Effects of aniline and several aniline derivatives on sex hormone regulation and reproduction of adult zebrafish

Abstract

Aniline (ANL) and several aniline derivatives, e.g., 3,4-dichloroaniline (3,4-DCA), 1-naphthylamine (1-NPA), 4,4-methylenedianiline (4,4-MDA), and 4- chloroaniline (4-CA) have been widely used in the production of herbicides, pharmaceuticals, cosmetic, dyes, rubber, and adhesives products. These chemicals can easily be released into the environment through industrial and municipal discharges or as degradation byproducts of those materials. Because of the wide range of use and possible exposure to human and environment, these compounds are considered priority pollutants in environmental risk assessment. There are reports on the environmental and human exposure of anilines. Experimental studies and epidemiological observations suggest possible adverse effects of anilines on endocrine and reproduction endpoints. ANL is rapidly metabolized to paracetamol and exhibits similar fetal anti-androgenic effects in male mice. It was also reported to disrupt testosterone level in the H295R cell line. 3,4-DCA was found to disrupt sex hormone level in tilapia fish in a previous study. Regarding the reproduction, a decrease of the fecundity of fish exposed to ANL and 4-CA was also reported. However, only ANL in cell line and mice and 3,4-DCA in fish were studied to understand endocrine disruption. Moreover, these studies were mostly descriptive and did not focus on the mode of action and effect on the reproduction relating to sex hormone regulation system. So, knowledge of the sex hormone disruption potentials and toxicological effects on reproduction are limited. The aim of this study was to investigate sex hormone disruption potentials of aniline and several aniline derivatives with the effects on the regulatory pathway and effect on the reproduction of zebrafish. To fulfil the objective of this study we carried out a series of study consisting of two main parts. In the first part (Chapter 2), the objective was to screen the sex hormone disruption effects of aniline and several aniline derivatives using the H295R cell line and zebrafish (Danio rerio). Sex hormone levels and steroidogenesis was investigated by using four important steroidogenic genes. In the H295R cell line, 3,4-DCA, 1-NPA and 4,4-MDA significantly decreased T level with the increase of E2/T ratio. E2 level was not changed after 1-NPA and 3,4-DCA exposure but significantly decreased after 4,4-MDA exposure. The mRNA expression of CYP17 was down-regulated and CYP19A was up-regulated by both 1-NPA and 3,4-DCA in the cells. All four investigated genes, StAR, CYP17, 3β-HSD and CYP19A were down-regulated in 4,4-MDA-exposed cells. In adult male zebrafish upon 14 days exposure to targeted compounds, significant decreases in plasma T and E2 level were observed after 3,4-DCA exposure, but for 1-NPA exposure, the pattern was similar with an inconsistent result in the lower level of exposure concentration. Among the major gonadal steroidogenic genes, star and cyp17 genes were down-regulated after exposure to 3,4-DCA, while for 1-NPA exposure 3β-hsd was down-regulated with a significantly decreasing trend of cyp17 in zebrafish. The steroidogenic genes, i.e., star, cyp17 and 3β-hsd, are associated with the enzymes which play an important role in the production of T, and cyp19a gene in the conversion of T to E2. The transcriptional alteration of these genes may lead to alteration of steroid hormones, and possibly to reproduction changes. Anilines were investigated to understand the effect on sex steroid hormone production and other biological processes, using in-vitro and in-vivo experiments. In the second part (Chapter 3), we aimed to understand the effect of 3,4-DCA and 4,4-MDA on sex hormone regulation through hypothalamus gonadal (HPG) axis with sex hormone and reproduction of adult zebrafish. Both male and female zebrafish were exposed for short term (21 days). A significant decrease of the egg number was observed upon exposure to both the compound. Plasma concentration of both testosterone (T) and 17β-estradiol (E2) level was significantly decreased. In 3,4-DCA, E2/T ratio was decreased in both sex fish whereas E2/T ratio was increased in male and decreased in the female fish upon exposure to 4,4-MDA. The sex hormone changes was supported by the regulatory changes of the genes along the HPG axis in both sex fish. For example, down-regulation of star and cyp19a genes in the gonad and upregulation of hypothalamic genes such as gnrh2 or gnrh3 genes transcription were observed. Moreover, down-regulation of ptgs2 gene was detected, suggesting disruption of oocyte maturation and ovulation by the exposure. Our observations indicate that 3,4-DCA and 4,4-MDA may influence steroid hormone production by altering the major regulatory genes in the HPG axis and eventually influence the reproduction of zebrafish. Consequences of longer-term exposure to this aniline warrant further investigation. Based on the results of the series of experiments of our study, we can demonstrate that, some of the aniline derivatives e.g., 3,4-DCA, 1-NPA, 4,4-MDA are potential for alteration of sex hormone levels and disrupt the steroidogenesis and as a consequence of effect on the sex hormone balance, 3,4-DCA and 4,4-MDA have influence on reproduction of zebrafish. 1-NPA was not investigated for the effect on HPG axis and reproduction. It should be done in future for understanding the mode of action and reproductive effect by 1-NPA. Moreover, the effect on longer-term exposure to these anilines warrants further investigation. The findings of this study will be useful for the assessment of the ecological risk of these compounds. It can also keep a great contribution to manage and regulate the safety of anilines in the environment.