Format

Send to

Choose Destination
Chem Res Toxicol. 2007 Jun;20(6):859-67. Epub 2007 May 5.

Protein targets of reactive electrophiles in human liver microsomes.

Author information

1
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

Erratum in

  • Chem Res Toxicol. 2008 Aug;21(8):1642.

Abstract

Liver microsomes are widely used to study xenobiotic metabolism in vitro, and covalent binding to microsomal proteins serves as a surrogate marker for toxicity mediated by reactive metabolites. We have applied liquid chromatography-tandem mass spectrometry (LC-MS-MS) to identify protein targets of the biotin-tagged model electrophiles 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) in human liver microsomes. The biotin-tagged peptides resulting from in-gel tryptic digestion were enriched by biotin-avidin chromatography and LC-MS-MS was used to identify 376 microsomal cysteine thiol targets of BMCC and IAB in 263 proteins. Protein adduction was selective and reproducible, and only 90 specific cysteine sites in 70 proteins (approximately 25% of the total) were adducted by both electrophiles. Differences in adduction selectivity correlated with different biological effects of the compounds, as IAB- but not BMCC-induced ER stress in HEK293 cells. Targeted LC-MS-MS analysis of microsomal glutathione-S-transferase cysteine 50, a target of both IAB and BMCC, detected time-dependent adduction by the reactive acetaminophen metabolite N-acetyl-p-benzoquinoneimine during microsomal incubations. The results indicate that electrophiles selectively adduct microsomal proteins, but display differing target selectivities that correlate with differences in toxicity. Analysis of selected microsomal protein adduction reactions thus could provide a more specific indication of potential toxicity than bulk covalent binding of radiolabeled compounds.

PMID:
17480101
PMCID:
PMC2556149
DOI:
10.1021/tx700031r
[Indexed for MEDLINE]
Free PMC Article

Publication type, MeSH terms, Substances, Grant support

Supplemental Content

Full text links

Icon for American Chemical Society Icon for PubMed Central
Loading ...
Support Center