Anti-estrogenic effect of semicarbazide in female zebrafish (Danio rerio) and its potential mechanisms

• Female zebrafish were exposed to semicarbazide for 96 hours or 28 days.
• Semicarbazide exposure reduced plasma 17β-estradiol levels at 96 hour and 28 day.
• Ovarian histology was affected after 28-day semicarbazide exposure.
• Semicarbazide-exposed zebrafish showed altered reproductive gene transcription.
• Semicarbazide exerted an anti-estrogenic effect in female zebrafish.

Semicarbazide (SMC), a member of the hydrazine family, has various toxic effects and has been detected in organisms, aquatic environments, and food. SMC exposure inhibited the transcription of hepatic vitellogenin and estrogen receptors in female zebrafish (Danio rerio), suggesting that it had anti-estrogenic properties. In order to elucidate the mechanisms underlying these, we exposed female zebrafish to SMC and used enzyme-linked immunosorbent assays to examine plasma 17β-estradiol (E2) and testosterone (T) levels. Gonad histology was analyzed and the mRNA expression of genes involved in the reproductive axis, the gamma-aminobutyric acid (GABA) shunt, and leptin was quantified by real-time PCR. Zebrafish were exposed to 1, 10, 100, or 1000 μg/L SMC in a semi-static system for 96 hours or 28 days. Plasma E2 levels were significantly decreased and ovarian maturation was inhibited by SMC, suggesting that its anti-estrogenic effect was exerted by reducing endogenous E2 levels. This was likely due to the SMC-mediated inhibition of cytochrome P450 (CYP) 19A mRNA levels, because this enzyme catalyzes the conversion of T to E2 in the gonads. In addition, down-regulation of the mRNA expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, steroidogenic acute regulatory protein, CYP17, and 17beta-hydroxysteroid dehydrogenase was observed; this was predicted to reduce T concentrations and further contribute to the reduced E2 levels. SMC-induced changes in the expression of these steroidogenic genes correlated with decreased transcription of gonadotropic hormones (follicle-stimulating hormone and luteinizing hormone) and significantly elevated leptin expression. Furthermore, SMC also altered expression of the key enzyme in gamma-aminobutyric acid (GABA) synthesis, GABA receptors, and salmon gonadotropin-releasing hormone, thus affecting gonadotropin expression. Overall, SMC acted at multiple sites related to reproduction to reduce plasma E2 levels, consequently exerting an anti-estrogenic effect in female zebrafish. These effects were observed at environmentally relevant concentrations and highlight the importance of controlling SMC contamination.