CYP1A2-and CYP3A4-mediated In Vitro Metabolism Of Thioxanthones Type Photoinitiators

As one kind of photoinitiators,thioxanthones(TXs)are applied to the industries of UV ink,UV coating,UV glue and printing and are especially used in food packaging as important components of UV ink.During their usage,their residues were detected in various environmental media such as milk,juice,fruit nectars and drinks,indoor dust and sewage sludge.They caused potential risk toward human health and ecoenvironment,increasingly arising public concern.However,till now studies on the biotransformation and human health risk of TXs were still limited.Using four typical TXs(TX,2-ITX,DETX and 2-Cl-TX)as the targets,we investigated the CYP enzyme-mediated metabolism of TXs.After the incubation with human liver microsome(Human liver microsome,HLM),the metabolites of 2-Cl-TX were analyzed.CYP1A2 and CYP3A4 are dominant to the metabolism of 2-Cl-TX.The estrogenic effects of TXs before and after metabolism were compared.We also investigated the effects of TXs on the mRNA expression and enzyme activities of CYP1A2 and CYP3A4.The obtained results were summarized as followings:(1)In vitro HLM-mediated metabolism of 2-Cl-TX experiment was carried out.2-Cl-TX was metabolized by HLM and two main products(M1 and M2)were detected by Q-TOF.Human CYP1A2 enzyme and CYP3A4 enzyme parcitipated the metabolism of 2-Cl-TX and affected the amount of M1 and M2.M1 would decrease and M2 would increase when CYP1A2 was inhibited.M1 would disappear and M2 would decrease significantly,when CYP3A4 was inhibited.Combined with Q-TOF and SMARTCyp,the structures of products were predicted.M1 was a sulfoxide produced by oxidation of sulfur atoms;M2 was a hydroxylated derivative.(2)Estrogenic effects of TX,2-ITX,DETX and 2-Cl-TX were investigated by recombinant human estrogen receptor(h-ER)yeast two-hybrid assay.Four TXs showed no siginificant estrogenic effects,indicating no metabolic activation.(3)The mRNA expression level of CYP1A2 and CYP3A4 enzyme in HepG2 cells was analyzed by Quantitative Real-time PCR.At the concentratae of 0.5 μM,2-Cl-TX significantly up-regulated the mRNA expression level of both CYP1A2 and CYP3A4 enzyme in a dose-effect dependent manner.The degree of up-regulation of CYP1A2 mRNA expression was higher than that of CYP3A4.(4)The effects of four TXs on enzyme activity of human CYP1A2 and CYP3A4 were analyzed by P450-GloTM Assay.These four TXs could inhibit the activity of CYP1A2 and CYP13A4 enzyme and showed a dose-effect dependent manner.Four TXs are the significant inhibitors of CYP1A2 and CYP3A4 enzyme.TXs have stronger inhibition to the activity of CYP3A4 enzyme than that of CYP1A2 and this inhibition potency toward CYP1A2 and CYP3A4 ranks 2-Cl-TX>2-ITX>DETX>TX.This study provided reference for the biotransformation and metabolic toxicity,and provided support for the determination of TXs and its metabolites,facilitating the health risk assessment of TXs.The toxicity of metabolites should be taken into consideration in future risk assessment of pollutants.The investigation on interactions between TXs and CYP1A2 and CYP3A4 enzymes provided reference for studies of the interactions between related compounds and CYP enzyme.