Assay of vtg, ERs and PPARs as endpoint for the rapid in vitro screening of the harmful effect of Di-(2-ethylhexyl)-phthalate (DEHP) and phthalic acid (PA) in zebrafish primary hepatocyte cultures

In the last years the concern about the negative effects of phthalates on reproduction significantly increased. Considering that, at date data available dealing with the adverse outcome of Di-(2-ethylhexyl)- phthalate (DEHP) on the reproduction of several species are still contrasting, in this study, the effects induced by DEHP (0.05, 0.1, 1, 10 and 100 nM) and its active metabolite, phthalic acid (PA) (0.01, 0.1, 1 and 10 lM), were analyzed in zebrafish, Danio rerio, primary hepatocyte cultures, using target molecules involved in fish reproduction (vitellogenin – vtg and estrogen receptors – ERa, b1 and b2) and metabolism (peroxisome proliferators activated receptors – PPAR a, b, c). The use of in vitro culture, in fact, has the potential to significantly reduce the number of animals sacrificed for research allowing a precise control of the physical and chemical parameters that is often not possible in vivo. Moreover, since many toxicological studies revealed a sex specific response to toxicants, male and female primary hepatocyte cultures were set up to elucidate the possible gender specific effects of two common environmental phthalates. The increase of vtg levels observed in the culture media of male or female hepatocytes strongly evidenced the phthalates E2-like action. Moreover, the data obtained suggested that the observed different ERs isoforms modulation is otherwise associated with the vtg increase, depending on fish gender. Regarding PPARs, a similar trend of expression was found in both males and females. In conclusion, this study enforces the role of vtg as biomarker for evaluate the presence of environmental doses of DEHP and PA. Considering the similar gender modulation observed for vtg and PPARs, these molecules could be used for the rapid screening of the presence of DEHP and PA. Noteworthy the gender specific modulation observed for ERs opens a debate on the estrogenic mechanism of action of DEHP and PA and their role on vtg induction.