced

the male.

**1.5.6.1 Vinclozolin** 

**1.5.6 Pesticides, fungicides and herbicides** 

Male Reproductive Disorders: Identification and Mechanism of Action 485

The U.S Environmental Protection Agency (EPA) defines a pesticide as "any substance or mixture of substances intended for preventing, destroying, repelling, or lessening the damage of any pest," which may include plants, weeds, animals, insects, and fungus. Many epidemiological studies use the generic term *pesticides* to refer to a broad range of structurally unrelated compounds with different mechanisms of action, biological targets, and target pests. Epidemiological studies that evaluate the effects of these chemicals on male reproductive parameters often lack direct, quantitative measures of exposure. A study of participants from The Study for Future Families evaluated semen quality and pesticide exposures in male partners of pregnant women attending prenatal clinics in Missouri and Minnesota (Swan et al., 2003) Urinary levels of metabolites from the pesticides alachlor, diazinon, atrazine, and metolochlor were detected more often in men from Missouri, representative of the pesticides used in the agricultural practices of this state. Pesticide metabolites of chlorpyrifos/chlorpyrifos methyl (3,5,6-trichloropyridinol) and methyl parathion (4-nitrophenol) were detected more frequently in men from Minnesota. For the Missouri group, there was an association between low semen quality and urinary levels of chlorpyrifos and parathion metabolites. Further, increased levels of herbicides alachlor and metoachlor were associated with decreased sperm morphology and concentration. In contrast, there was no association between levels of any of these pesticides and their metabolites and semen parameters within the Minnesota group (Swan et al., 2003). A followup study focused on the men from Missouri, using a nested case-control design (cases: men with low semen parameters; controls: men with normal parameters). Urinary levels of metabolites of eight currently used pesticides were measured and correlated with semen quality. Men with elevated metabolite levels of alachlor and atrazine (herbicides) and diazinon (2-isopropoxy-4-methyl-pyrimidinol insecticide) were significantly more likely to have poor semen quality than controls (Swan, 2006). This study provide evidence that environmental exposures differ between regions, even within the same country. Different agricultural practices will create regional variation in the amounts and types of pesticides used, leading to differences in biological effects. A study in male infertility patients in Massachusetts measured urinary metabolites of carbaryl/naphthalene and chlorpyrifos. Sperm concentration, motility, and, to a lesser extent, morphology were reduced in men with elevated exposure to carbaryl/naphthalene (as measured by urinary levels of the metabolite 1-naphthol) and to chlorpyrifos (as measured by urinary levels of the metabolite 3,5,6-trichloro-2-pyridinol [TCPY]) (Meeker et al., 2004). The mechanism of action of carbaryl may be related to the production of reactive oxygen species (ROS) rather than endocrine disruption. Carbaryl produced lipid peroxidation at low concentrations, which in turn induced the sperm plasma membrane to lose its fluidity and integrity, thereby impairing sperm motility (Meeker et al., 2004). Generally, the studies reviewed here demonstrated a relationship between pesticide exposure and reduced semen quality. However, toxicology studies using animal models are essential to understand the biological mechanisms underlying the adverse reproductive affects caused by pesticide exposure in

Vinclozolin is a dicarboximide fungicide that has two active metabolites, M1 and M2, which have anti-androgenic properties. In vivo and in vitro experiments demonstrate that these compounds act as potent androgen receptor antagonists, and administration to pregnant

cryptorchidism, hypospadias and testicular cancer) are associated with TDS. The in utero administration of DBP to rodents during the sensitive period of tissue morphogenesis permanently alters the testis and produces foci of testicular dysgenesis (immature seminiferous tubules with undifferentiated Sertoli cells, SCO tubules, Leydig cell hyperplasia, morphologically distorted tubules and the presence of abnormal germ cells) which persist in the adult animal (Fisher et al., 2003). The downstream consequences of altered Sertoli cell (and subsequently Leydig cell) function may be a key cause of many of the observed changes in both human TDS and the rat TDS-like model due to the central role of this cell type in driving testis morphogenesis in both rodents and humans. Several population studies evaluated phthalate ester exposure and semen quality. A randomized controlled study of men with unexplained infertility reported a negative correlation between seminal plasma phthalate ester concentration and sperm morphology (Rozati et al., 2002). Environmental phthalate levels measured by urinary metabolite, were reported to be associated with increased DNA damage in sperm (Duty et al., 2003). The studies measuring phthalate levels and semen quality seem to suggest an effect on sperm morphology and motility, rather than on total sperm numbers. Hauser et al.,(2005) suggest a mechanism by which PCB exposure may extend the bioavailability of phthalate metabolites, which in turn adversely affect semen quality. As human exposure consists of phthalate mixtures, along with xenobiotics, studies designed to test or measure single phthalate esters fail to appropriately characterize risks associated with these chemicals.

#### **1.5.5 Phytoestrogens**

Phytoestrogens are nonsteroidal plant-derived compounds with potent estrogenic activity. There are four main groups of phytoestrogens: isoflavonoids, flavonoids, coumestans, and lignans. Phytoestrogens exert their action via multiple mechanisms. Phytoestrogens interact with both ERα and ERβ, thereby inducing weak estrogenic and antiestrogenic actions (Kuiper et al., 1998). Coumestrol and genistein, two phytoestrogens, exhibit a higher affinity for ERß than for ERα (Whitten & Naftolin, 1998). Some phytoestrogens exert an inhibitory action on steroidogenic enzymes (Strauss et al., 1998). For example, isoflavonoids and lignans inhibit 5α-reductase activity, thereby reducing the conversion of testosterone to the active form DHT. A number of phytoestrogens, including lignans, isoflavonoids daidzein and equol, enterolactone, and genistein, were found to induce SHBG production in the liver (Adlercreutz et al., 1987). There are few studies measuring the effects of phytoestrogens on semen parameters in men. The effects of short-term phytoestrogen supplementation on semen quality and endocrine function were examined in a group of young, healthy males. Subjects received 500 mg supplements containing 40 mg of phytoestrogens isoflavones genistein, daidzein, and glycitein daily for 2 months and donated semen and blood for 2 months before and 4 months after supplementation (Mitchell et al., 2001). Testicular volume was not influenced by phytoestrogen supplementation; nor did serum E2, testosterone, FSH, or LH differ between the supplement-taking group and the control group who did not take supplements. Finally, phytoestrogen supplementation did not produce changes in seminal volume, sperm concentration, sperm count, and sperm motility (Mitchell et al., 2001). A case report described therapeutic phytoestrogen supplementation (80 mg/day for 6 months) to an oligospermic man, which did sufficiently improve semen parameters such that intrauterine insemination was performed and the couple was able to conceive (Casini et al., 2006). To date, evidence linking dietary consumption of phytoestrogens and reduced semen quality is insufficient and requires further study.
