Exposure To Phenols And Male Reproductive Impairment:Epidemiological And Metabolomics Study

Widespread human exposure to phenols has been documented recently, and some phenols which are potential endocrine disruptors have demonstrated adverse effects on male reproduction in animal and in vitro studies. However, implications about exposure to phenols and male infertility are scarce in humans. Both environmental and genetic factors play an important role in male infertility, therefore, gene–environment interaction is an important topic regarding susceptibility to chemical toxicity on the research agenda. Additionally, metabolomics, a newly emerging “omics” technology, has been proven to be a useful tool in the discovery of new biomarkers for mechanisms of toxicity. Thus, in this study, we focused on the topic of exposure to phenols and male reproductive impairment. We integrated the data of chemical exposure and genetic background in the population study, and also conducted mechanistic research based on epidemiological findings. First, we developed a method to determine nine environmental phenols in human urine using ultra-high-performance liquid chromatography–tandem mass spectrometry. Then, we determined the phenol exposure levels in the general population in China, and explored the relationships between phenol exposure and male infertility as well as abnormal semen parameters. We further identify whether some Y chromosome haplogroups modify the susceptibility to the representative phenol(4-t-octylphenol) exposure on idiopathic male infertility. According to the epidemiological findings, we used metabolomics approach to study the representative phenol(bisphenol A) reproductive toxicity in a rat model by analyzing both testes and urine samples. Our study provides the first evidence suggesting adverse effects of phenol exposure on male reproductive health in humans and also provides new insights into phenols-induced reproductive toxicity.Part I. Exposure to phenols and male reproductive impairment: Epidemiological studyChapter I.Determination of nine environmental phenols in human urine by ultra-high-performance liquid chromatography–tandem mass spectrometry Background Health risks caused by widespread human exposure to phenols have recently become hot issues of public concern. Few study reported the method which could simultaneously determine internal exposure levels of several phenols in humans. In this study, a method was developed to determine nine environmental phenols, including bisphenol A, 2,3,4-trichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, triclosan(2,4,4’-trichloro-2’-hydroxyphenylether), 4-t-octylphenol, 4-n-octylphenol, 4-n-nonylphenol and benzophenone-3(2-hydroxy-4-methoxybenzophenone) in human urine using ultra-high-performance liquid chromatography–tandem mass spectrometry. Method The human urine was incubated with β-glucuronidase/sulfatase overnight. The analytes in human urine were extracted and preconcentrated with solid-phase extraction, and then quantified with ultra-high-performance liquid chromatography-electrospray ionization(negative ion mode)–tandem mass spectrometry using multiple reaction monitoring mode.Limits of detection of the nine phenols ranged from 0.02 to 0.90 μg/l. Intra-day precisions of the nine phenols ranged from 2% to 41%. Inter-day precisions of the nine phenols ranged from 3% to 38%. Recoveries of the nine phenols ranged from 78% to 138%. Linear ranges of the nine phenols ranged from 0.03-50 μg/l. This method was further validated by the determination of phenols in 325 human urine samples that generated data regarding the exposure of various phenols in the Chinese general population. ResultsConclusions This study presents a sensitive, accurate and reproducible method suitable for the determination of nine environmental phenols in human urine with ultra-high-performance liquid chromatography–tandem mass spectrometry. The application of this method was the first pilot survey of the exposure of various phenols in the Chinese general population. This method is easily popularized and useful for collecting data regarding the exposure of various urinary phenols in the general population, and for exploring the health risks of various phenols in epidemiological studies.Chapter II.Association of exposure to various phenols with idiopathic male infertility and abnormal semen parametersBackground Widespread human exposure to phenols has been documented recently, and some phenols which are potential endocrine disruptors have demonstrated adverse effects on male reproduction in animal and in vitro studies. However, implications about exposure to phenols and male infertility are scarce in humans. Case–control study ofidiopathic infertile men and fertile controls was conducted to study the association of exposure to various phenols with idiopathic male infertility and abnormal semen parameters. Method Urinary levels of bisphenol A, benzophenone-3, pentachlorophenol, triclosan, 4-t-octylphenol, 4-n-octylphenol and 4-n-nonylphenol in 877 idiopathic infertile men and 713 fertile controls and semen parameters were measured. Multivariate logistic regression and tests for trend were performed to analyze the relationships between phenol exposure levels and idiopathic male infertility as well as abnormal semen parameters. Results Among these phenols, triclosan showed highest urinary levels, followed by bisphenol A, pentachlorophenol, benzophenone-3, 4-t-octylphenol, 4-n-nonylphenol and 4-n-octylphenol. After multivariate adjustment, we found 4-t-octylphenol, 4-n-octylphenol and 4-n-nonylphenol exposure was associated with idiopathic male infertility(p-value for trend: <0.0001, 0.014 and 0.001, respectively). Aside from these associations, bisphenol A, 4-t-octylphenol and 4-n-nonylphenol exposure was also associated with abnormal semen parameters. Moreover, we observed significant associations between sum alkylphenols exposure and idiopathic male infertility. Conclusions To the best of our knowledge, we present the first human study that explored the relationships between exposure to various environmental phenols and idiopathic male infertility by monitoring urinary phenols with large samples. These findings provide the first evidence suggesting adverse effects of phenols on male reproductive health in humans.Chapter III.The genetic susceptibility to representative phenol exposure on male infertilityBackground Both environmental and genetic factors play an important role in male infertility. Our study found that there was strong association between exposure to 4-t-octylphenol and male infertility in the Chinese general population, therefore, there is a great need to study the genetic susceptibility to 4-t-octylphenol exposure on male infertility to advance our knowledge of the potential male reproductive toxicity of 4-tert-octylphenol in humans. Y chromosome haplogroup is related to male reproduction and also can be used to judge the genetic background of individuals. In this case-control study, we identify whether some Y chromosome haplogroups modify the susceptibility to the representative phenol(4-t-octylphenol) exposure on idiopathic male infertility. Method We determined urinary 4-t-octylphenol concentrations in 774 idiopathic infertile men and 520 fertile controls. Y chromosome haplogroups were defined using the SNa Pshot assays. Likelihood ratio tests were used to explore the interactions between Y haplogroups and 4-t-octylphenol exposure on idiopathic male infertility, and false discovery rate(FDR) was used to adjust for multiple testing. Results Han Chinese in different Y chromosome haplogroups were all susceptible to 4-t-octylphenol exposure on idiopathic male infertility. We observed that the haplogroup O3* modified the susceptibility to 4-t-octylphenol exposure on idiopathic male infertility after FDR adjustment(pinter= 1.1e-3). Conclusions We firstly described the distribution of the susceptibility to the 4-t-octylphenolexposure on idiopathic male infertility in various Y chromosome haplogroups in a Han Chinese population, and found that Han Chinese are generally susceptible to 4-t-octylphenol exposure on idiopathic male infertility. Also, we found that individuals in haplogroup O3* presented a significantly decreased risk of idiopathic male infertility with increasing 4-t-octylphenol exposure levels. Our study emphasizes the study on the functional characteristics of individuals in different Y chromosome haplogroups as well as the study on Y chromosome haplogroups and susceptibility to the chemical exposure.Part II. Metabolomics reveals new mechanisms of male reproductive toxicity of representative phenol Background Epidemiological study indicates that bisphenol A may impair spermatogenesis, however, the underlying mechanism is still poorly understood. Metabolomics has been proven to be a useful tool in the discovery of new biomarkers for mechanisms of toxicity. To date, the study which focuses on bisphenol A reproductive toxicity using a metabolomic approach has yet to be reported. In this study, we used metabolomics approach to study the representative phenol(bisphenol A) reproductive toxicity in a rat model by analyzing both testes and urine samples. Method 18 Sprague-Dawley rats were orally administrated doses of BPA at the levels of 0, 0.0005 mg/kg/d and 50 mg/kg/d for 8 weeks. We used an unbiased liquid chromatography-quadrupole time-of-flight-based metabolomics approach to discover, identify, and analyze the variation of testicular metabolites, and the rat urine was alsoanalyzed by this system. We identified testicular biomarkers based on accurate masisotopic pattern and the information from online database and local standardAccording to testicular metabolomics suggestions, testicular superoxide dismutasglutathione peroxidase and catalaselevels in testes were assayed using commercispectrophotometric kits. Additionally, a capillary electrophoresis in tandem witelectrospray ionization time-of-flight mass spectrometry based non-targeted higthroughput metabolomic analysis was performed in rat urine, during which thurinary metabolites were identified based on local standards. RT-PCR and westerblotting were further conducted to analyze hepatic expression of methioninadenosyltransferase Iα(Mat1a) and methionine adenosyltransferase IIα(Mat2a). Falsdiscovery rate was used to adjust for multiple testing, and bioinformatics analysis waconducted using KEGG.Results Urinary bisphenol A-glucuronide was increased in the BPA exposed group. In the testes metabolomic profile, two n-6 fatty acids, linoleic acid(LA) and arachidonic acid(AA) were identified as potential testicular biomarkers. Decreased levels of LA and increased levels of AA as well as AA/LA ratio were observed in the testes of the exposed group. Testicular superoxide dismutase declined significantly in the exposed group compared with that in the non-exposed group, and the glutathione peroxidase as well as catalase also showed a decreasing trend in BPA treated group. In the urine metabolomic profile, we found BPA increased synthesis of methylated products including methylated purines and methylated amino acids, elevated purine nucleotide catabolism, elevated urea cycle, elevated water-soluble vitamin excretion and increased flux through the choline metabolism pathway. We further identified both increased m RNA and protein levels of Mat1 a and Mat2 a encoding critical enzyme involved in the choline metabolism in rat liver.Conclusions Our study identified two testicular biomarkers in relation to bisphenol A toxicity, and found bisphenol A caused testicular n-6 fatty acid composition variation and decreased testicular antioxidant enzyme activity. Elevated choline metabolism may be underlying mechanism of highly methylated environment caused by BPA exposure, which is related to protein degradation and DNA damage. This study provides new insights into BPA-induced reproductive toxicity, and emphasizes that metabolomics brings the promise of biomarkers identification for the discovery of mechanisms underlying reproductive toxicity...