**1.5.6 Pesticides, fungicides and herbicides**

484 Toxicity and Drug Testing

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

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

appropriately characterize risks associated with these chemicals.

quality is insufficient and requires further study.

**1.5.5 Phytoestrogens** 

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 the male.

#### **1.5.6.1 Vinclozolin**

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

Environmental Toxicants Induced

**1.5.8.1 Antihypertensive** 

**1.5.8.2 Hormones** 

**1.5.8.3 Antiandrogens** 

**1.5.8.4 Antibiotics** 

**1.5.8 Medications and male reproductive toxicity** 

affect libido and erectile function (Benoff et al., 1994).

libido or erectile function (Nudell et al., 2002).

parameters (Overstreet et al., 1999).

Male Reproductive Disorders: Identification and Mechanism of Action 487

There are a variety of prescription medications that can lead to male infertility, often temporary but sometimes permanent. Arthritis medication, depression drugs, high blood pressure medication, drugs for digestive problems as well as antibiotics and cancer drugs are just a few of the medications that can lead to interferences with sperm production, sexual function and ejaculation (Nudell et al., 2002). Here is a look at some of the common

Although most men who are treated for hypertension are older, the recent focus on the importance of blood pressure control has led to greater numbers of younger patients on antihypertensives. Many of these medications are commonly associated with erectile dysfunction but most do not directly affect fertility. One exception is spironolactone, which acts as an anti-androgen and has been associated with impaired semen quality. Calcium channel blockers (e.g. nifedipine) have been reported to cause reversible functional defects in sperm, impairing their ability to fertilize eggs without affecting sperm production or standard semen analysis parameters; however, not all investigators report these types of effects. Diuretics can affect function by decreasing penile blood flow, and beta-blockers may

Diethylstilbestrol (DES) was given to pregnant women in the 1950s, and reports of epididymal cysts and cryptorchidism (undescended testes) in males with prenatal DES exposure have raised concerns about fertility; however, follow-up studies on adult men with prenatal DES exposure have revealed no adverse effects on fertility (Wilcox et al., 1995). Exogenous androgens are well known to induce hypogonadotropic hypogonadism. This may be induced directly by testosterone supplementation or by use of synthetic anabolic steroids, leading to azoospermia. This hypogonadism is usually reversible but may take 3 to 6 months, and some patients do not recover pituitary function. It is important to remember that testosterone replacement therapy in younger men may lead to infertility. Dehydroepiandrosterone (DHEA) is a natural steroid prohormone precursor of androsterone, testosterone, and estrogen. DHEA is commonly taken and easily available over the counter. Antiandrogens and estrogens can adversely affect fertility by altering the HPG axis or decreasing libido or erectile function, while progesterones act by decreasing

Finasteride and dutasteride are antiandrogens that act by inhibiting 5-alpha-reductase. Finasteride has also been used to treat male-pattern baldness. These drugs increase the risk of low ejaculate volumes and libido, as well as cause erectile and ejaculatory dysfunction; however, men taking low doses of finasteride for hair loss have shown no changes in semen

Many antibiotics have been reported to exert adverse effects on male fertility; however, there are few human data on the majority of these medications. High doses of nitrofurantoin have been reported to cause early maturation arrest at the primary spermatocyte stage but

medications and drugs that can cause a man to experience fertility problems.

rats results in abnormalities of androgen-regulated sexual differentiation similar to those induced by flutamide, e.g. reduced anogenital distance, nipple retention, hypospadias, undescended testes and small or absent accessory glands (Gray et al., 2001). Studies have tried to define the 'sensitive window' for exposure to vinclozolin, and have determined that administration to pregnant rats during gestational day (GD) 14–19 induced reproductive tract malformations, with treatment over GD16–17 causing the most severe malformations (Wolf et al., 2000). This illustrates that the whole period of male reproductive tract differentiation is sensitive to the effects of anti-androgens.

#### **1.5.6.2 Linuron**

Linuron is a urea-based herbicide which acts as a weak androgen receptor antagonist in vitro and in vivo, and disrupts androgen-dependent male reproductive tract development after gestational exposure (Gray et al., 2001). When administered to pregnant rats (GD 14– 18; 100 mg/ kg/day) the male pups displayed a reduced anogenital distance and retention of areolas (Gray et al., 1999). Linuron failed to induce either hypospadias or undescended testes, suggesting that linuron affects testosterone-but not DHT-mediated development, though how this occurs is not known (McIntyre et al., 2002).
