br Materials and methods br Results br Discussion
Materials and methods
Discussion In addition, the responses of CYP450s induced by metalaxyl are species-specific in the four cells. For example, metalaxyl lead to induction of CYP1A1, CYP1A2, and CYP2B1 in HepG2 cells, CYP1A2 and CYP2B1 in H4IIE cells, CYP1A1 and CYP2B1 in LMH cells, and CYP2B1 in L8824 cells. Generally, increased CYP450 enzymes induced by a xenobiotic should promote the catalysis of the xenobiotic to intermediates (Nebert et al., 1990). Furthermore, CYP450 enzyme-specific catalysis of xenobiotic indicates different metabolic pathways due to the variation of CYP450 enzymes in sort and structure (Guengerich, 2008). Therefore, the observation in the present study may suggest that metalaxyl is metabolized by different CYP450 enzymes in the four cells through various catalytical pathways. The suggestions were, at least in part, confirmed by the hindrance of attenuation of metalaxyl in the medium when the specific inhibitors were added. The biotransformation of metalaxyl has been reported in several species of organisms and the metabolites were identified in these studies (Abass et al., 2007, Li et al., 2013, WHO, 2002). Arguably, the metabolites of metalaxyl varied among the species. Li et al. (2013) reported that the major metabolite of metalaxyl was metalaxyl kd025 in in tomato and cucumber. In rat in vivo, the predominant metabolite was didemethylmetalaxyl (WHO, 2002). Two hydroxymetalaxyl derivatives were identified as the major metabolites in human liver microsomes (WHO, 2002). Although the metabolites were not detected in the present study due to the application of cell models instead of Whole organisms and the complexity of metabolites of metalaxyl in different species, it is speculated that there were various intermediates of metalaxyl existing in the medium of the four cells. Therefore, CYP450 enzyme-specific catalysis is believed to be one of the important contributors to the diversity of metabolites of metalaxyl. In addition, there were only six CYP450 enzymes were investigated in our study, although CYP450s consists of a large of subfamily enzymes. It is hardly to exclude other CYP450 enzymes involved in the metabolism of metalaxyl in these cells. In the present study, it was found that HepG2, H4IIE and LMH cells were inclined to remove S-metalaxyl and lead to decrease of ERs of metalaxyl, while L8824 cells were inclined to attenuate R-metalaxyl and resulted in an inverse shift of ER. These findings indicate the enantioselective accumulation of metalaxyl is species-specific. Enantioselective accumulation of metalaxyl has also been reported in plants and varied among species (Li et al., 2013, Wang et al., 2014). However, the enantioselective species-specific accumulation of metalaxyl was seldom compared in animals in previous studies. It was reported that the metabolism of S-metalaxyl was faster than that of R-metalaxyl in rat and rabbit hepatic microsomes (Zhang et al., 2012). On the contrary, Xu et al. (2011) found that invertebrate earthworm was inclined to accumulate S-metalaxyl. Comprehensively considering together, it is speculated that the metabolism of S-metalaxyl may be faster than R-metalaxyl in higher animals such as mammals and birds, and slower than R-metalaxyl in lower animals such as invertebrates and fish. The enantioselective species-specific accumulation of metalaxyl is believed to lead to the difference in toxicity outcomes among organisms due to the different toxicities of the enantiomers. In most of previous studies, the toxicity of R- metalaxyl was found to be greater than S- metalaxyl either in targeted and non-targeted organisms (Chen and Liu, 2008, Yao et al., 2009, Zadra et al., 2002) Therefore, the accumulation of metalaxyl might show more potential of toxicity in mammals and birds than in fish since mammals and birds are inclined to metabolize S-metalaxyl. But a reverse toxicity for the enantiomers of metalaxyl was also reported in earthworm in which R-metalaxyl possess less toxicity than S-metalaxyl (Xu et al., 2011), indicating that enantioselective toxicity of metalaxyl might also be species-specific. Therefore, it is suggested that enantioselective species-specific accumulation and enantioselective species-specific toxicity should be both considered when assessing the risk of residue of metalaxyl in organisms.