Capturing labile sulfenamide and sulfinamide serum albumin adducts of carcinogenic arylamines by chemical oxidation

Abstract

Aromatic amines and heterocyclic aromatic amines (HAAs) are a class of structurally related carcinogens that are formed during the combustion of tobacco or during the high temperature cooking of meats. These procarcinogens undergo metabolic activation by N-oxidation of the exocyclic amine group to produce N-hydroxylated metabolites, which are critical intermediates implicated in toxicity and DNA damage. The arylhydroxylamines and their oxidized arylnitroso derivatives can also react with cysteine (Cys) residues of glutathione or proteins to form, respectively, sulfenamide and sulfinamide adducts. However, sulfur-nitrogen linked adducted proteins are often difficult to detect because they are unstable and undergo hydrolysis during proteolytic digestion. Synthetic N-oxidized intermediates of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogenic HAA produced in cooked meats, and 4-aminobiphenyl, a carcinogenic aromatic amine present in tobacco smoke, were reacted with human serum albumin (SA) and formed labile sulfenamide or sulfinamide adducts at the Cys(34) residue. Oxidation of the carcinogen-modified SA with m-chloroperoxybenzoic acid (m-CPBA) produced the arylsulfonamide adducts, which were stable to heat and the chemical reduction conditions employed to denature SA. The sulfonamide adducts of PhIP and 4-ABP were identified, by liquid chromatography/mass spectrometry, in proteolytic digests of denatured SA. Thus, selective oxidation of arylamine-modified SA produces stable arylsulfonamide-SA adducts, which may serve as biomarkers of these tobacco and dietary carcinogens.

Figure 1

(A) UV spectra of modified LQQC*PF peptides and ESI/MS product ion spectra of (B) LQQC*PF sulfonic acid ([M + H]⁺ at m/z 783.3), (C) LQQC*PF (C-[S=O]-PhIP) sulfinamide ([M+H]⁺ at m/z 973.3), and (D) LQQC*PF (C-[SO₂]-PhIP) sulfonamide ([M+H]⁺ at m/z 989.5).

Figure 2

UPLC-ESI/MS² chromatograms of Cys peptide adducts recovered from 100 ng of SA digest of (A) HONH-PhIP-modified SA, following trypsin/chymotrypsin digestion; (B) HONH-PhIP-modified SA, which was denatured by heating the SA in 6 M guanidine–HCl with DTT, followed by alkylation with IAA, prior to trypsin/chymotrypsin digestion; and (C) HONH-PhIP-modified SA was oxidized with m-CPBA prior to denaturation and digestion with trypsin/chymotrypsin. Targeted UPLC-ESI/MS² was done on the following peptides: LQQC*PFEDHVK (C-[S=O]-PhIP) as triply charged species ([M+3H]³⁺ at m/z 527.9); LQQC*PFEDHVK (C-[SO₂]-PhIP) ([M+3H]³⁺ at m/z 533.2); LQQC*PF (C-[S=O]-PhIP) ([M+H] ⁺ at m/z 973.3); and LQQC*PF (C-[SO₂]-PhIP) ([M+H]⁺ at m/z 989.4). The scale of the signals for all 3 treatments were normalized to the response of LQQC*PFEDHVK (C-[SO₂]-PhIP) in panel C.

Figure 3

Estimates of LQQC*PFEDHVK adducts recovered from trypsin/chymotrypsin digestion of m-CPBA-oxidized or nonoxidized HONH-PhIP-modified SA, without or with denaturation. Adduct formation was estimated by UPLC-ESI/MS², employing LQQC*PF (C-[SO₂]-[²H₅]-PhIP) sulfonamide and LQQC*PF (C-[S=O]-[²H₅]-PhIP) sulfinamide as internal standards. The ionization efficiencies of the mixed cleavage peptide adducts LQQC*PFEDHVK were assumed to be comparable to the ionization efficiencies of the internal standards of [²H₅]-PhIP-modified LQQC*PF. Total PhIP bound to SA was determined by UV measurement at 320 nm.

Figure 4

Reconstructed ion chromatograms of N-acetoxy-PhIP-modified SA monitoring: PhIP ([M+H]⁺, m/z 225.1 → 210.1); 5-HO-PhIP ([M+H]⁺, m/z 241.1 → 210.1); and LQQC*PFEDHVK (C-[SO₂]-PhIP) ([M+3H]³⁺, m/z 533.2 → 225.2, 521.8, 670.5) in (A) Non-oxidized SA and (B) m-CPBA-oxidized SA, digested with trypsin/chymotrypsin. The scales of the signals were fixed to the maximum response of the respective analytes between the nonoxidized and oxidized samples.

Figure 5

UPLC-ESI/MS² chromatograms of Cys peptide adducts recovered from HONH-4-ABP-modified SA following digestion with trypsin/chymotrypsin (left panel) and corresponding product ion mass spectra of (A) LQQC*PFEDHVK (C-[S=O]-4-ABP) sulfinamide ([M+3H]³⁺ at m/z 509.6), (B) LQQC*PFEDHVK (C-[SO₂]-4-ABP) sulfonamide ([M+3H]³⁺ at m/z 514.9), (C) LQQC*PF (C-[S=O]-4-ABP) sulfinamide ([M+H]⁺ at m/z 918.4, and (D) LQQC*PF (C-[SO₂]-4-ABP) sulfonamide ([M+H]⁺ at m/z 934.4).

Figure 6

UPLC-ESI/MS² chromatograms of (A) a putative C*PF (C-[S=O]-4-ABP) sulfinamide adducts ([M+H]⁺ at m/z 549.2), (B) C*PF (C-[SO₂]-4-ABP) sulfonamide ([M+H]⁺ at m/z 565.2), and (C) 4-ABP ([M+H]⁺, m/z 170.1 → 153.1) recovered from HONH-4-ABP-modified SA digested with Pronase E, leucine aminopeptidase, and prolidase. The scale of the signal of the putative C-[S=O]-4-ABP adduct, which was not detected, was normalized to the scale of response of (C-[SO₂]-4-ABP). D) Product ion mass spectrum of C*PF (C-[SO₂]-4-ABP) sulfonamide adduct.

Scheme 1

Formation of SA-Cys sulfenamide, sulfinamide, and sulfonamide adducts with N-oxidized metabolites of PhIP. The genotoxic metabolite HONH-PhIP undergoes oxidation to form Nitroso-PhIP, which forms a sulfinamide adduct, following rearrangement of the semimercaptal. HONH-PhIP forms reactive sulfate or acetate esters following conjugation reactions with phase II enzymes. These esters of HONH-PhIP react with SA to form the sulfenamide adduct at Cys. The superoxide generated during the oxidation of HONH-PhIP to Nitroso-PhIP, or other reactive oxygen species (ROS), can convert the labile sulfenamide and sulfinamide adducts of SA-Cys-PhIP to the stable sulfonamide linkage.