Exposure profiling of reactive compounds in complex mixtures

Abstract

Humans are constantly exposed to mixtures, such as tobacco smoke, exhaust from diesel, gasoline or new bio-fuels, containing several 1000 compounds, including many known human carcinogens. Covalent binding of reactive compounds or their metabolites to DNA and formation of stable adducts is believed to be the causal link between exposure and carcinogenesis. DNA and protein adducts are well established biomarkers for the internal dose of reactive compounds or their metabolites and are an integral part of science-based risk assessment. However, technical limitations have prevented comprehensive detection of a broad spectrum of adducts simultaneously. Therefore, most studies have focused on measurement of abundant individual adducts. These studies have produced valuable insight into the metabolism of individual carcinogens, but they are insufficient for risk assessment of exposure to complex mixtures. To overcome this limitation, we present herein proof-of-principle for comprehensive exposure assessment, using N-terminal valine adduct profiles as a biomarker. The reported method is based on our previously established immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with modification to enrich all N-terminal valine alkylated peptides. The method was evaluated using alkylated peptide standards and globin reacted in vitro with alkylating agents (1,2-epoxy-3-butene, 1,2:3,4-diepoxybutane, propylene oxide, styrene oxide, N-ethyl-N-nitrosourea and methyl methanesulfonate), known to form N-terminal valine adducts. To demonstrate proof-of-principle, the method was successfully applied to globin from mice treated with four model compounds. The results suggest that this novel approach might be suitable for in vivo biomonitoring.

Keywords: 1,2 epoxy-3-butene; 1,2:3,4-diepoxybutane; 1,3-butadiene; 1-hydroxy (or 2-hydroxy)-propyl-valine; 1-phenyl-2-hydroxyethyl-valine or 2-phenyl-2-hydroxyethyl-valine; 2,3,4-trihydroxybutyl-valine; 3,4-epoxy-1,2-butanediol; BD; Biomarkers; Biomonitoring; DEB; EB; EB-diol; ENU; ENU-Val; Et-Val; FA; H(2)N-Val; HB-Val; HP-Val; Hb; IA; LC–MS/MS; MMS; Me-Val; Mixtures; Multiple exposure detection; N,N-(2,3-dihydroxy-1,4-butadiyl)-valine; N-(2-hydroxy-3-buten-1-yl)-valine; N-ethyl-N-nitrosourea; N-terminal valine adducts; PO; SO; SO-Val; THB-Val; carbamoylated-valine; ethyl-valine; formic acid; hemoglobin; immunoaffinity; liquid chromatography–tandem mass spectrometry; methyl-methanesulfonate; methyl-valine; non-alkylated-valine; propylene oxide; pyr-Val; styrene oxide.

Figure 1

Scheme of adduct profiling assay. The alkylated N-terminal peptides of the α-chain are isolated from trypsin hydrolyzed globin by series of depletion and selection IA chromatography, prior to analysis by LC-MS/MS as described in Materials and Methods.

Figure 2

Overview of selected model compounds and their corresponding N-terminal valine adducts.

Figure 3

Characterization of mouse SO-Val (1-11) standard. Mouse 1-11 peptide standard was reacted with SO in 0.1 M NH₄HCO₃ at pH 6.5 for 72 h. MS/MS fragmentation suggests single alkylation at the N-terminal valine.

Figure 4

Extracted ion chromatograms of mouse globin treated in vitro with a mixture of EB, DEB, PO and SO, MMS and ENU. The treated globin was processed as described in Materials and Methods and analyzed by LC-MS/MS. [¹³C₅]SO-Val was utilized as internal standard.

Figure 5

Representative extracted ion-chromatogram of exposed mouse globin. B6C3F1 mice were treated with EB, SO, MMS and ENU and globin was isolated and analyzed as described in Materials and Methods. [¹³C₅]SO-Val was utilized as internal standard.

Figure 6

Proposed formation of the ENU-derived carbamoyl adduct (ENU-Val).