Interaction of benzo[a]pyrene diol epoxide isomers with human serum albumin: Site specific characterisation of adducts and associated kinetics.

Hitesh V Motwani,Margareta Törnqvist,Emelie Westberg

doi:10.1038/srep36243

Abstract

Carcinogenicity of benzo[a]pyrene {B[a]P, a polycyclic aromatic hydrocarbon (PAH)} involves DNA-modification by B[a]P diol epoxide (BPDE) metabolites. Adducts to serum albumin (SA) are not repaired, unlike DNA adducts, and therefore considered advantageous in assessment of in vivo dose of BPDEs. In the present work, kinetic experiments were performed in relation to the dose (i.e. concentration over time) of different BPDE isomers, where human SA (hSA) was incubated with respective BPDEs under physiological conditions. A liquid chromatography (LC) tandem mass spectrometry methodology was employed for characterising respective BPDE-adducts at histidine and lysine. This strategy allowed to structurally distinguish between the adducts from racemic anti- and syn-BPDE and between (+)- and (−)-anti-BPDE, which has not been attained earlier. The adduct levels quantified by LC-UV and the estimated rate of disappearance of BPDEs in presence of hSA gave an insight into the reactivity of the diol epoxides towards the N-sites on SA. The structure specific method and dosimetry described in this work could be used for accurate estimation of in vivo dose of the BPDEs following exposure to B[a]P, primarily in dose response studies of genotoxicity, e.g. in mice, to aid in quantitative risk assessment of PAHs.

Highlights

Adducts formed through covalent binding of low molecular weight electrophilic compounds to nucleophilic sites on blood proteins are often used as a surrogate biomarker for the corresponding DNA adducts[9]
One approach to measure the BPDE adducts to serum albumin (SA) or Hb is to detach the adduct by hydrolysis to form corresponding tetrols, which are detected by gas chromatography (GC) or liquid chromatography (LC) in combination with fluorescence or mass spectrometry (MS)
We developed a method for LC-MS/MS measurement of histidine and lysine adducts from (±)-anti-BPDE in human SA (hSA) based on enzymatic digestion[21]

Summary

Introduction

Adducts formed through covalent binding of low molecular weight electrophilic compounds to nucleophilic sites on blood proteins are often used as a surrogate biomarker for the corresponding DNA adducts[9]. Carboxylic ester adducts in albumin with aspartate and glutamate, considered favourable for binding the (+)-anti-BPDE18, could be released as tetrols following mild acidic hydrolysis in vitro. This approach has shown to be sensitive enough for application to studies of PAH-exposed humans, for instance occupationally exposed workers[17]. The ester adducts are prone to undergo hydrolysis in vivo These circumstances make it difficult to calculate dose in vivo from measured tetrols and they are not suitable as biomarkers for in vivo dose assessment. Methods to measure histidine adducts from (±)-anti-BPDE in human SA (hSA) were studied. It has not been possible to use these methods in their present form to measure in vivo dose of the individual BPDE isomers, e.g. in mice

Methods

Results

Conclusion