Abstract
Electrophilic diol epoxide metabolites are involved in the carcinogenicity of benzo[a]pyrene, one of the widely studied polycyclic aromatic hydrocarbons (PAHs). The exposure of humans to this PAH can be assessed by measuring stable blood protein adducts, such as to histidine and lysine in serum albumin, from their reactive metabolites. In this respect, measurement of the adducts originating from the genotoxic (+)-anti-benzo[a]pyrene diol epoxide is of interest. However, these are difficult to measure at such low levels as are expected in humans generally exposed to benzo[a]pyrene from air pollution and the diet. The analytical methods detecting PAH-biomarkers still suffer from low selectivity and/or detectability to enable generation of data for calculation of in vivo doses of specific stereoisomers, for evaluation of risk factors and assessing risk from exposures to PAH. Here, we suggest an analytical methodology based on high-pressure liquid chromatography (HPLC) coupled to high-resolution tandem mass spectrometry (MS) to lower the detection limits as well as to increase the selectivity with improvements in both chromatographic separation and mass determination. Method development was performed using serum albumin alkylated in vitro by benzo[a]pyrene diol epoxide isomers. The (+)-anti-benzo[a]pyrene diol epoxide adducts could be chromatographically resolved by using an HPLC column with a pentafluorophenyl stationary phase. Interferences were further diminished by the high mass accuracy and resolving power of Orbitrap MS. The achieved method detection limit for the (+)-anti-benzo[a]pyrene diol epoxide adduct to histidine was approximately 4 amol/mg serum albumin. This adduct as well as the adducts to histidine from (−)-anti- and (+/−)-syn-benzo[a]pyrene diol epoxide were quantified in the samples from benzo[a]pyrene-exposed mice. Corresponding adducts to lysine were also quantified. In human serum albumin, the anti-benzo[a]pyrene diol epoxide adducts to histidine were detected in only two out of twelve samples and at a level of approximately 0.1 fmol/mg.
Highlights
Polycyclic aromatic hydrocarbons (PAHs) are widely present in the environment as a result of incomplete combustion of organic matter
The Benzo[a]pyrene diol epoxides (BPDEs) adducts to His and Lys in serum albumin (SA) were monitored with Orbitrap mass spectrometry (MS) at instrumental settings described in Materials and Methods, Section 2.4
The present study has demonstrated an high-pressure liquid chromatography (HPLC)/HRMS/MS methodology to detect and identify BPDE-His adducts at the sub-fmol/mg SA level in both benzo[a]pyreneexposed mice and in humans
Summary
Polycyclic aromatic hydrocarbons (PAHs) are widely present in the environment as a result of incomplete combustion of organic matter. One of the most well studied PAHs is benzo[a]pyrene, a PAH consisting of five fused benzene rings and classified as a human carcinogen [5]. PAHs can be metabolized by cytochrome P450 and epoxide hydrolase to the corresponding diol epoxides. These electrophilic metabolites have a potential to cause mutations depending on their ability to react with DNA [2,5,6] and are able to react with nucleophilic sites of amino acids in blood proteins. Benzo[a]pyrene diol epoxides (BPDEs) are relatively well-studied in this respect. The research group of Tannenbaum et al showed that adducts from BPDE are formed to serum albumin (SA) of humans [7,8] by reaction with the carboxylate side chains of glutamic and aspartic acid and the imidazole ring of histidine (His) 146, as well as with the carboxylate groups in hemoglobin (Hb) [9]
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