Expression of Xenobiotic Biomarkers CYP1 Family in Preputial Tissue of Patients with Hypospadias and Phimosis and Its Association with DNA Methylation Level of SRD5A2 Minimal Promoter

Abstract

analyzed preputial tissues from patients with hypospadias (n = 23) and phimosis (n = 16). The atypical CYP1 family genes, CYP1A1 and CYP1B1, are potential biomarkers of environmental chemical exposure. We then compared the expression levels of CYP1A1 and CYP1B1 between hypospadias and phimosis samples by quantitative RT-PCR analysis. The mRNA expression levels of SRD5A2 and AR also were measured, because the androgen-related genes involved in the onset of disorders of male reproductive system. A significantly higher CYP1B1 expression level and a lower AR expression level were observed in the hypospadias groups than in the phimosis group. Positive correlations (P < 0.001) between the mRNA expression levels of the CYP1 family and SRD5A2 were found in patients with hypospadias but not in those with phimosis. Moreover, the methylation levels of the four genes were determined by bisulfite genomic sequencing. Although the SRD5A2 promoter region showed moderate methylation, no methylation was detected in CYP1A1, CYP1B1, or AR. There was no significant difference in SRD5A2 promoter methylation level between hypospadias and phimosis patients. Negative correlations were found between the methylation level of SRD5A2, especially at the − 221 Sp1 site, and the CYP1 family mRNA expression levels (CYP1A1, p = 0.002; CYP1B1, p = 0.007) in hypospadias patients, but not in phimosis patients. The significant positive association of mRNA expression level and the negative association of methylation level of the SRD5A2 gene with the mRNA expression levels of CYP1 family genes in the preputial tissue seem to indicate the chemical exposure of patients with hypospadias.During the past century, the prevalence of male reproductive system abnormalities, especially hypospadias, has increased markedly in developed countries, such as the United States, Abstract Several epidemiological studies have suggested that the incidence of male reproductive organ malformations, including hypospadias or cryptorchidism, has increased due to fetal-stage exposure to environmental pollutants. However, the association of chemical exposure with the expression of target regulatory genes in the tissues of patients has not yet been reported. Because experimental approaches or clinical trials in human studies are limited, especially those using fetal and/or infants, it is difficult to obtain clear physiological evidence of mechanisms underlying male reproductive malformations. Thus, the lack of physiological evidence makes this issue controversial. We Seiichiroh Ohsako and Toshiki Aiba have contributed equally to this work. Electronic supplementary materialThe online version of this article

Exploring disease-specific methylated CpGs in human male genital abnormalities by using methylated-site display-amplified fragment length polymorphism (MSD-AFLP)

“…Endocrine-disrupting chemicals in the environment, including heavy metals, have been reported to cause epigenetic changes, diseases such as cancer, and reproductive and developmental abnormalities [14,15,16]. There are many reports describing the relationship between hypospadias and epigenetic changes, which appear to be caused by exposure to environmental endocrine-disrupting chemicals [8, 9, 14], therefore this study analyzed urinary elements of hypospadias patients. An epidemiological survey in India showed that the risk of hypospadias increases when the levels of cadmium and lead in blood are high [15].…”

“…Genes involved with male hormones, such as androgen receptor ( AR ) and 5α-reductase type 2 ( SRD5A2 ), are associated with the onset of hypospadias as indicated by genetic polymorphisms [5,6,7]. In addition, the causative gene of hypospadias, MAMLD , was discovered when it was found that MAMLD possesses a nonsense mutation in a 46, XY disorder of sexual development (DSD) [8, 9]. Several MAMLD1 mutations have been shown to cause hypospadias owing to the production of dysfunctional proteins or unstable mRNAs [6].…”

“…A previous study showed that methylation levels of DNA in the upstream region of the AR transcription start site are higher and AR mRNA expression levels are lower in foreskin from hypospadias patients than in healthy individuals [7]. DNA methylation and mRNA expression levels in foreskin from hypospadias and phimosis patients have also been compared [8]. Although AR mRNA expression levels were significantly lower in hypospadias patients than in control phimosis patients, bisulfite genomic sequencing could not detect any methylated CpGs in the CpG-islands examined [8].…”

“…DNA methylation and mRNA expression levels in foreskin from hypospadias and phimosis patients have also been compared [8]. Although AR mRNA expression levels were significantly lower in hypospadias patients than in control phimosis patients, bisulfite genomic sequencing could not detect any methylated CpGs in the CpG-islands examined [8]. Although the methylation levels of the SRD5A2 promoter region shows no significant difference between hypospadias and phimosis patients, a negative correlation was detected in hypospadias patients between the DNA methylation level of the SP1 site of the SRD5A2 minimum promoter and the mRNA expression level of CYP1 family members (CYP1A1 and CYP1B1) Phimosis samples lack such a correlation, implicating chemical exposure as a cause of hypospadias [8].…”

Etiology of Hypospadias: A Comparative Review of Genetic Factors and Developmental Processes Between Human and Animal Models

“…Higher methylation level of AR gene in the foreskin, resulting in a decreased expression of the AR protein, might be involved in the pathogenesis of hypospadias. 21 Ohsako et al 22 reported a negative association of methylation level of the SRD5A2 gene with the mRNA expression levels of CYP1 family genes in the preputial tissue of patients with hypospadias and suggested it is likely to indicate the involvement of chemical exposure and epigenetic change in the onset of hypospadias. Significant associations were observed between methylation levels of individual CpG sites and hypospadias, and DNA methylation patterns have been used in identifying and evaluating new candidate genes that may be involved in the etiology of hypospadias.…”