Attomole detection of in vivo protein targets of benzene in mice: evidence for a highly reactive metabolite

Modified proteins were detected in liver and bone marrow of mice following treatment with [(14)C]benzene. Stained sections were excised from one-dimensional and two-dimensional gels and converted to graphite to enable (14)C/(13)C ratios to be measured by accelerator mass spectrometry. Protein adducts of benzene or its metabolites were indicated by elevated levels of (14)C. A number of proteins were identified by in-gel proteolysis and conventional mass spectrometric methods with the low molecular weight proteins identified including hemoglobin and several histones. The incorporation of (14)C was largely proportional to the density of gel staining, giving little evidence that these proteins were specific targets for selective labeling. This was also true for individual histones subfractionated with Triton-acid-urea gels. A representative histone, H4, was isolated and digested with endopeptidase Asp-N, and the resulting peptides were separated by high performance liquid chromatography. (14)C levels in collected fractions were determined, and the peptides were identified by conventional mass spectrometry. The modifications were distributed throughout the protein, and no particular amino acids or groups of amino acids were identified as selective targets. Thus chemical attack by one or more benzene metabolites upon histones was identified and confirmed, but the resulting modifications appeared to be largely nonspecific. This implies high reactivity toward proteins, enabling such attack to occur at multiple sites within multiple targets. It is not known to what extent, if any, the modification of the core histones may contribute to the carcinogenicity of benzene.