**3.3 Biomarkers of genetic damage**

500 Toxicity and Drug Testing

5. Sperm motility (% of motile and velocity), Sperm viability (Vital stain and

6. Sperm function assays (Acrosome reaction, Hemizona assay of sperm binding

7. Sperm genetic analysis (Sperm chromatin stability assay, Comet assay. Assessment of chromosomal aneuploidy and Nuclear microdeletions). 8. Marker chemicals from accessory glands (Epididymis is represented by glyceryl-

10. Personal reproductive history (Pubertal development, Paternity (Pregnancy

evaluating marker chemicals secreted by each respective gland (Schrader, 1997). For example, the epididymis is represented by glycerylphosphorylcholine, the seminal vesicles by fructose, and the prostate gland by zinc. Measures of semen pH and volume provide additional general information on the nature of seminal plasma, reflecting post testicular effects. A toxicant or its metabolite may act directly on accessory sex glands to alter the quantity or quality of their secretions. Alternatively, the toxicant may enter the seminal plasma and affect the sperm or may be carried to the site of fertilization by the sperm and affect the ova or conceptus. The presence of toxicants or their metabolites in seminal plasma can be analyzed using atomic absorption spectrophotometry or gas chromotography/mass spectrometry. Impact on the neuroendocrine system is another mechanism whereby toxicants can disturb the male reproductive system. To establish the extent of endocrine dysfunction, hormone levels can be measured in blood and urine. The profile recommended by NIOSH to evaluate endocrine dysfunction associated with reproductive toxicity consists of assessing serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and prolactin (Schrader, 1997). Because of the pulsatile secretion of LH, testosterone, and to a much lesser extent FSH, and the variability in the evaluation of reproductive hormones, it is recommended that three blood samples be drawn at set intervals in the early morning and the results pooled or averaged for clinical assessment. In epidemiologic field studies, however, multiple blood samples are impractical and may decrease participation rates. Alternatively, LH and FSH can be measured in urine, providing indices of gonadotropin levels that are relatively unaffected by pulsatile secretion. However, if an exposure can affect hepatic metabolism of sex steroid hormones (Apostoli et al., 1996), urinary measures of excreted testosterone metabolite (androsterone) or estradiol metabolite (estrone-3-glucuronide) are not recommended. Moreover, future assessment of reproductive hormones may extend to inhibin, activin, and follistatin, polypeptides that are

Table 2. Assessment of Male Reproductive Capacity in Humans (Moline et al., 2000).

phosphorylcholine, Seminal vesicles by fructose, and the Prostate gland by zinc).

timing and outcomes), Sexual functions (Erection, Ejaculation, Orgasm and

1. Testosterone (T) , Prolactin, LH, FSH, and Inhibin-B concentrations.

Sl. No. Methods of Assessment:

2. Semen volume and pH. 3. Sperm density/Sperm count.

4. Sperm morphology and morphometry

Hyper Osmotic Swelling (HOS)).

and sperm penetration assay).

9. Nocturnal penile measurements.

Libido)).

Biomarkers of chromosomal and genetic damage are increasingly used in the search to understand abnormal reproductive health outcomes, in part because of the possibility that there may be identifiable genetic polymorphisms which make an individual more susceptible to the adverse reproductive effects from exogenous substances. These assays provide promising and sensitive approaches for investigating germinal and potentially heritable effects of exposures to agents and for confirming epidemiologic observations on smaller numbers of individuals. Efficient technology for examining chromosomal abnormalities in sperm has only been developed recently. Chromosomal abnormalities are primarily of two types: numerical and structural. Both kinds can be attributed in some cases to paternal factors. Karyotype studies have shown that although oocytes demonstrate a higher frequency of numerical chromosomal abnormalities, human sperm demonstrate a higher frequency of structural abnormalities with less frequent numerical abnormalities (Moosani et al., 1995). In assessing sperm exposure to toxicants, it is therefore imperative to assess DNA structural integrity and not just chromosomal count. Aneuploidy is a chromosomal abnormality that causes pregnancy loss, perinatal death, congenital defects, and mental retardation. Aneuploidy, a disorder of chromosome count, is observed in approximately 1 in 300 newborns. It is speculated that of all species, humans experience the highest frequency of aneuploidy at conception, with estimates ranging from 20 to 50% (Moosani et al., 1995). Spontaneous abortions occur in at least 10-15% of all clinically recognized pregnancies. Of these, 35% contain chromosomal aneuploidy. Despite such a high frequency, there is little information about what causes this abnormality in humans. Paternal origins of aneuploidy and other genetic abnormalities can be analyzed by studying chromosome complements in human sperm. Two types of analyses provide data on chromosomal abnormalities in human sperm: sperm karyotype analysis and fluorescence *in situ* hybridization (FISH) (Moosani et al., 1995). Each technique has advantages and disadvantages. Sperm karyotyping is performed after sperm have fused with hamster oocytes. It provides precise information on numerical and gross structural abnormalities of all chromosomes from a given spermatozoon. However, only a limited number of sperm can be evaluated in each assay, and only those sperm that fertilize the oocytes are analyzable. Furthermore, this assay is technically difficult, labor intensive, expensive, and requires the use of animals. Also, it is better suited for clinical than for field studies because it must be performed on fresh semen. FISH, on the other hand, relies on the use of chromosomespecific probes to detect extra chromosomes (aneuploidy) or chromosome breaks or

Environmental Toxicants Induced

Sharpe & Irvine, BMJ, 2004).

Male Reproductive Disorders: Identification and Mechanism of Action 503

Fig. 4. Possible Pathways of endocrine disruption by environmental chemicals. DDE= 1, 1 dichloro-2, 2-bis (p-chlorophenyl) ethylene; DDT= dichlorodiphenyltrichloroethane; PAHs= polycyclic aromatic hydrocarbons; PCBs= polychlorinated biphenyls. (Modified from

(retinoid X receptor)-α and p21, and down-regulation of pRB, cyclin D, CDK2, cyclin E, and CDK4 (Ryu et al., 2007). Neonatal estrogen treatment (DES, ethinyl estradiol [EE]) increased apoptosis at all stages of spermatogenesis in the rat (Atanassova et al., 1999). Thus, apoptosis is a likely mechanism for some mechanisms of endocrine disruption in the testis (Jana et al., 2010b). Testicular androgenic signalling may be impaired via several mechanisms, including decreased Leydig cell population, impaired Leydig cell steroidogenesis, and dysregulation of the HPT axis. Abnormal development and maturation of the Leydig cell population reduces the steroidogenic potential of the testis. *In utero* exposure to phthalate esters is associated with morphological abnormalities of the male reproductive tract, including decreased anogenital distance, cryptorchidism, hypospadias, diminished Leydig cell population, and decreased testicular testosterone (Mylchreest et al., 2000; Fisher et al., 2003). Phthalate esters such as DEHP are proposed to exert antiandrogenic and estrogenic mechanisms of action and disrupt hormone synthesis via an AhR pathway (Ge et al., 2007). Low-dose BPA exposure in utero reduced the size of the epididymis and decreased anogenital distance and increased prostate size (Gupta, 2000) in adult mice. Leydig cell maturation and development include expression of genes related to endocrine signaling (LH receptors, AR) and steroidogenesis. Steroidogenesis is dependent

rearrangements in sperm. It is performed directly on sperm cells, eliminating the need for the use of animals. Although information is gained only for several chromosomes at a time, slides can be reprobed to increase the number of chromosomes evaluated. Furthermore, FISH can be conducted on archived sperm (either frozen or dried on slides), making it ideal for use in field studies. However, because the incidence of sperm aneuploidy is low, many cells (up to 10,000 per semen sample) must be evaluated, which requires significant scoring times. In comparison to karyotype analysis, however, FISH is relatively inexpensive and technically simpler, and data are obtained on all sperm, not just the ones that are capable of fertilization. These two techniques complement each other, with FISH providing information on large numbers of cells and karyotyping providing more precise and detailed information (Robbins et al., 1997).

#### **3.4 Develop biomarkers of exposures and male reproductive health for research and clinical use**

Resources must be invested in developing more advanced biomarkers of exposure to reproductive toxicants and of male reproductive health outcomes. Advanced biomarkers would allow for the development of toxicant-specific tests (e.g., polycyclic aromatic hydrocarbon-DNA adducts) and the detection of subclinical changes that might have significant health implications but which now go unnoticed by current measures. New biomarkers of semen quality are advantageous in that they can both describe male reproductive capacity and indicate toxic effects independent of the female partner's reproductive health. New tests could more accurately measure sperm function, fertilization potential, and the transmission of an intact male genome. Genetic testing may provide valuable tools for researchers and clinicians. For example, the sperm chromatin stability assay and FISH are used to assess genetic structure after exposure to a potential toxicant. Recently, single nucleotide polymorphisms have been used in the assessment of geneenvironment interactions.

#### **4. Mechanism of male reproductive toxicity**

The disruption of spermatogenesis may be represented by four mechanisms, including (1) epigenetic changes to the genome, (2) apoptosis of germ cells, (3) dysregulation of androgenic signaling, and (4) disruption of Sertoli and other spermatogenesis support (Phillips & Tanphaichitr, 2008) (**Figure 4**). The first mechanism is relatively novel and was only demonstrated in vitro by one group thus far. Rats exposed in vitro to anti-androgenic pesticides vinclozolin or methoxychlor demonstrated heritable changes in methylation status of genomic DNA. These epigenetic effects included impaired male fertility and were evident in the F3 and F4 generations (Anway et al., 2005). Reduced sperm number or altered sperm morphology may be indicative of problems during spermatogenesis and spermiogenesis and may be produced by the direct loss of developing spermatocytes. Adult rats exposed *in utero* to flutamide, an antiandrogen, exhibit hypo-spermatogenesis associated with increased apoptosis of adult germ cells (Maire et al., 2005). Anti-androgenic exposure is associated with elevation of pro-apoptotic molecules, including Fas-L (Maire et al., 2005), caspase-3 and caspase-6, Bax, Bak, and Bid, and a decrease in anti-apoptotic molecules Bcl2 and Bclw (Bozec et al., 2004) in rat models. Di(2-ethylhexyl) phthalate (DEHP) exposure in rats induces testicular apoptosis via a mechanism involving ERK1/2 induced up-regulation of PPAR (peroxisome proliferators-activated receptor) -γ, RXR

rearrangements in sperm. It is performed directly on sperm cells, eliminating the need for the use of animals. Although information is gained only for several chromosomes at a time, slides can be reprobed to increase the number of chromosomes evaluated. Furthermore, FISH can be conducted on archived sperm (either frozen or dried on slides), making it ideal for use in field studies. However, because the incidence of sperm aneuploidy is low, many cells (up to 10,000 per semen sample) must be evaluated, which requires significant scoring times. In comparison to karyotype analysis, however, FISH is relatively inexpensive and technically simpler, and data are obtained on all sperm, not just the ones that are capable of fertilization. These two techniques complement each other, with FISH providing information on large numbers of cells and karyotyping providing more precise and detailed

**3.4 Develop biomarkers of exposures and male reproductive health for research and** 

Resources must be invested in developing more advanced biomarkers of exposure to reproductive toxicants and of male reproductive health outcomes. Advanced biomarkers would allow for the development of toxicant-specific tests (e.g., polycyclic aromatic hydrocarbon-DNA adducts) and the detection of subclinical changes that might have significant health implications but which now go unnoticed by current measures. New biomarkers of semen quality are advantageous in that they can both describe male reproductive capacity and indicate toxic effects independent of the female partner's reproductive health. New tests could more accurately measure sperm function, fertilization potential, and the transmission of an intact male genome. Genetic testing may provide valuable tools for researchers and clinicians. For example, the sperm chromatin stability assay and FISH are used to assess genetic structure after exposure to a potential toxicant. Recently, single nucleotide polymorphisms have been used in the assessment of gene-

The disruption of spermatogenesis may be represented by four mechanisms, including (1) epigenetic changes to the genome, (2) apoptosis of germ cells, (3) dysregulation of androgenic signaling, and (4) disruption of Sertoli and other spermatogenesis support (Phillips & Tanphaichitr, 2008) (**Figure 4**). The first mechanism is relatively novel and was only demonstrated in vitro by one group thus far. Rats exposed in vitro to anti-androgenic pesticides vinclozolin or methoxychlor demonstrated heritable changes in methylation status of genomic DNA. These epigenetic effects included impaired male fertility and were evident in the F3 and F4 generations (Anway et al., 2005). Reduced sperm number or altered sperm morphology may be indicative of problems during spermatogenesis and spermiogenesis and may be produced by the direct loss of developing spermatocytes. Adult rats exposed *in utero* to flutamide, an antiandrogen, exhibit hypo-spermatogenesis associated with increased apoptosis of adult germ cells (Maire et al., 2005). Anti-androgenic exposure is associated with elevation of pro-apoptotic molecules, including Fas-L (Maire et al., 2005), caspase-3 and caspase-6, Bax, Bak, and Bid, and a decrease in anti-apoptotic molecules Bcl2 and Bclw (Bozec et al., 2004) in rat models. Di(2-ethylhexyl) phthalate (DEHP) exposure in rats induces testicular apoptosis via a mechanism involving ERK1/2 induced up-regulation of PPAR (peroxisome proliferators-activated receptor) -γ, RXR

information (Robbins et al., 1997).

environment interactions.

**4. Mechanism of male reproductive toxicity** 

**clinical use** 

Fig. 4. Possible Pathways of endocrine disruption by environmental chemicals. DDE= 1, 1 dichloro-2, 2-bis (p-chlorophenyl) ethylene; DDT= dichlorodiphenyltrichloroethane; PAHs= polycyclic aromatic hydrocarbons; PCBs= polychlorinated biphenyls. (Modified from Sharpe & Irvine, BMJ, 2004).

(retinoid X receptor)-α and p21, and down-regulation of pRB, cyclin D, CDK2, cyclin E, and CDK4 (Ryu et al., 2007). Neonatal estrogen treatment (DES, ethinyl estradiol [EE]) increased apoptosis at all stages of spermatogenesis in the rat (Atanassova et al., 1999). Thus, apoptosis is a likely mechanism for some mechanisms of endocrine disruption in the testis (Jana et al., 2010b). Testicular androgenic signalling may be impaired via several mechanisms, including decreased Leydig cell population, impaired Leydig cell steroidogenesis, and dysregulation of the HPT axis. Abnormal development and maturation of the Leydig cell population reduces the steroidogenic potential of the testis. *In utero* exposure to phthalate esters is associated with morphological abnormalities of the male reproductive tract, including decreased anogenital distance, cryptorchidism, hypospadias, diminished Leydig cell population, and decreased testicular testosterone (Mylchreest et al., 2000; Fisher et al., 2003). Phthalate esters such as DEHP are proposed to exert antiandrogenic and estrogenic mechanisms of action and disrupt hormone synthesis via an AhR pathway (Ge et al., 2007). Low-dose BPA exposure in utero reduced the size of the epididymis and decreased anogenital distance and increased prostate size (Gupta, 2000) in adult mice. Leydig cell maturation and development include expression of genes related to endocrine signaling (LH receptors, AR) and steroidogenesis. Steroidogenesis is dependent

Environmental Toxicants Induced

p-dioxin (Dhanabalan & Mathur, 2009).

Male Reproductive Disorders: Identification and Mechanism of Action 505

increased synthesis of sex hormone binding globulin (SHBG) and other plasma binding proteins (Haffner, 1996) and disruption of testicular androgen signaling by AhR ligands, PAH and nicotine, contained in tobacco smoke (Kizu et al., 2003) and many others. Further that, increasing evidence suggests an induction of oxidative stress in the testis represents another common response after exposure to environmental toxicants (Jana et al., 2010a & 2010b). Increase in oxidative stress can be seen in ≤80% of clinically proven infertile men, and exposure to environmental toxicants is a major factor contributing to such an increase (Tremellen, 2008). Environmental toxicants that have been shown to induce oxidative stress in the testis are highly heterogeneous, with different chemical structures, and include cadmium (Liu et al., 2009), bisphenol A (Kabuto et al., 2004) and 2,3,7,8-tetrachlorodibenzo-

Interestingly, these environmental toxicants commonly increase oxidative stress by down regulating the production of antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. In turn, excessive amounts of reactive oxygen species (ROS) are produced. ROS damage the lipids, proteins, carbohydrates and DNA in cells (Jana et al., 2010a & 2010b; **Figure 5**). Importantly, these observations were confirmed in studies illustrating that co-administration of antioxidants such as vitamin E with environmental toxicants could alleviate the pathophysiological effects (e.g. reduction in sperm count) of toxicants in the testis (Latchoumycandane & Mathur, 2002). These findings demonstrate that oxidative stress induced by environmental toxicants is one of the major contributing factors to male infertility. In fact, oxidative stress has long been linked to male infertility; although most studies have focused on its roles in causing abnormalities in germ cells and apoptosis (Sikka, 2001; Turner and Lysiak, 2008). Recent studies have shown that environmental toxicant-induced oxidative stress can cause male infertility by disrupting the cell junctions and adhesion between Sertoli–Sertoli cells and/or Sertoli–germ cells via the phosphatidylinositol 3-kinase (PI3K)/c-Src/focal adhesion kinase (FAK) signaling pathway (Wong & Cheng, 2011). Oxidative stress is known to increase epithelial and endothelial permeability by disrupting tight junctions (TJ) and adherens junctions (AJ) between cells (Sandoval and Witt, 2008). Activation of the PI3K/c-Src signalling pathway in response to oxidative stress induced by environmental toxicants could be a common mechanism by which the toxicants trigger damage to the testis. Early evidence shows that the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the testis are caused by an induction in c-Src kinase activity. Furthermore, significant increase in the c-Src level has also been detected in the testis after cadmium exposure in rodents, indicating that c-Src is activated in response to multiple environmental toxicants (Wong et al., 2004; Wong & Cheng, 2011) **(Figure 6).** The MAPK pathways have emerged as a common signaling platform for multiple environmental toxicants (**Figure 6**). Three MAPKs (extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38) have been shown to be activated in the testis after exposure to environmental toxicants. MAPKs are involved in regulating normal reproductive functions in the testis, which include spermatogenesis (e.g. cell-cycle progression, meiosis, BTB dynamics, cell adhesion dynamics and spermiogenesis), steroidogenesis, sperm hyperactivation and acrosome reaction (Almog & Naor 2010). As a result, unregulated activation of MAPKs by environmental toxicants imposes an array of pathophysiological effects on Sertoli cells, germ cells and Leydig cells in the testis. These include an increase in DNA damage and apoptosis, disruption of cell junctions and steroidogenesis (Li et al., 2009; Wong & Cheng, 2011). MAPKs are activated by oxidative

on availability of cholesterol to the cytochrome P-450 cholesterol side chain cleavage (P450scc) enzyme complex within the mitochondria, the rate-limiting and regulated step in steroidogenesis (Miller, 1988). Steroidogenic acute regulatory protein (StAR) is proposed as the candidate protein for the acute regulation of steroidogenesis. StAR transports cholesterol into the inner membrane, where steroidogenic enzymes catalyze consecutive reactions to convert cholesterol to testosterone in Leydig cells (Clark et al., 1994, Jana et al., 2008; 2010a; 2010b). *In utero* exposure to exogenous estrogens (EE, DES, genistein, and BPA)

downregulate expression of a number of testicular genes including *Cyp17, Cyp11a,* and *StAR* expression in the rat and mouse (Fielden et al., 2002). The expression of a number of genes related to steroidogenesis (*Scarb1*, *Star*, *Cyp11a1*, *HSD3b1*, and *CYP17a1*) was altered following in utero exposure to di(*n*-butyl) phthalate (Barlow et al., 2003). Testicular androgen signalling may also be impaired through suppression of normal HPT regulation of Leydig cell steroidogenesis. Disruption of the HPT axis, thereby reducing testicular testosterone levels, was demonstrated in the rat following exposure to a range of endocrine disrupters. Exposure to estrogens DES and EE also impaired HPT signalling in the rat, reducing plasma testosterone and increasing plasma FSH (Atanassova et al., 1999). Both Leydig and Sertoli cells contain the enzyme aromatase and convert androgens to estrogens, thereby providing an intratesticular source of estrogens (O'Donnell et al., 2001). Atrazine, a herbicide with antiandrogenic and estrogenic properties, was found to produce a number of adverse reproductive effects in the rat. Atrazine has a low affinity for androgen and estrogen receptors, reduces androgen synthesis, and enhances estrogen production via the induction of aromatase (Sanderson et al., 2000). Thus, testicular physiology is sensitive to perturbations of androgenic and estrogenic signalling, such that xenobiotic exposures might result in reduced fertility. Sertoli cell number is directly representative of the spermatogenic potential of the testis. Ablation of the Sertoli cell population, or loss of Sertoli cell function, is therefore another mechanism by which endocrine disrupters may impair spermatogenesis. Exposure to prenatal DES or EE reduced adult population of Sertoli cells in rats (Atanassova et al., 1999) and supports the hypothesis that *in utero* exposure to estrogens contributes to impaired spermatogenesis in the adult. It is also noteworthy that human spermatogenesis is much less efficient than in rodents, such that small decreases in the population of Sertoli cells would be expected to have large effects on male fertility in human. Estrogenic disruption of the testis is perhaps the most well characterized example of endocrine disruption of spermatogenesis; however, it is also worthy to note that estrogens play important roles in development of hormone responsive tissue, including Leydig cells and development and differentiation of the fetal male reproductive tract (Tsai-Morris et al., 1986). ERs, both alpha and beta (ERα and ERß), are found throughout the male reproductive tract and represent a transcriptional mechanism by which endocrine disrupters may alter gene expression. In rodents, ERα is expressed by all developmental stages of Leydig cells (fetal, neonatal/pubertal/adult), seminiferous tubules, efferent ductules, and epididymis but not Sertoli cells. ERß is expressed in all stages of rodent Leydig and Sertoli cell development and in efferent ductules and epididymis (O'Donnell et al., 2001). The existence of plasma membrane ERs along with the different tissue distribution, C-terminal ligandbinding domain and N-terminal transactivation domain of ERα and ERß provide possible explanations for the differential effects of so-called weak estrogens like BPA (Wozniak et al., 2005). These are but a few of the mechanisms by which endocrine disrupters might impair spermatogenesis. Other mechanisms include dysregulation of bioavailable androgens via

on availability of cholesterol to the cytochrome P-450 cholesterol side chain cleavage (P450scc) enzyme complex within the mitochondria, the rate-limiting and regulated step in steroidogenesis (Miller, 1988). Steroidogenic acute regulatory protein (StAR) is proposed as the candidate protein for the acute regulation of steroidogenesis. StAR transports cholesterol into the inner membrane, where steroidogenic enzymes catalyze consecutive reactions to convert cholesterol to testosterone in Leydig cells (Clark et al., 1994, Jana et al., 2008; 2010a;

downregulate expression of a number of testicular genes including *Cyp17, Cyp11a,* and *StAR* expression in the rat and mouse (Fielden et al., 2002). The expression of a number of genes related to steroidogenesis (*Scarb1*, *Star*, *Cyp11a1*, *HSD3b1*, and *CYP17a1*) was altered following in utero exposure to di(*n*-butyl) phthalate (Barlow et al., 2003). Testicular androgen signalling may also be impaired through suppression of normal HPT regulation of Leydig cell steroidogenesis. Disruption of the HPT axis, thereby reducing testicular testosterone levels, was demonstrated in the rat following exposure to a range of endocrine disrupters. Exposure to estrogens DES and EE also impaired HPT signalling in the rat, reducing plasma testosterone and increasing plasma FSH (Atanassova et al., 1999). Both Leydig and Sertoli cells contain the enzyme aromatase and convert androgens to estrogens, thereby providing an intratesticular source of estrogens (O'Donnell et al., 2001). Atrazine, a herbicide with antiandrogenic and estrogenic properties, was found to produce a number of adverse reproductive effects in the rat. Atrazine has a low affinity for androgen and estrogen receptors, reduces androgen synthesis, and enhances estrogen production via the induction of aromatase (Sanderson et al., 2000). Thus, testicular physiology is sensitive to perturbations of androgenic and estrogenic signalling, such that xenobiotic exposures might result in reduced fertility. Sertoli cell number is directly representative of the spermatogenic potential of the testis. Ablation of the Sertoli cell population, or loss of Sertoli cell function, is therefore another mechanism by which endocrine disrupters may impair spermatogenesis. Exposure to prenatal DES or EE reduced adult population of Sertoli cells in rats (Atanassova et al., 1999) and supports the hypothesis that *in utero* exposure to estrogens contributes to impaired spermatogenesis in the adult. It is also noteworthy that human spermatogenesis is much less efficient than in rodents, such that small decreases in the population of Sertoli cells would be expected to have large effects on male fertility in human. Estrogenic disruption of the testis is perhaps the most well characterized example of endocrine disruption of spermatogenesis; however, it is also worthy to note that estrogens play important roles in development of hormone responsive tissue, including Leydig cells and development and differentiation of the fetal male reproductive tract (Tsai-Morris et al., 1986). ERs, both alpha and beta (ERα and ERß), are found throughout the male reproductive tract and represent a transcriptional mechanism by which endocrine disrupters may alter gene expression. In rodents, ERα is expressed by all developmental stages of Leydig cells (fetal, neonatal/pubertal/adult), seminiferous tubules, efferent ductules, and epididymis but not Sertoli cells. ERß is expressed in all stages of rodent Leydig and Sertoli cell development and in efferent ductules and epididymis (O'Donnell et al., 2001). The existence of plasma membrane ERs along with the different tissue distribution, C-terminal ligandbinding domain and N-terminal transactivation domain of ERα and ERß provide possible explanations for the differential effects of so-called weak estrogens like BPA (Wozniak et al., 2005). These are but a few of the mechanisms by which endocrine disrupters might impair spermatogenesis. Other mechanisms include dysregulation of bioavailable androgens via

2010b). *In utero* exposure to exogenous estrogens (EE, DES, genistein, and BPA)

increased synthesis of sex hormone binding globulin (SHBG) and other plasma binding proteins (Haffner, 1996) and disruption of testicular androgen signaling by AhR ligands, PAH and nicotine, contained in tobacco smoke (Kizu et al., 2003) and many others. Further that, increasing evidence suggests an induction of oxidative stress in the testis represents another common response after exposure to environmental toxicants (Jana et al., 2010a & 2010b). Increase in oxidative stress can be seen in ≤80% of clinically proven infertile men, and exposure to environmental toxicants is a major factor contributing to such an increase (Tremellen, 2008). Environmental toxicants that have been shown to induce oxidative stress in the testis are highly heterogeneous, with different chemical structures, and include cadmium (Liu et al., 2009), bisphenol A (Kabuto et al., 2004) and 2,3,7,8-tetrachlorodibenzop-dioxin (Dhanabalan & Mathur, 2009).

Interestingly, these environmental toxicants commonly increase oxidative stress by down regulating the production of antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. In turn, excessive amounts of reactive oxygen species (ROS) are produced. ROS damage the lipids, proteins, carbohydrates and DNA in cells (Jana et al., 2010a & 2010b; **Figure 5**). Importantly, these observations were confirmed in studies illustrating that co-administration of antioxidants such as vitamin E with environmental toxicants could alleviate the pathophysiological effects (e.g. reduction in sperm count) of toxicants in the testis (Latchoumycandane & Mathur, 2002). These findings demonstrate that oxidative stress induced by environmental toxicants is one of the major contributing factors to male infertility. In fact, oxidative stress has long been linked to male infertility; although most studies have focused on its roles in causing abnormalities in germ cells and apoptosis (Sikka, 2001; Turner and Lysiak, 2008). Recent studies have shown that environmental toxicant-induced oxidative stress can cause male infertility by disrupting the cell junctions and adhesion between Sertoli–Sertoli cells and/or Sertoli–germ cells via the phosphatidylinositol 3-kinase (PI3K)/c-Src/focal adhesion kinase (FAK) signaling pathway (Wong & Cheng, 2011). Oxidative stress is known to increase epithelial and endothelial permeability by disrupting tight junctions (TJ) and adherens junctions (AJ) between cells (Sandoval and Witt, 2008). Activation of the PI3K/c-Src signalling pathway in response to oxidative stress induced by environmental toxicants could be a common mechanism by which the toxicants trigger damage to the testis. Early evidence shows that the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the testis are caused by an induction in c-Src kinase activity. Furthermore, significant increase in the c-Src level has also been detected in the testis after cadmium exposure in rodents, indicating that c-Src is activated in response to multiple environmental toxicants (Wong et al., 2004; Wong & Cheng, 2011) **(Figure 6).**

The MAPK pathways have emerged as a common signaling platform for multiple environmental toxicants (**Figure 6**). Three MAPKs (extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38) have been shown to be activated in the testis after exposure to environmental toxicants. MAPKs are involved in regulating normal reproductive functions in the testis, which include spermatogenesis (e.g. cell-cycle progression, meiosis, BTB dynamics, cell adhesion dynamics and spermiogenesis), steroidogenesis, sperm hyperactivation and acrosome reaction (Almog & Naor 2010). As a result, unregulated activation of MAPKs by environmental toxicants imposes an array of pathophysiological effects on Sertoli cells, germ cells and Leydig cells in the testis. These include an increase in DNA damage and apoptosis, disruption of cell junctions and steroidogenesis (Li et al., 2009; Wong & Cheng, 2011). MAPKs are activated by oxidative

Environmental Toxicants Induced

systemic toxicity in vivo.

Male Reproductive Disorders: Identification and Mechanism of Action 507

Although this type of study is inherently difficult to undertake, it is crucial for a full understanding of the impact of environmental toxicants on the reproductive system. However, much work is needed to understand the precise molecular events and mechanism(s) regulated by environmental toxicants to target c-Src, FAK, MAPK and polarity proteins in the testis. Only then can we identify specific phosphorylation targets or isoforms so that small-molecule agonists and/or antagonists can be designed to limit

Fig. 5. Primary pathologies of male reproductive system in connection with environmental

toxins, oxidative stress and infertility.

stress induced by environmental toxicants in cells and tissues. For example, blocking oxidative stress by free-radical scavengers (e.g. N-acetyl cysteine), reverses cadmiuminduced MAPK activation (Chen, et al., 2008). This phenomenon is partly regulated by the inhibition of Ser/Thr protein phosphatases 2A (PP2A) and 5 (PP5) by oxidative stress, which results in an increase in phosphorylation of MAPK (Chen, et al., 2008). In addition, activation of ERK can lead to phosphorylation of c-Src, FAK and paxillin under oxidative stress, implying that MAPKs might be one of the upstream targets to activate these nonreceptor tyrosine kinases (Li et al., 2009) **(Figure 6).** Activation of MAPKs by environmental toxicants also upregulates the expression of proinflammatory cytokines such as nuclear factor kB (NFkB), and tumor necrosis factor-α (TNFα) in macrophages and monocytes (Lecureur et al., 2005), which can diffuse from microvessels in the interstitial space and disrupt the BTB because they are known to perturb the Sertoli cell TJ-permeability barrier (Li et al., 2009). Similarly, cadmium and pollutants from motorcycle exhausts (e.g. polycyclic aromatic hydrocarbons) increase the expression of transforming growth factor-β (TGF-β) and interleukin-6 (IL-6) in the testis, respectively (Lui et al., 2009). TNFα, TGF-β and IL-1α are known to disrupt Sertoli–Sertoli and Sertoli–germ cell junctions via downregulation (Li et al., 2006) and/or redistribution of junctional proteins (Wong & Cheng, 2005) such as occludin, ZO-1 and N-cadherin in the seminiferous epithelium. Consequently, the loss of integral membrane proteins at the cell–cell interface causes disruption of the BTB and adhesion of germ cells in the seminiferous epithelium, which lead to the premature release of germ cells from the epithelium and hence infertility (Li et al., 2006; Li et al., 2009; Wong & Cheng, 2011). Furthermore, proinflammatory cytokines (e.g. IL-6 and TNFα) activate leukocytes to produce ROS, which amplifies the deleterious effects of environmental toxicant-induced oxidative stress (Tremellen, 2008). The male reproductive system has emerged as one of the major targets of environmental toxicants. Although acute exposure to toxicants contributes to apoptosis and the necrosis of testicular cells, chronic and sub-lethal exposure is prevailing in the general public (Hauser & Sokol 2008). Due to the unusually long half-lives of some of these toxicants in the mammalian body (e.g. cadmium has a mean half-life of >15 years), chronic and low-level exposure to humans could cause long-term unwanted health effects. The disruptive effects of environmental toxicants on cell junctions mediated by non-receptor tyrosine kinases (e.g. c-Src and FAK) and cytokines through oxidative stress because such damage is often observed in low-level exposure before apoptosis occurs (Li et al., 2006; Li et al., 2009). Significantly, these signalling pathways converge to utilize polarity proteins to regulate intercellular junctions. Polarity proteins (which are known to control cell adhesion in the testis) thus emerge as novel targets for therapeutic intervention to limit environmental toxicant-induced infertility. Although it is equally important to study the epigenetic (e.g. vinclozolin) and endocrine-disruptive (e.g. BPA, dioxin, cadmium) effects of environmental toxicants, it is increasingly clear that these toxicants are imposing an immediate deleterious effect in the testis via disruption of cell junctions between testicular cells due to increase in oxidative stress. In addition, endocrinedisrupting toxicants that affect estrogen levels might cause a disturbed balance of ROS and oxidative stress because estrogen is an important free-radical scavenger in humans, besides being essential for spermatogenesis (Carreau and Hess, 2010).

Recent studies have emphasized the importance of assessing the effects of a mixture of environmental toxicants on male reproductive function because humans are exposed to an array of chemicals that might antagonize or agonize each other (Hauser & Sokol 2008).

stress induced by environmental toxicants in cells and tissues. For example, blocking oxidative stress by free-radical scavengers (e.g. N-acetyl cysteine), reverses cadmiuminduced MAPK activation (Chen, et al., 2008). This phenomenon is partly regulated by the inhibition of Ser/Thr protein phosphatases 2A (PP2A) and 5 (PP5) by oxidative stress, which results in an increase in phosphorylation of MAPK (Chen, et al., 2008). In addition, activation of ERK can lead to phosphorylation of c-Src, FAK and paxillin under oxidative stress, implying that MAPKs might be one of the upstream targets to activate these nonreceptor tyrosine kinases (Li et al., 2009) **(Figure 6).** Activation of MAPKs by environmental toxicants also upregulates the expression of proinflammatory cytokines such as nuclear factor kB (NFkB), and tumor necrosis factor-α (TNFα) in macrophages and monocytes (Lecureur et al., 2005), which can diffuse from microvessels in the interstitial space and disrupt the BTB because they are known to perturb the Sertoli cell TJ-permeability barrier (Li et al., 2009). Similarly, cadmium and pollutants from motorcycle exhausts (e.g. polycyclic aromatic hydrocarbons) increase the expression of transforming growth factor-β (TGF-β) and interleukin-6 (IL-6) in the testis, respectively (Lui et al., 2009). TNFα, TGF-β and IL-1α are known to disrupt Sertoli–Sertoli and Sertoli–germ cell junctions via downregulation (Li et al., 2006) and/or redistribution of junctional proteins (Wong & Cheng, 2005) such as occludin, ZO-1 and N-cadherin in the seminiferous epithelium. Consequently, the loss of integral membrane proteins at the cell–cell interface causes disruption of the BTB and adhesion of germ cells in the seminiferous epithelium, which lead to the premature release of germ cells from the epithelium and hence infertility (Li et al., 2006; Li et al., 2009; Wong & Cheng, 2011). Furthermore, proinflammatory cytokines (e.g. IL-6 and TNFα) activate leukocytes to produce ROS, which amplifies the deleterious effects of environmental toxicant-induced oxidative stress (Tremellen, 2008). The male reproductive system has emerged as one of the major targets of environmental toxicants. Although acute exposure to toxicants contributes to apoptosis and the necrosis of testicular cells, chronic and sub-lethal exposure is prevailing in the general public (Hauser & Sokol 2008). Due to the unusually long half-lives of some of these toxicants in the mammalian body (e.g. cadmium has a mean half-life of >15 years), chronic and low-level exposure to humans could cause long-term unwanted health effects. The disruptive effects of environmental toxicants on cell junctions mediated by non-receptor tyrosine kinases (e.g. c-Src and FAK) and cytokines through oxidative stress because such damage is often observed in low-level exposure before apoptosis occurs (Li et al., 2006; Li et al., 2009). Significantly, these signalling pathways converge to utilize polarity proteins to regulate intercellular junctions. Polarity proteins (which are known to control cell adhesion in the testis) thus emerge as novel targets for therapeutic intervention to limit environmental toxicant-induced infertility. Although it is equally important to study the epigenetic (e.g. vinclozolin) and endocrine-disruptive (e.g. BPA, dioxin, cadmium) effects of environmental toxicants, it is increasingly clear that these toxicants are imposing an immediate deleterious effect in the testis via disruption of cell junctions between testicular cells due to increase in oxidative stress. In addition, endocrinedisrupting toxicants that affect estrogen levels might cause a disturbed balance of ROS and oxidative stress because estrogen is an important free-radical scavenger in humans, besides

being essential for spermatogenesis (Carreau and Hess, 2010).

Recent studies have emphasized the importance of assessing the effects of a mixture of environmental toxicants on male reproductive function because humans are exposed to an array of chemicals that might antagonize or agonize each other (Hauser & Sokol 2008). Although this type of study is inherently difficult to undertake, it is crucial for a full understanding of the impact of environmental toxicants on the reproductive system. However, much work is needed to understand the precise molecular events and mechanism(s) regulated by environmental toxicants to target c-Src, FAK, MAPK and polarity proteins in the testis. Only then can we identify specific phosphorylation targets or isoforms so that small-molecule agonists and/or antagonists can be designed to limit systemic toxicity in vivo.

Fig. 5. Primary pathologies of male reproductive system in connection with environmental toxins, oxidative stress and infertility.

Environmental Toxicants Induced

effects on male reproductive health.

pp.1135–1144.

*Endocrinology*, Vol 3: pp. 51.

308, pp. 1466–1469.

Vol. 46, pp.204-208.

**6. Acknowledgment** 

**7. References** 

Male Reproductive Disorders: Identification and Mechanism of Action 509

definition of EDCs. It is now accepted that there are a plethora of ways in which the environmental chemicals can potentially act on the endocrine as well as male reproductive systems. Though supportive data must need to determine whether human male reproductive health is declining or not. However; the hypothesis of a 'testicular dysgenesis syndrome' is an important advancement and may aid our understanding of the underlying aetiology of these disorders. Within the reproductive tract, the male is exquisitely vulnerable to the effects of anti-androgens during development due the dependence on the synthesis and action of androgens for the masculinization of the male reproductive tract. The ability of phthalates to suppress androgen synthesis during development and to induce testicular dysgenesis together with cryptorchidism and hypospadias has close parallels with human TDS. However, the crucial question regarding whether the level of environmental chemicals is sufficient to impact on human male reproductive health remains unanswered, although advances will be made from studying the effects of multi-component EDC mixtures in both in vitro and in vivo test systems. Moreover, it has been observed that in wildlife, there is a increasing rates of testicular cancer, to the debate regarding trends in sperm counts, there has been increasing concern that hazardous substances in the environment adversely affect male reproductive health. The ultimate benefits of this chapter that should serve as a framework for future studies to improve our knowledge in this area. By better defining the problems, learning about the mechanisms responsible for adverse effects, and developing panels of relevant biomarkers, we will make progress toward preventing future adverse

The authors acknowledge the financial support provided by the Bose Institute, Kolkata.

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Fig. 6. Molecular signalling pathways of testicular toxicity by environmental toxicants through the induction of oxidative stress. Oxidative stress induced by environmental toxicants activates the PI3K/ C-src/FAK pathway, which subsequently controls the phosphorylation of TJ and/or AJ proteins. This leads to the internalization of TJ and AJ proteins at the cell–cell interface. In addition, environmental toxicants induce the production of cytokines which are also regulated by the activation of MAPK through oxidative stress. Cytokines stimulate the production of reactive oxygen species (ROS) from leukocytes to further increase oxidative stress. Cytokines and the activation of MAPK together result in endocytic vesicle- mediated internalization of TJ and AJ proteins. Polarity proteins such as Par6 are also involved in mediating the action of cytokines to recruit the E3 ubiquitin ligase Smurf1 for the poly ubiquitination and degradation of RhoA, which is important for the disruption of cell junctions. This illustrates that crosstalk exists between the PI3K/C-Src/FAK and cytokines/ MAPK pathways via polarity proteins as their common downstream signalling mediators. The disruption of cell junctions ultimately leads to the germ cell apoptosis and necrosis and as a result sperm count and quality of semen are reduced. (Modified from Wong & Cheng, Trends in Pharmacological Sciences, 2011).
