Endocrine disrupting chemicals (bisphenol A, 4-nonylphenol, 4-tert-octylphenol) modulate expression of two distinct cytochrome P450 aromatase genes differently in gender types of the hermaphroditic fish Rivulus marmoratus

Abstract

To understand the effect of endocrine-disrupting chemicals (EDCs) on cytochrome P450 aromatase ( rm- cyp19) gene expression between gender types in the hermaphroditic fish Rivulus marmoratus, we cloned two distinct rm- cyp19 genes using RT-PCR with degenerative primers, obtained full-length cDNAs using 5′- and 3′-RACE-PCR methods, and completely sequenced them. The brain aromatase ( rm- cyp19b) cDNA consisted of 2,124 bp including the open reading frame (ORF), which encoded a putative protein of 505 amino acids. The ovarian aromatase ( rm-cyp19a) cDNA consisted of 2,075 bp, including the ORF encoding a putative protein of 516 amino acids. Expression patterns of rm- cyp19b and rm-cyp19a mRNAs were investigated in embryos of different developmental stages and in seven different tissues of adult fish. The rm- cyp19b gene in hermaphrodite and secondary male R. marmoratus was predominantly expressed in the brain, while the rm- cyp19a gene was expressed gender-specifically in the gonad. The expression of rm- cyp19b mRNA increased from stage 1 (2d post fertilization) to stage 4 (12d post fertilization) in a developmental stage-dependent manner but steeply decreased in the hatching stage. Compared to the rm- cyp19b gene, the abundance of ovarian aromatase rm- cyp19a transcripts was very low, and its expression was first detected at stage 3 and then decreased gradually to the hatching stage. Alteration of rm- cyp19b and rm- cyp19a gene expression was further analyzed in the brain and gonad by real-time RT-PCR 96 h after EDC exposure in hermaphrodites and secondary males. The brain aromatase rm- cyp19b gene was up-regulated in the brain after 4-nonylphenol (4-NP)-exposure, while the ovarian aromatase rm- cyp19a gene was significantly down-regulated in the gonad. In 300 μg/L 4-tert octylphenol (4-tert-OP), or 600 μg/L bisphenol A-exposed brain and gonad, both rm- cyp19b and rm- cyp19a genes were up-regulated. In the case of secondary males, the rm- cyp19b gene was highly expressed in the 4-NP-exposed brain, while expression of the rm- cyp19a gene was not detected in the gonad. These results indicate that the expression of rm- cyp19a and rm- cyp19b genes is differently modulated according to estrogenic compounds and gender type of R. marmoratus.