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INVESTIGATION OF THE RADICAL-MEDIATED PRODUCTION OF BENZENE OXIDE PROTEIN ADDUCTS IN VITRO AND IN VIVO
Citation:
Troester, M. A., A B. Lindstrom, AND S. M. Rappaport. INVESTIGATION OF THE RADICAL-MEDIATED PRODUCTION OF BENZENE OXIDE PROTEIN ADDUCTS IN VITRO AND IN VIVO. Presented at Society of Toxicology Meeting, San Francisco, CA, March 25-29, 2001.
Impact/Purpose:
The objective of this task is to develop state-of-the-art methods for measuring xenobiotic compounds, to include the isolation of the analyte from the appropriate matrix (extraction), preconcentration (typically sorbent-based), and analysis via GC/MS and/or LC/MS. Once established, these methods will be applied in small scale pilot studies or demonstration projects. Particular emphasis will be placed on methods which are readily transferable to other laboratories, including those within the Human Exposure and Atmospheric Sciences Division (HEASD), the National Exposure Research Laboratory (NERL), other EPA Laboratories, Program Offices, Regions, and academic institutions.
Specific objectives of this task include the following:
1) Development of GC/MS and LC/MS methods for the measurement of key xenobiotic compounds and their metabolites (to include the pyrethroid pesticides, perfluorinated organic compounds, and the BFRs) in relevant environmental and biological matrices.
2) Development of efficient low cost methods for the extraction and clean up of these compounds collected from relevant matrices.
3) Determination of xenobiotic compound and metabolite concentrations in samples derived from laboratory and field monitoring studies to help assess exposures and evaluate associated risks.
Description:
High background levels of benzene oxide (BO) adducts with hemoglobin and albumin (BO-Hb and BO-Alb) have been measured in unexposed humans and animals. To test the influence of radical-mediated pathways on production of these BO-protein adducts, we employed Fenton chemistry to generate free radicals in vitro. Incubations containing human Hb, ascorbate, and H2O2 produced levels of BO-Hb that were approximately 13 times higher than control incubations. Addition of sodium benzoate to these incubations led to even greater levels of BO-Hb while the iron chelator Desferrioxamine (1 mM) diminished production of BO-Hb. Results suggest a radical-mediated pathway for production of BO-Hb in vitro. We then tested the radical-mediated production of BO-protein adducts in vivo by administering a single dose of 0 to 1600 mg of carbon tetrachloride (CCl4) (a known producer of free radicals in vivo) per kg body weight to 24 male F344 rats, with and without co-exposure to sodium benzoate. Comparison of adduct concentration by dose of CCl4 using an analysis of variance (ANOVA) procedure suggests that there was no significant increase in the concentration of BO-Alb (p = 0.576) or BO-Hb (p = 0.204) as a function of CCl4 dose. Simultaneous treatment with CCl4 and aqueous sodium benzoate (5 mmol/kg body weight) also failed to significantly increase levels of BO-Alb (p = 0.167) or BO-Hb (p = 0.771) as a function of CCl4 dose. Thus, we were unable to demonstrate radical-mediated production of background BO-Alb and BO-Hb in vivo.
Supported in part by NIEHS Superfund Basic Research Program Grant P42-ES05948 and in part by the United States Environmental Protection Agency. It has been subjected to U.S. EPA review and approved for publication.