Attomole Detection of in Vivo Protein Targets of Benzene in Mice

Abstract

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 twodimensional 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.

Current perspectives of 14 C-isotope measurement in biomedical accelerator mass spectrometry

“…Protein binding in liver and kidney exhibited approximate linearity with dose whereas in the bladder non-linearity was observed. No DNA adducts were detected in the bladder supporting the hypothesis for this compound that bladder cancer induction arises through regenerative hyperplasia rather than through a genotoxic mechanism [27].…”

“…In addition to binding to DNA, substantial protein modification was seen after benzene exposure [27]. Liver and bone marrow proteins were run out on one dimensional and two dimensional gels, bands excised and analysed by AMS.…”

Naphthalene metabolism in relation to target tissue anatomy, physiology, cytotoxicity and tumorigenic mechanism of action

“…Validation in this context would focus on estimation of key parameters that are feasible to measure in a 1-year time-frame in a series of short-term experiments using human volunteers under an IRBand USEPA-HSRB-approved study protocol. These experiments would involve controlled administration of approximately 1 part per trillion of 14 C-radiolabeled naphthalene in air (i.e., a concentration that is a fraction of ambient levels of naphthalene typically found in indoor air, as discussed in Griego et al, 2008 by inhalation for 6 h. Naphthalene and metabolites in blood and urine at these exposure levels can be analyzed readily by accelerator mass spectrometry (AMS) (see, e.g., Bogen et al, 1998;Dingley et al, 1998;Williams et al, 2002;Cupid et al, 2004). Measured samples should include at least hourly blood samples.…”

Protein Damage by Reactive Electrophiles: Targets and Consequences

“…These studies also provided some degree of cross-validation, as many of the proteins identified were targets of multiple electrophiles. Williams et al (51) applied highly sensitive accelerator mass spectrometry (AMS) to detect 14 C enrichment in proteins from liver and bone marrow of mice after treatment with [ 14 C]benzene. The overall approach was analogous to that described above, except that AMS analysis detected attomolar levels of 14 C. The detection of radiolabels associated with histones in bone marrow samples led them to analyze proteolytic digests by AMS, and 14 C label was found to be widely distributed across the protein sequences analyzed.…”