Browsing by keyword "Chromatography, Liquid"

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    • Glucuronidation of the oxidative cytochrome P450-mediated phenolic metabolites of the endocrine disruptor pesticide: methoxychlor by human hepatic UDP-glucuronosyl transferases
      Hazai, Eszter; Gagne, Peter V.; Kupfer, David (2004-06-19)
      Methoxychlor, a currently used pesticide, is a proestrogen exhibiting estrogenic activity in mammals in vivo. Methoxychlor undergoes oxidative metabolism by cytochromes P450, yielding 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M) and 1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane (bis-OH-M) as main metabolites. Since humans may be exposed to these estrogenic metabolites, which are potential substrates of UDP-glucuronosyltransferases (UGTs), their glucuronide conjugation was investigated with human liver preparations and individual UGTs. Incubation of both mono-OH-M and bis-OH-M with human liver microsomes formed monoglucuronides. The structures of the glucuronides were identified by liquid chromatography/tandem mass spectometry. Examination of cDNA-expressed recombinant human hepatic UGTs revealed that several catalyze glucuronidation of both compounds. Among the cDNA-expressed UGT1A enzymes, UGT1A9 seemed to be the main catalyst of formation of mono-OH-M-glucuronide, whereas UGT1A3 seemed to be the most active in bis-OH-M-glucuronide formation. Furthermore, the chiral selectivity of mono-OH-M glucuronidation was examined. The results of the incubation of single enantiomers generally agreed with the chiral analyses of mono-OH-M derived from the glucuronidase digestion of the glucuronides of the racemic mono-OH-M. There was a relatively slight but consistent enantioselective preference of individual UGT1A1, UGT1A3, UGT1A9, and UGT2B15 enzymes for glucuronidation of the S- over the R-mono-OH-M, whereas in human liver microsomes differences were observed among donors in generating the respective R/S-mono-OH-M ratio. Since it was previously shown that human liver microsomes demethylate methoxychlor mainly into S-mono-OH-M, the observation that UGT1A isoforms preferentially glucuronidate the S-mono-OH-M suggests a suitable mechanism for eliminating this major enantiomer. This enantiomeric preference, however, is not extended to all samples of human liver microsomes that we tested.

    • p38 MAPK-mediated regulation of Xbp1s is crucial for glucose homeostasis
      Lee, Jaemin; Sun, Cheng; Zhou, Yingjiang; Lee, Justin; Gokalp, Deniz; Herrema, Hilde; Park, Sang Won.; Davis, Roger J.; Ozcan, Umut (2011-09-04)
      Here we show that p38 mitogen-activated protein kinase (p38 MAPK) phosphorylates the spliced form of X-box binding protein 1 (Xbp1s) on its Thr48 and Ser61 residues and greatly enhances its nuclear migration in mice, whereas mutation of either residue to alanine substantially reduces its nuclear translocation and activity. We also show that p38 MAPK activity is markedly reduced in the livers of obese mice compared with lean mice. Further, we show that activation of p38 MAPK by expression of constitutively active MAP kinase kinase 6 (MKK6Glu) greatly enhances nuclear translocation of Xbp1s, reduces endoplasmic reticulum stress and establishes euglycemia in severely obese and diabetic mice. Hence, our results define a crucial role for phosphorylation on Thr48 and Ser61 of Xbp1s in the maintenance of glucose homeostasis in obesity, and they suggest that p38 MAPK activation in the livers of obese mice could lead to a new therapeutic approach to the treatment of type 2 diabetes.