Lin Ao

Association between urinary polycyclic aromatic hydrocarbon metabolites and sperm DNA damage: a population study in Chongqing, China.

Background Polycyclic aromatic hydrocarbons (PAHs), a class of the most ubiquitous environmental contaminants, may reduce male reproductive functions, but the data from human population studies are very limited. Objectives We designed this study to determine whether environmental exposure to PAHs contributes to the alteration in semen quality, sperm DNA damage, and apoptosis in the general male human population. Methods We measured urinary levels of four PAH metabolites and assessed semen quality, sperm apoptotic markers with Annexin V assay, and sperm DNA damage with comet assay in 232 men from Chongqing, China. Results We found that increased urinary 2-hydroxynaphthalene (2-OHNa) levels were associated with increased comet parameters, including the percentage of DNA in the tail (tail%) [β coefficient = 13.26% per log unit 2-OHNa (micrograms per gram creatinine); 95% confidence interval (CI), 7.97–18.55]; tail length (12.25; 95% CI, 0.01–24.52), and tail distribution (7.55; 95% CI, 1.28–18.83). The urinary level of 1-hydroxypyrene was associated only with increased tail% (5.32; 95% CI, 0.47–10.17). Additionally, the increased levels of four urinary PAH metabolites were significantly associated with decreased vital Annexin V negative sperm counts. However, there was no significant association between urinary PAH metabolite levels and human semen parameters or morphology of the sperm samples. Conclusions Our data indicate that the environmental level of PAH exposure is associated with increased sperm DNA damage but not with semen quality. These findings suggest that exposure to PAHs may disrupt sperm DNA and thereby interfere with human male fertility.

Urinary phthalate metabolites and male reproductive function parameters in Chongqing general population, China.

This study was designed to investigate the phthalates exposure levels in general population in Chongqing City of China, and to determine the possible associations between phthalate exposure and male reproductive function parameters. We recruited 232 general men through Chongqing Family Planning Research Institute and Reproductive Center of Chongqing. In a single spot urine sample from each man, phthalate metabolites, including mono-butyl phthalate (MBP), mono-ethyl phthalate (MEP), mono-(2-ethylhexyl) phthalate (MEHP), mono-benzyl phthalate (MBzP), phthalic acid (PA), and total PA were analyzed using solid phase extraction and coupled with high-performance liquid chromatography and detection by tandem mass spectrometry. Semen parameters were dichotomized based on World Health Organization reference values. Sperm DNA damage were analyzed using the alkaline single-cell gel electrophoresis assay. Reproductive hormones were determined in serum by the radioimmunoassay kit. We observed a weak association between urinary MBP concentration and sperm concentration in Chongqing general population. MBP levels above the median were 1.97 times (95% confidence interval [CI] 0.97-4.04) more likely to have sperm concentration below the reference value. There were no other associations between phthalate metabolites and reproductive function parameters after adjusted for potential risk factors. Our study suggested that general population in Chongqing area of China exposure to the environmental level of phthalate have weak or without adverse effects on the reproduction.

The combined toxicity of dibutyl phthalate and benzo(a)pyrene on the reproductive system of male Sprague Dawley rats in vivo.

Our previous studies revealed more than 100 pollutants, most of which were endocrine disruptors (EDs) in two Chinese rivers, the Jialing and the Yangtze near Chongqing. Most EDs, such as dibutyl phthalate (DBP) and benzo(a)pyrene (BaP), are known to act individually as reproductive toxicants. However, little is known about the combined toxicity of DBP and BaP. In the current study, male Sprague Dawley rats were subchronically exposed to single doses of DBP (250 mg/kg), single doses of BaP (5 mg/kg) and combined doses of DBP and BaP. Significant adverse effects were observed on the reproductive system, including decreased sperm count, increased production of abnormal sperm, changes in serum testosterone levels and irregular arrangements of the seminiferous epithelium. Biochemical analyses showed that the activities of superoxide dismutase and glutathione peroxidase decreased after exposure to these EDs. Therefore, our data suggest that exposure to DBP and BaP, in either separate or combined doses, can affect the reproductive system of male rats adversely via oxidative stress-related mechanisms. No significant additive effect was observed after combined exposure. These results indicate that exposure to mixtures of EDs have unexpected and elusive effects. Our findings provide preliminary but important data for assessing water safety in China.

Association between mobile phone use and semen quality: a systemic review and meta-analysis

Possible hazardous health effects of radiofrequency electromagnetic radiations emitted from mobile phone on the reproductive system have raised public concern in recent years. This systemic review and meta‐analysis was prepared following standard procedures of the Cochrane Collaboration and the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses statement and checklist. Relevant studies published up to May 2013 were identified from five major international and Chinese literature databases: Medline/PubMed, EMBASE, CNKI, the VIP database and the Cochrane Central Register of Controlled Trials in the Cochrane Library. Eighteen studies with 3947 men and 186 rats were included in the systemic review, of which 12 studies (four human studies, four in vitro studies and four animal studies) with 1533 men and 97 rats were used in the meta‐analyses. Systemic review showed that results of most of the human studies and in vitro laboratory studies indicated mobile phone use or radiofrequency exposure had negative effects on the various semen parameters studied. However, meta‐analysis indicated that mobile phone use had no adverse effects on semen parameters in human studies. In the in vitro studies, meta‐analysis indicated that radiofrequency radiation had detrimental effect on sperm motility and viability in vitro [pooled mean difference (MDs) (95% CI): −4.11 (−8.08, −0.13), −3.82 (−7.00, −0.65) for sperm motility and viability respectively]. As for animal studies, radiofrequency exposure had harmful effects on sperm concentration and motility [pooled MDs (95% CI): −8.75 (−17.37, −0.12), −17.72 (−32.79, −2.65) for sperm concentration and motility respectively]. Evidence from current studies suggests potential harmful effects of mobile phone use on semen parameters. A further multicentred and standardized study is needed to assess the risk of mobile phone use on the reproductive system.

CpG island hypermethylation of multiple tumor suppressor genes associated with loss of their protein expression during rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine

The epigenetic mechanisms underlying the tumorigenesis caused by polycyclic aromatic hydrocarbons and nitrosamine compounds such as 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are currently unknown. We reported previously that dynamic changes in DNA methylation occurred during MCA/DEN-induced rat lung carcinogenesis. Here, we used the same animal model to further study the evolution of methylation alterations in tumor suppressor genes (TSGs) DAPK1, FHIT, RASSF1A, and SOCS-3. We found that none of these genes were methylated in either normal or hyperplasia tissue. However, as the severity of the cancer progressed through squamous metaplasia and dysplasia to carcinoma in situ (CIS) and infiltrating carcinoma, so methylation became more prevalent. Particularly dramatic increases in the level of methylation, the average number of methylated genes, and the incidence of concurrent methylation in three genes were observed in CIS and infiltrating carcinoma. Similar but less profound changes were seen in squamous metaplasia and dysplasia. Furthermore, methylation status was closely correlated to loss of protein expression for these genes, with protein levels markedly declining along the continuum of carcinogenesis. These results suggest that progressive CpG island hypermethylation leading to inactivation of TSGs might be a vital molecular mechanism in the pathogenesis of MCA/DEN-induced multistep rat lung carcinogenesis.

Lifestyles Associated With Human Semen Quality: Results From MARHCS Cohort Study in Chongqing, China.

AbstractDecline of semen quality in past decades is suggested to be potentially associated with environmental and sociopsychobehavioral factors, but data from population-based cohort studies is limited.The male reproductive health in Chongqing College students (MARHCS) study was established in June 2013 as a perspective cohort study that recruited voluntary male healthy college students from 3 universities in Chongqing. The primary objectives of the MARHCS study are to investigate the associations of male reproductive health in young adults with sociopsychobehavioral factors, as well as changes of environmental exposure due to the relocation from rural campus (in University Town) to metro-campus (in central downtown). A 93-item questionnaire was used to collect sociopsychobehavioral information in manner of interviewer–interviewing, and blood, urine and semen samples were collected at the same time.The study was initiated with 796 healthy young men screened from 872 participants, with a median age of 20. About 81.8% of this population met the WHO 2010 criteria on semen quality given to the 6 routine parameters. Decreases of 12.7%, 19.8%, and 17.0%, and decreases of 7.7%, 17.6%, and 14.7% in total sperm count and sperm concentration, respectively, were found to be associated with the tertiles of accumulated smoking amount. Fried food consumption (1–2 times/wk or ≥3 times/wk vs nonconsumers) was found to be associated with decreased total sperm count (10.2% or 24.5%) and sperm concentration (13.7% or 17.2%), respectively. Coffee consumption was found to be associated with increased progressive and nonprogressive motility of 8.9% or 15.4% for subjects consuming 1–2 cups/wk or ≥3 cups/wk of coffee, respectively. Cola consumption appeared an association with decreased semen volume at 4.1% or 12.5% for 1–2 bottles/wk or ≥3 bottles/wk.A cohort to investigate the effects of environmental/sociopsychobehavioral factors act on semen quality was successfully set up. We found smoking, coffee/cola/fried foods consumption to be significantly associated with semen quality from the baseline investigation.

Epigenetic regulation of sox30 is associated with testis development in mice.

DNA methylation is involved in tissue-specific and developmentally regulated gene expression. Here, we screened a novel methylation gene Sox30, whose methylation might contribute to its regulation and testis development in mice. Sox30 is a member of Sox transcription factors, and is considered to be involved in spermatogonial differentiation and spermatogenesis. However, the precise function and regulatory expression pattern remain unclear. In the present study, we found that Sox30 is highly expressed in adult testes but not in ovaries. Sox30 expression begins in early development, and in the testes, it is specifically increased coincidentally with development until adulthood. Moreover, Sox30 is expressed not only in testis germ cells, but also in sertoli cells. Sox30 is hypo-methylated in testis, epididymis and lung of adult mice, in which Sox30 is expressed. By contrast, Sox30 is hypermethylated in ovary, heart, brain, liver, kidney, spleen, pancreas, muscle, intestine, pituitary gland, blood and hippocampus of adult mice, in which the Sox30 is absent. Importantly, decreased methylation at CpG islands of Sox30 is observed in mouse developmental testes after birth, which is associated with enhanced Sox30 expression. However, the hypermethylated status of Sox30 is maintained in ovaries that does not express Sox30 during this period. Further, following demethylation treatment using 5-aza-dC, Sox30 expression is restored in GC2, TM3 and TM4 cell lines. This observation convincingly confirms that methylation really contributes to Sox30 silencing. In summary, we show that Sox30 expression is under the control of DNA methylation status, and this expression pattern is associated with testis development in mice.

Epigenetic silencing of cell cycle regulatory genes during 3‐methylcholanthrene and diethylnitrosamine‐induced multistep rat lung cancer

The rat lung cancers induced by 3‐methylcholanthrene (MCA) and diethylnitrosamine (DEN) are considered to be a good model for illustrating genetic alterations in human lung precancerous and cancerous lesions. Recently, we had reported that the model can also be used to investigate the step‐by‐step dynamic changes in DNA methylation during lung carcinogenesis. In this study, we have used the same animal model to further study the evolution of methylation alterations of cell cycle regulatory genes CDKN1B (p27) and CDKN1C (p57). Our results showed epigenetic alterations in p27 and p57. Promoter hypermethylation of p27 was detected in one sample of carcinoma in situ (CIS) and two samples of infiltrating carcinoma, all three of which lacked expression of the p27 protein. Methylation of the p57 promoter correlated with the loss of protein expression in lung pathologic lesions, with a gradual increase in methylation frequency from 0 sample in the normal epithelium and hyperplasia, to 11.1% in squamous metaplasia, 18.9% in dysplasia, 26.7% in CIS, and finally 36.0% in infiltrating carcinoma samples. Immunohistochemical analysis showed that p27 and p57 protein expression decreased as lung carcinogenesis progressed. Moreover, weak expression of p27 and p57 in methylated primary tumor cell lines increased markedly after treatment with 5‐aza‐2′‐deoxycytidine (5‐aza‐dC), confirming that methylation was indeed responsible for the gene downregulation. These results suggest that the progression of rat lung carcinogenesis induced by MCA/DEN is associated with dynamic changes in promoter hypermethylation of cell cycle regulatory genes, including p27 and p57, accounting for their defective expression.

Molecular analysis of DNA repair gene methylation and protein expression during chemical-induced rat lung carcinogenesis.

A defective ratio between DNA damage and repair may result in the occurrence of a malignant phenotype. Previous studies have found that many genetic alterations in DNA repair genes occur frequently in lung cancer. However, the epigenetic mechanisms underlying this tumorigenesis are not clear. Herein, we have used a chemical-induced rat lung carcinogenesis model to study the evolution of methylation alterations of DNA repair genes BRCA1, ERCC1, XRCC1, and MLH1. Methylation-specific PCR and immunohistochemistry were used to analyze gene methylation status and protein expression during the progression of lung carcinogenesis. Promoter hypermethylation of BRCA1 was only detected in three samples of infiltrating carcinoma. CpG island hypermethylation of ERCC1, XRCC1, and MLH1 was found to increase gradually throughout lung carcinogenesis progression. Both the prevalence of at least one methylated gene and the average number of methylated genes were heightened in squamous metaplasia and dysplasia compared with normal tissue and hyperplasia, and was further increased in carcinoma in situ (CIS) and infiltrating carcinoma. Immunohistochemical analysis showed that BRCA1 and MLH1 protein expression decreased progressively during the stages of lung carcinogenesis, whereas ERCC1 and XRCC1 expression were only found in later stages. Although methylation levels were elevated for ERCC1 and XRCC1 during carcinogenesis, an inverse correlation with protein expression was found only for BRCA1 and MLH1. These results suggest that a continuous accumulation of DNA repair gene hypermethylation and the consequent protein alterations might be a vital molecular mechanism during the process of multistep chemical-induced rat lung carcinogenesis.

The Interaction of Mitochondrial Biogenesis and Fission/Fusion Mediated by PGC-1α Regulates Rotenone-Induced Dopaminergic Neurotoxicity

Parkinson’s disease is a common neurodegenerative disease in the elderly, and mitochondrial defects underlie the pathogenesis of PD. Impairment of mitochondrial homeostasis results in reactive oxygen species formation, which in turn can potentiate the accumulation of dysfunctional mitochondria, forming a vicious cycle in the neuron. Mitochondrial fission/fusion and biogenesis play important roles in maintaining mitochondrial homeostasis. It has been reported that PGC-1α is a powerful transcription factor that is widely involved in the regulation of mitochondrial biogenesis, oxidative stress, and other processes. Therefore, we explored mitochondrial biogenesis, mitochondrial fission/fusion, and especially PGC-1α as the key point in the signaling mechanism of their interaction in rotenone-induced dopamine neurotoxicity. The results showed that mitochondrial number and mass were reduced significantly, accompanied by alterations in proteins known to regulate mitochondrial fission/fusion (MFN2, OPA1, Drp1, and Fis1) and mitochondrial biogenesis (PGC-1α and mtTFA). Further experiments proved that inhibition of mitochondrial fission or promotion of mitochondrial fusion has protective effects in rotenone-induced neurotoxicity and also promotes mitochondrial biogenesis. By establishing cell models of PGC-1α overexpression and reduced expression, we found that PGC-1α can regulate MFN2 and Drp1 protein expression and phosphorylation to influence mitochondrial fission/fusion. In summary, it can be concluded that PGC-1α-mediated cross talk between mitochondrial biogenesis and fission/fusion contributes to rotenone-induced dopaminergic neurodegeneration.