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

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.

Detection of Benzo[a]pyrene Diol Epoxide Adducts to Histidine and Lysine in Serum Albumin In Vivo by High-Resolution-Tandem Mass Spectrometry

“…In the early work by Tannenbaum et al, it was shown that (−)-anti enantiomer of BPDE binds to N τ in His in SA [7] and that (+) and (−) enantiomers of anti-BPDE exhibit different reactivity towards the nucleophilic sites in SA [8,13]. We have shown in vitro that (+)-anti-BPDE as well as (+/−)-syn-BPDE form adducts to His in SA [20,21]. It has earlier been shown that the (+)-anti enantiomer of BPDE is the most tumorigenic metabolite [13,22].…”

“…From our previous studies, it is indicated that His146 in SA in the two species have similar reactivity. In vitro alkylation with (±)-anti BPDE gives approximately the same adduct levels to His in mouse SA and in human SA, as well as the same ratio (see Section 2.3) of adduct levels from (+)-and (−)-anti-BPDE [20,21]. Further, the peptide sequence in SA close to His146 is identical in the two species, which indicates there is no large difference in the adduct forming capacity of His 146 [21].…”

“…Samples of human SA had previously been alkylated in vitro with (±)-anti-BPDE and (±)-syn-BPDE, respectively [20]. The general procedure was that BPDE solution in tetrahydrofuran was mixed with human SA (80 mg) in water (2 mL) to give a final solution of 0.5 µg BPDE per mg SA.…”

“…We have previously measured stable adducts from BPDE isomers in human SA in vitro, where cleavage of the protein was performed using hydrazine [18] or enzymatic digestion [19,20], followed by high-performance liquid chromatography (HPLC)-MS analysis. The major site of adduct formation in SA was found to be His146, while adducts to lysine (Lys) 195 were detected to a minor extent.…”

In Vitro Co-Exposure to CeO2 Nanomaterials from Diesel Engine Exhaust and Benzo(a)Pyrene Induces Additive DNA Damage in Sperm and Cumulus Cells but Not in Oocytes

“…We estimated that 30 ± 6% of the B a P was removed from the solution just by 1h settling and 57 ± 11% by 1h centrifugation. This could highlight the incomplete dissolution but also to the chemical instability of B a P in these abiotic conditions related to its high affinity for serum components (i.e., as albumin in Ferticult ® ) [ 47 , 48 , 49 , 50 ]. UV-vis spectrometry was used to estimate the affinity of B a P for the surface of the aged CeO 2 NMs in abiotic conditions.…”