A new approach for measuring protein adducts from benzo[a]pyrene diolepoxide by high performance liquid chromatography/tandem mass spectrometry

Abstract

The long-range goal of the present study is the development of a general approach for in vivo dosimetry of reactive metabolites of polycyclic aromatic hydrocarbons (PAHs), to be used as a tool in cancer risk assessment. With benzo[a]pyrene (BaP) chosen as indicator and a model of PAHs this study aims at the development of a method for the determination of adducts to histidine (His) in hemoglobin (Hb) and serum albumin (SA) of reactive metabolites of BaP. The predominantly mutagenic metabolite of BaP has been shown to be a diolepoxide isomer, +(anti)r-7, t-8-dihydroxy-t-9,10-epoxy-7,8, 9,10-tetrahydrobenzo[a]pyrene (+BPDE). In comparison with other methods for protein degradation, hydrazinolysis was found to be sufficiently effective and mild. The His adduct isolated after protein hydrazinolysis, with protection by tert-butyloxycarbonyl (Boc) of the hydrazide and alpha-amino groups, was shown to be N(im)- +/- (r-7, t-8, t-9-trihydroxy-7, 8, 9, 10-tetrahydrobenzo[a]pyren-c-10-yl)-N(alpha), N(2)-bis(tert-butyloxycarbonyl)-L-histidinehydrazide. Isomers of this compound, used as references, were synthesized and characterized by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Adducts in Hb and SA from in vitro treatment with BPDE were characterized after hydrazinolysis by HPLC-UV/MS, muHPLC/MS/MS and gas chromatography/mass spectrometry (GC/MS). Approximately 70 and 10% of the isolated BPDE adducts from SA and Hb, respectively, were His adducts. Other products were released as BaP tetrols and BaP triols. For the purpose of enrichment/purification of BPDE-His adducts, C(18) and cation exchange solid phase extraction (SPE) were utilized. The sensitivity obtained by this new approach, based on hydrazinolysis of protein, enrichment by SPE and analysis with muHPLC/MS/MS (APCI), is in the low-fmole range.