**3. Potential mechanisms for idiopathic male infertility**

Male infertility is a complex, multifactorial disorder and often its etiology remains poorly understood. Increasing volume of data suggests that oxidative

**87**

**Figure 1.**

*resistance-associated protein.*

scientific society [10].

*Polymorphism of Xenobiotic Detoxification Genes and Male Infertility*

stress is the most probable cause of idiopathic male infertility. Oxidative stress is mediated by reactive oxygen species (ROS), if their level exceeds antioxidant capacity of the organism. An increased ROS level is observed in 40–80% of infertile men and in 11–78.5% of infertile men with normal sperm count [8]. Elevated ROS levels can be explained by several reasons such as increased leukocytes' activity due to inflammation in the genital tract, varicocele or presence of immature spermatozoa as well as external causes like exposure to noxious chemical compounds, radiation and lifestyle factors [9]. A small amount of ROS is necessary in some physiological processes such as capacitation [10], while excessive ROS may damage sperms. Spermatozoa have restricted volume of cytoplasm, therefore the quantity of ROS-metabolizing enzymes is also limited and make them more vulnerable to ROS compared to other cells [9]. ROS cause sperm damages in several ways: first, ROS are capable of interacting with the sperm plasma membrane, which is rich in polyunsaturated fatty acids, promoting the decrease of its flexibility and, hence, tail motility [11]. Second, ROS may lead to the sperm's acrosome membrane peroxidation and decline in acrosin activity, thus making fertilization less probable [12]. Lastly, an increased ROS level is associated with the increase in sperm DNA fragmentation (that is used as an assessment tool for unexplained male infertility) and diminished sperm motility [13]. Moreover, considering that ROS are intensively produced in the mitochondria of stressed spermatozoids, they may cause mutations of mitochondrial DNA of different cells which participate in spermatogenesis. As a result, sperm maturation and functioning could be violated. Thus, the development of effective antioxidant treatment and antistress strategies has become one of the paramount tasks for the

*The three-step detoxification of xenobiotics. Lipophilic substances subjected to metabolic activation by phase I enzyme cytochrome P450 (CYP) acquire reactive center for phase II conjugation reactions that lead to formation of hydrophilic compounds, following elimination by phase III transporters or passive transport. Most of the noxious effects are caused by reactive electrophiles after phase I activation, but some xenobiotics pose a threat before metabolic activation. CYP—cytochrome P450; NATs—N-acetyltransferases; GSTs—glutathione S-transferases; SULT—sulfotransferase; UGT—UDP-glucuronosyltransferase; NQO—NAD(P)H quinone oxidoreductase; OATP2—organic anion transporting polypeptide 2; P-gp— P-glycoprotein; MRP—multidrug* 

*DOI: http://dx.doi.org/10.5772/intechopen.79233*

### *Polymorphism of Xenobiotic Detoxification Genes and Male Infertility DOI: http://dx.doi.org/10.5772/intechopen.79233*

### **Figure 1.**

*Male Reproductive Health*

detoxifying enzymes.

**2. Xenobiotic metabolism**

and drugs in human [6].

and tissue specificities [7].

susceptibility to chemically induced damages. Thus, xenobiotic-mediated adverse effects of male reproductive system are associated with the polymorphisms in xenobiotic detoxification genes. Different variants of one gene are categorized as polymorphism if their frequency in the population exceeds 1% [5]. In contrast to mutations, polymorphisms have indirect connections with particular seminal fluid abnormalities but may be indispensable during the investigation of multifactorial diseases. Polymorphic variants may change the expression or function of encoded protein, leading to its conformational changes that would result in different pharmacokinetics, chemical reaction capacities and efficiency of the xenobioticdetoxifying enzymes. In this chapter, we discuss polymorphisms in the main enzymes capable of maintaining the oxidants/antioxidants balance in reproductive tissues, focusing mainly on phase I cytochrome P4501A1 (CYP1A1) and phase II (GSTM1, GSTT1, GSTP1 and arylamine N-acetyltransferase 2 (NAT2))

The foreign environmental chemicals are known as xenobiotics (Gk. *xenos*— "stranger") and include drugs, drug metabolites and environmental pollutants (such as synthetic pesticides, herbicides and industrial pollutants). Xenobiotics may cause damages in the innate state (alkyl iodides, acyl halides and nitrogen

1.Phase I enzymes initiate the detoxification process, during which the lipophilic xenobiotics become more polar and acquire sites for subsequent conjugation reactions. Phase I enzymes comprise mainly the cytochrome P450 (CYP) superfamily of microsomal enzymes, which include the 36 gene families. It is considered that CYP1, CYP2, CYP3, CYP4 and CYP7 families play the key roles in hepatic and extra-hepatic metabolism and in the elimination of xenobiotics

2.Phase II enzymes catalyze the conjugation process. These enzymes can interact with xenobiotics either directly or, more generally, interact with the metabolites produced by phase I enzymes. Phase II enzymes include a lot of superfamilies, namely, the sulfotransferases (SULT), UDP-glucuronosyltransferases (UGT), DT-diaphorase or NAD(P)H quinone oxidoreductase (NQO) or NAD(P)H menadione reductase (NMO), glutathione S-transferases (GST) and N-acetyltransferases (NAT). Each superfamily consists of families and subfamilies of genes encoding the various isoforms with different substrate

3.Phase III elimination via transporters or passive transport. Phase III transporters involve P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), which belongs to the subfamily of the ATP binding cassette (ABC) transporters and organic anion transporting polypeptide 2 (OATP2).

Male infertility is a complex, multifactorial disorder and often its etiology remains poorly understood. Increasing volume of data suggests that oxidative

**3. Potential mechanisms for idiopathic male infertility**

mustards) or after activation in the metabolizing process.

Xenobiotic metabolism is performed in three stages (**Figure 1**):

**86**

*The three-step detoxification of xenobiotics. Lipophilic substances subjected to metabolic activation by phase I enzyme cytochrome P450 (CYP) acquire reactive center for phase II conjugation reactions that lead to formation of hydrophilic compounds, following elimination by phase III transporters or passive transport. Most of the noxious effects are caused by reactive electrophiles after phase I activation, but some xenobiotics pose a threat before metabolic activation. CYP—cytochrome P450; NATs—N-acetyltransferases; GSTs—glutathione S-transferases; SULT—sulfotransferase; UGT—UDP-glucuronosyltransferase; NQO—NAD(P)H quinone oxidoreductase; OATP2—organic anion transporting polypeptide 2; P-gp— P-glycoprotein; MRP—multidrug resistance-associated protein.*

stress is the most probable cause of idiopathic male infertility. Oxidative stress is mediated by reactive oxygen species (ROS), if their level exceeds antioxidant capacity of the organism. An increased ROS level is observed in 40–80% of infertile men and in 11–78.5% of infertile men with normal sperm count [8]. Elevated ROS levels can be explained by several reasons such as increased leukocytes' activity due to inflammation in the genital tract, varicocele or presence of immature spermatozoa as well as external causes like exposure to noxious chemical compounds, radiation and lifestyle factors [9]. A small amount of ROS is necessary in some physiological processes such as capacitation [10], while excessive ROS may damage sperms. Spermatozoa have restricted volume of cytoplasm, therefore the quantity of ROS-metabolizing enzymes is also limited and make them more vulnerable to ROS compared to other cells [9]. ROS cause sperm damages in several ways: first, ROS are capable of interacting with the sperm plasma membrane, which is rich in polyunsaturated fatty acids, promoting the decrease of its flexibility and, hence, tail motility [11]. Second, ROS may lead to the sperm's acrosome membrane peroxidation and decline in acrosin activity, thus making fertilization less probable [12]. Lastly, an increased ROS level is associated with the increase in sperm DNA fragmentation (that is used as an assessment tool for unexplained male infertility) and diminished sperm motility [13]. Moreover, considering that ROS are intensively produced in the mitochondria of stressed spermatozoids, they may cause mutations of mitochondrial DNA of different cells which participate in spermatogenesis. As a result, sperm maturation and functioning could be violated. Thus, the development of effective antioxidant treatment and antistress strategies has become one of the paramount tasks for the scientific society [10].

Mutations (that include both chromosomal and single-gene alterations) in several hundred genes are the other important and significant factor that potentially may lead to male infertility. For instance, Y-chromosome microdeletions (YCM), which influence genes responsible for spermatogenesis, are one of the best-studied types of mutations that may cause male infertility. Thus, recent meta-analysis has shown that oligospermic men with sperm concentration > 1 million/mL had significantly higher rates of YCM compared to normospermic men [14]. Among the other examples of mutations, there are the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene mutations, which lead to the absence of vas deferens; deletions of the autosomal Doublesex and Mab-3 Related Transcription factor 1 (DMRT1) gene in the azoospermic men (which is critical for germ cells development), ranging in size from 54 kb to over 2 Mb; and alterations in autosomal PLEC1 (plectin) and microRNA 661 (MIR661) genes in an azoospermic and oligozoospermic men and various single-nucleotide polymorphisms (SNPs) [15]. The latter is one of the most promising candidates for the elucidation of "hidden" genetic factors responsible for idiopathic male infertility. For example, rs1801133 (677C > T) variant in the methylenetetrahydrof>olate reductase (MTHFR) (NAD (P) H) gene found in males with impaired spermatogenesis [16], rs4986938 (1406 + 1872G > A) polymorphism of estrogen receptor 2 (ESR2) gene, rs2070744 (786C > T in the promoter region) and rs61722009 (27 bp Variable Number Tandem Repeat (VNTR) polymorphisms in the intron 4, also known as 4a4b polymorphisms) variants of NOS3 (nitric oxide synthase 3) are the potential predisposal factors of male infertility [15].

Still, male infertility depends not only on mutations alone but also on the complex system of gene–environmental interactions and epigenetic factors. A bright example of environmental factors, which may cause seminal fluid abnormalities in the corresponding genetic background, is xenobiotics, which is discussed in the following sections.

### **4. Association of xenobiotics with male infertility**

Xenobiotics may interact with macromolecules in reversible (forming noncovalent bindings, for example, ion pairing, hydrogen bonding, hydrophobic interactions, etc.) or irreversible ways through covalent bond formation between electrophilic xenobiotics or their active metabolites and endogenous molecules. The first variant may lead to the alteration in enzyme and transporter activity, ion channels blockade, activation of specific receptors and violation in DNA transcription, if the specific structure of the xenobiotic fits to macromolecule-binding sites. Irreversible covalent interaction does not demand such fitness of structures and leads to the formation of endogenous adducts of nucleophiles (such as nucleophilic amino acid or nucleic acids). As a result, such reactions may cause genetic mutations, carcinogenesis (if the violated gene participates in regulation of cell reproduction and differentiation) and protein malfunctions, which consequently may promote cell death and tissue toxicity.

It is worth mentioning that xenobiotics injure the male reproductive system not only by themselves and their metabolites but also via defensive reactions of the organism, such as innate and specific acquired immune responses. Thus, locally available reactive metabolites may cause disruption of testis immunoprivilege through the immunocompetent cells activation. This would lead to organ-specific immunopathology. Xenobiotics may have a toxic effect due to the malfunction of both the first and the second phase enzymes because of their hyperactive or inhibited functioning. In the case of increased phase I enzyme activity, or decreased

**89**

*Polymorphism of Xenobiotic Detoxification Genes and Male Infertility*

phase II enzyme activity, electrophilic intermediates of the xenobiotic metabolism are accumulated in the cells and mediate the abovementioned damages. At the same time, the reduced activity of the first phase enzymes leads to metabolism retardation and accumulation of noxious chemical compounds in different tissues of the organism, including in those of the reproductive system. For example, decreased activity of GST (phase II enzyme that transfers glutathione (GSH) to activated environmental chemicals) will probably lead to the formation of reactive intermediates that will mediate cell damage mechanisms. On the other hand, phase I enzyme CYP1A1 is able to metabolize the polycyclic aromatic hydrocarbons (PAHs) to intermediate substances, which prompt genotoxic and mutagenic effects before they are further detoxified by phase II enzymes. This indicates that the increased activity of CYP1A1 may contribute to the accumulation of these compounds in the organism and to the elevation of the PAH-DNA adducts level. Although the liver performs the main functions of detoxification, reactive metabolites can also be generated in a particular organ, mediating organ specific toxicity. For example, experiments on laboratory animals such as rats and mice showed the presence of both phase I and phase II enzymes in their testicles. [17, 18]. The possibility of PAHs (benzo(a)pyrene) detoxification was also shown on the rats' Leydig cells [19]. The xenobiotic-detoxification enzymes are much needed in the reproductive system, since some endocrine-deteriorating agents (phthalates, dioxins, polychlorinated biphenyls (PCBs) and pesticides) have been shown to exert a negative influence on the reproductive tissues [20]. Thus, stable and lipophilic chlorinated hydrocarbons are capable of penetrating into the male reproductive tract, promoting idiopathic sterility and other reproductive impairments. One of the well-studied examples of male testicular toxins is occupational xenobiotic nematocide 1,2-dibromo 3-chloropropane (DBCP) that causes a partially reversible damage to the seminiferous epithelium, leading to diminished sperm counts and sterility [21]. Another occupational and environmental toxin dichlorodiphenyldichloroethylene also reduces

*DOI: http://dx.doi.org/10.5772/intechopen.79233*

sperm count and mediates male infertility [22].

**5. Polymorphism of genes that affect spermatogenesis**

Except xenobiotic detoxification gene polymorphisms, another target group of genes that are most probably involved in development of male infertility is genes take part in spermatogenesis. This group includes genes with different functions such as endocrine regulation of spermatogenesis (i.e., androgen receptor (AR), follicle-stimulating hormone receptor (FSHR) and Estrogen receptor α and β), specific spermatogenic functions (i.e., deleted azoospermia-like (DAZL), Ubiquitin carboxyl-terminal hydrolase 26 (USP26), protamine-1 (PRM1) and gonadotrophin-regulated testicular helicase (GRTH)), regulation of cell functions such as metabolism, cell cycle and mutation repair (i.e., mtDNA polymerase γ (POLG) and Methylene tetrahydrofolate reductase (MTHFR)) and Y-chromosome haplogroups. Y-chromosome abnormalities are the best studied, while data about association of male infertility with the other spermatogenesis regulation genes remain contradictory. For example, the spermatogenesis locus azoospermia factor c (AZFc) region partial deletion (gr/gr deletions, which include DAZ (Deleted in azoospermia) genes) is regarded as classical Y-chromosome mutation. Its role in male infertility was proven by several studies, although it is present in normospermic men as well [16, 23]. Here are some examples of the other probable risk factors obtained by meta-analysis [23]. One of gene candidates, which is regarded as the most probable risk factor of male infertility, is MTHFR, which encodes methylenetetrahydrofolate reductase—one of the key enzymes in folate metabolism. The C → T substitution in

### *Polymorphism of Xenobiotic Detoxification Genes and Male Infertility DOI: http://dx.doi.org/10.5772/intechopen.79233*

*Male Reproductive Health*

infertility [15].

following sections.

Mutations (that include both chromosomal and single-gene alterations) in several hundred genes are the other important and significant factor that potentially may lead to male infertility. For instance, Y-chromosome microdeletions (YCM), which influence genes responsible for spermatogenesis, are one of the best-studied types of mutations that may cause male infertility. Thus, recent meta-analysis has shown that oligospermic men with sperm concentration > 1 million/mL had significantly higher rates of YCM compared to normospermic men [14]. Among the other examples of mutations, there are the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene mutations, which lead to the absence of vas deferens; deletions of the autosomal Doublesex and Mab-3 Related Transcription factor 1 (DMRT1) gene in the azoospermic men (which is critical for germ cells development), ranging in size from 54 kb to over 2 Mb; and alterations in autosomal PLEC1 (plectin) and microRNA 661 (MIR661) genes in an azoospermic and oligozoospermic men and various single-nucleotide polymorphisms (SNPs) [15]. The latter is one of the most promising candidates for the elucidation of "hidden" genetic factors responsible for idiopathic male infertility. For example, rs1801133 (677C > T) variant in the methylenetetrahydrof>olate reductase (MTHFR) (NAD (P) H) gene found in males with impaired spermatogenesis [16], rs4986938 (1406 + 1872G > A) polymorphism of estrogen receptor 2 (ESR2) gene, rs2070744 (786C > T in the promoter region) and rs61722009 (27 bp Variable Number Tandem Repeat (VNTR) polymorphisms in the intron 4, also known as 4a4b polymorphisms) variants of NOS3 (nitric oxide synthase 3) are the potential predisposal factors of male

Still, male infertility depends not only on mutations alone but also on the complex system of gene–environmental interactions and epigenetic factors. A bright example of environmental factors, which may cause seminal fluid abnormalities in the corresponding genetic background, is xenobiotics, which is discussed in the

Xenobiotics may interact with macromolecules in reversible (forming noncovalent bindings, for example, ion pairing, hydrogen bonding, hydrophobic interactions, etc.) or irreversible ways through covalent bond formation between electrophilic xenobiotics or their active metabolites and endogenous molecules. The first variant may lead to the alteration in enzyme and transporter activity, ion channels blockade, activation of specific receptors and violation in DNA transcription, if the specific structure of the xenobiotic fits to macromolecule-binding sites. Irreversible covalent interaction does not demand such fitness of structures and leads to the formation of endogenous adducts of nucleophiles (such as nucleophilic amino acid or nucleic acids). As a result, such reactions may cause genetic mutations, carcinogenesis (if the violated gene participates in regulation of cell reproduction and differentiation) and protein malfunctions, which consequently may

It is worth mentioning that xenobiotics injure the male reproductive system not only by themselves and their metabolites but also via defensive reactions of the organism, such as innate and specific acquired immune responses. Thus, locally available reactive metabolites may cause disruption of testis immunoprivilege through the immunocompetent cells activation. This would lead to organ-specific immunopathology. Xenobiotics may have a toxic effect due to the malfunction of both the first and the second phase enzymes because of their hyperactive or inhibited functioning. In the case of increased phase I enzyme activity, or decreased

**4. Association of xenobiotics with male infertility**

promote cell death and tissue toxicity.

**88**

phase II enzyme activity, electrophilic intermediates of the xenobiotic metabolism are accumulated in the cells and mediate the abovementioned damages. At the same time, the reduced activity of the first phase enzymes leads to metabolism retardation and accumulation of noxious chemical compounds in different tissues of the organism, including in those of the reproductive system. For example, decreased activity of GST (phase II enzyme that transfers glutathione (GSH) to activated environmental chemicals) will probably lead to the formation of reactive intermediates that will mediate cell damage mechanisms. On the other hand, phase I enzyme CYP1A1 is able to metabolize the polycyclic aromatic hydrocarbons (PAHs) to intermediate substances, which prompt genotoxic and mutagenic effects before they are further detoxified by phase II enzymes. This indicates that the increased activity of CYP1A1 may contribute to the accumulation of these compounds in the organism and to the elevation of the PAH-DNA adducts level. Although the liver performs the main functions of detoxification, reactive metabolites can also be generated in a particular organ, mediating organ specific toxicity. For example, experiments on laboratory animals such as rats and mice showed the presence of both phase I and phase II enzymes in their testicles. [17, 18]. The possibility of PAHs (benzo(a)pyrene) detoxification was also shown on the rats' Leydig cells [19]. The xenobiotic-detoxification enzymes are much needed in the reproductive system, since some endocrine-deteriorating agents (phthalates, dioxins, polychlorinated biphenyls (PCBs) and pesticides) have been shown to exert a negative influence on the reproductive tissues [20]. Thus, stable and lipophilic chlorinated hydrocarbons are capable of penetrating into the male reproductive tract, promoting idiopathic sterility and other reproductive impairments. One of the well-studied examples of male testicular toxins is occupational xenobiotic nematocide 1,2-dibromo 3-chloropropane (DBCP) that causes a partially reversible damage to the seminiferous epithelium, leading to diminished sperm counts and sterility [21]. Another occupational and environmental toxin dichlorodiphenyldichloroethylene also reduces sperm count and mediates male infertility [22].
