title: Integrated transcriptional and proteomic analysis with in vitro biochemical assay reveal the important role of CYP3A46 in T-2 toxin hydroxylation in porcine primary hepatocytes.
abstract: Both T-2 toxin and its metabolites are highly potent mycotoxins that can cause severe human and animal diseases upon exposure. Understanding the toxic mechanism and biotransformation process of T-2 toxin at a cellular level is essential for the development of counter-measures. We investigated the effect of T-2 toxin in porcine primary hepatocytes using porcine genome array and two-dimensional difference gel electrophoresis with matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry. Integrated transcriptional and proteomic analysis demonstrated that T-2 toxin adversely affected porcine hepatocytes by initiating lipid metabolism disorder, oxidative stress response, and apoptosis. In addition, xenobiotic metabolism genes, including cytochrome P450 3As (CYP3A46 and CYP3A39), carboxylesterase 1Cs (CES1C4 and CES1C5), and epoxide hydrolase (EPHX1), increased in T-2 toxin treatment cells. Using HepG2 cells to over-express the recombinant xenobiotic metabolism genes above and rapid resolution liquid chromatography/tandem mass spectrometry to detect metabolites of T-2 toxin, we determined that porcine CYP3A46 mainly catalyzed T-2 to form 3'-hydroxy-T-2, which was further confirmed by purified CYP3A46 protein. However, recombinant porcine CES1C5 and EPHX1 did not enhance hydrolysis and de-epoxidation of T-2 implying that other esterases and epoxide hydrolases may play dominant roles in those reactions.
authors.affiliation: Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Jianshe
authors.initials: J
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Jiang
authors.forename: Hongxia
authors.initials: H
authors.lastname: Zhang
authors.forename: Junping
authors.initials: J
authors.lastname: Wang
authors.forename: Hua
authors.initials: H
authors.lastname: Cai
authors.forename: Cheng
authors.initials: C
authors.lastname: Li
authors.forename: Kangbai
authors.initials: K
authors.lastname: Li
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Xuejiang
authors.initials: X
authors.lastname: Guo
authors.forename: Guangxun
authors.initials: G
authors.lastname: Zou
authors.forename: Dazhi
authors.initials: D
authors.lastname: Wang
authors.forename: Yiqun
authors.initials: Y
authors.lastname: Deng
authors.forename: Jiayin
authors.initials: J
authors.lastname: Dai
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D001681:Biological Assay; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004851:Epoxide Hydrolases / Q000235:genetics / Q000378:metabolism; D005670:Fusarium / Q000737:chemistry; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D050356:Lipid Metabolism / Q000187:drug effects; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D061251:Primary Cell Culture; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D034421:Sus scrofa; D013605:T-2 Toxin / Q000378:metabolism / Q000633:toxicity; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D001189:Aryl Hydrocarbon Hydroxylases; D002265:Carboxylic Ester Hydrolases; D004851:Epoxide Hydrolases; D013605:T-2 Toxin