Effects And Mechanisms Of Monocrotophos Pesticide On Sex Hormone Synthesis And Transformation In The Male Goldfish(carassius Auratus)

Our previous studies have demonstrated that monocrotophos (MCP) haspotential estrogenic properties, since it increased17β-estradiol (E2) levels anddecreased testosterone (T) contents in male goldfish (Carassius auratus) and guppy(Poecilia reticulata). Sex hormone biosynthesis involves a series of reactionscatalyzed by several steroidogenic enzymes, thus disruption on the process ofsteroidogenesis may be responsible for the imbalance of androgen and estrogencaused by MCP pesticide. In this study, following a21-day exposure to0.01mg/L,0.10mg/L, and1.00mg/L MCP in male goldfish, changes of cholesterol (thesubstrate for sex hormones synthesis) contents were investigated, and then the effectsof MCP exposure on mRNA levels of steroidogenic acute regulatory protein (StAR)and steroidogenic enzymes were studied based on cloning cDNA fragments of severalsteroidogenic enzymes. Finally, the contents of intermediate products and effectorhormones involved in the sex steroid biosynthesis pathway were detected after MCPexposure, to establish the correlation of steroidogenic enzyme expression and sexhormone levels. The aim of this study is to elucidate the mechanisms causingimbalance of sex steroids by MCP pesticide in male fish from the perspective of sexhormone synthesis and transformation. The results showed that:(1) After a21-day exposure to0.01mg/L,0.10mg/L and1.00mg/L MCP,concentration of total cholesterol, high density lipoproteins cholesterol (HDL-C), lowdensity lipoprotein cholesterol, and triglyceride in plasma of male goldfish were eachdetected using automatic biochemistry analyzer. MCP decreased the HDL-C levels inplasma, which may lead to a decline of the substrate of sex hormones took into thetestis and thus inhibit T synthesis.(2) After a21-day exposure to0.01mg/L,0.10mg/L and1.00mg/L MCP,real-time PCR was used to detect mRNA levels of StAR and several steroidogenicenzymes, including cholesterol side chain cleavage enzyme (P450scc),3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P45017alpha-hydroxylase(P450c17), and ELISA was adopted to determine levels of intermediate products involved in the steroid biosynthesis pathway, including pregnenolone, progesterone,17α-hydroxypregnenolone,17α-hydroxyprogesterone, dehydroepiandrosterone,androstenedione, estrone, and11β-hydroxy testosterone. MCP inhibited StAR mRNAexpression levels in testis of male goldfish, which may result in reducedintra-mitochondrial cholesterol pools; MCP decreased the abundance of P450scctranscripts, leading to a decline of pregnenolone; MCP reduced3β-HSD and P450c17mRNA expression levels, which resulted in a decline of androstenedione; thus it wasdeduced that the inhibition of MCP pesticide on the above mentioned steroidogenesispaths might be another reason that T in male fish was decreased.(3) After a21-day exposure to0.01mg/L,0.10mg/L and1.00mg/L MCP,real-time PCR was used to quantify mRNA levels of20β-hydroxysteroiddehydrogenase (20β-HSD), cytochrome P450aromatase (P450arom),17β-hydroxysteroid dehydrogenase type I (17β-HSD1),11β-hydroxylase (P45011β),and11β-hydroxysteroid dehydrogenase type II (11β-HSD2), and ELISA was adoptedto test levels of effector hormones involved in the sex steroid biosynthesis pathway,including17α,20β-dihydroxy-4-pregnen-3-one and11-ketotestosterone (11-KT). MCPraised P450arom and17β-HSD1mRNA levels, thus it was suggested that theenhancement of MCP pesticide on gene expression of these two steroidogenicenzymes contributed to the increase of E2, and the increase of P450arom expressionmight strengthen the decrease of androgen T; MCP increased20β-HSD mRNAexpression levels in testis of male goldfish, leading to an increase of17α,20β-dihydroxy-4-pregnen-3-one; however, although MCP decreased mRNAexpression of P45011βand11β-HSD2,11-KT contents were not affected according tothis study.In conclusion, the action mechanisms of monocrotophos pesticide on sexhormone synthesis and transformation in the male goldfish were discussed byevaluating the levels of cholesterol contents, mRNA levels of StAR and steroidogenicenzymes, and contents of intermediate products and effector hormones involved in thesteroid biosynthesis pathway. The results showed that MCP caused the imbalance ofestrogen and androgen mainly via (a) decreasing HDL-C concentrations in the plasma,inhibiting mRNA expression of steroidogenic enzymes related to androgen synthesis,and consequently reducing the androgen (mainly T) levels;(b) promoting mRNAexpression of steroidogenic enzymes related to estrogen synthesis, and then increasingestrogen (mainly E2) levels. Through systematic investigation on action machnisms of MCP pesticide which does not have a similar structure to E2, the present study canprovide a reference for a better understanding of the acting targets of non-typicalenvironmental estrogens.