Estrogenic activity of procymidone in primary cultured rainbow trout epatocytes

It has been shown that procymidone, a dicarboximide fungicide, alters sexual differentiation in vivo and in vitro. The aim of this study was to evaluate the estrogenic activity of this fungicide using the synthesis of vitellogenin (Vtg) in rainbow trout hepatocyte as a biological marker. The cells were treated for 24 h with procymidone 150 μM, using 17β-estradiol 20 μM as a positive control. The doses were chosen on the basis of cell viability (Neutral Red and MTT tests) and solubility. The results show that procymidone leads to a qualitative and quantitative increase in Vtg synthesis. In Western immonoblots, the 170 and 30 kDa bands, which respectively correspond to the monomeric form of Vtg and posvitine, were brighter in cells treated with procymidone and 17β-estradiol than those corresponding to the negative controls (cells treated for 24 h with DMSO 0.1% alone); ELISA showed that the cells treated with the fungicide and 17β-estradiol had a 48 and 76%, respectively, higher Vtg concentration than the negative controls (P<0.01). Western blotting also revealed the induction of HSP27 (27 KDa), which further confirms the estrogenic acitivity of procymidone as it is known that the 3′ region of HSP27/28 containing the gene mRNAs is induced by estrogen treatment. Procymidone increased also the production of both HSP70 protein (70 KDa) and free oxygen radicals. This last finding is in agreement with the toxic mechanism of dicarboximide fungicides. It can therefore be presumed that the estrogenic activity of procymidone in primary cultured trout hepatocytes is related to oxidative damage which, as many other studies have shown, can increase the levels of estrogens such as 17β-estradiol, and thus increase Vtg synthesis