Mass Spectrometric Characterization of Human Serum Albumin Adducts Formed with N-Oxidized Metabolites of 2-Amino-1-methylphenylimidazo[4,5-b]pyridine in Human Plasma and Hepatocytes

Abstract

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogenic heterocyclic aromatic amine formed in cooked meats, is metabolically activated to electrophilic intermediates that form covalent adducts with DNA and protein. We previously identified an adduct of PhIP formed at the Cys34 residue of human serum albumin following reaction of albumin with the genotoxic metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP). The major adducted peptide recovered from a tryptic/chymotryptic digest was identified as the missed-cleavage peptide LQQC*[SO2PhIP]PFEDHVK, a [Cysteine-S-yl-PhIP]-S-dioxide linked adduct. In this investigation, we have characterized the albumin adduction products of N-sulfooxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-sulfooxy-PhIP), which is thought to be a major genotoxic metabolite of PhIP formed in vivo. Targeted and data-dependent scanning methods showed that N-sulfooxy-PhIP adducted to the Cys34 of albumin in human plasma to form LQQC*[SO2PhIP]PFEDHVK at levels that were 8 to 10-fold greater than the adduct levels formed with N-(acetyloxy)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-acetoxy-PhIP) or HONH-PhIP. We also discovered that N-sulfooxy-PhIP forms an adduct at the sole tryptophan (Trp214) residue of albumin in the sequence AW*[PhIP]AVAR. However, stable adducts of PhIP with albumin were not detected in human hepatocytes. Instead, PhIP and 2-amino-1-methyl-6-(5-hydroxy)-phenylimidazo[4,5-b]pyridine (5-HO-PhIP), a solvolysis product of the proposed nitrenium ion of PhIP, were recovered during the proteolysis, suggesting a labile sulfenamide linkage had formed between an N-oxidized intermediate of PhIP and Cys34 of albumin. A stable adduct was formed at the Tyr411 residue of albumin in hepatocytes, and identified as a deaminated product of PhIP, Y*[desaminoPhIP]TK, where the 4-HO-tyrosine group bound to the C-2 imidazole atom of PhIP.

Novel Transgenic Mouse Model for Studying Human Serum Albumin as a Biomarker of Carcinogenic Exposure

“…The mass spectrum of 411 Y *[desaminoPhIP] TK 413 (Figure 9C) is in agreement with the spectrum previously reported. 21,22 In contrast, an adduct of NO 2 -PhIP was identified at 411 Y *[desaminoPhIP] TQK 414 in mouse albumin of both transgenic and wild-type mice (Figures 9D and 9E). The reconstructed ion mass chromatogram of NO 2 -PhIP adducted mouse peptide 411 Y *[desaminoPhIP] TQK 414 and its corresponding mass spectrum are shown in Figures 9D–9F.…”

“…The tuning parameters for the MS were previously reported. 21 The capillary temperature was set at 270 °C; ionization voltage, 2 kV in positive ion mode; ion trap mass spectrometry (ITMS) acquired with 1 μ scan; maximum injection time, 10 ms; activation time, 10 ms; activation Q, 0.35; AGC (automated gain control), 30000. Peptide adducts were identified manually, facilitated by using MyriMatch 2.1.140 software that was developed by Dr. David Tabb at Vanderbilt University.…”

“…Peptide adducts were identified manually, facilitated by using MyriMatch 2.1.140 software that was developed by Dr. David Tabb at Vanderbilt University. 21,27 For the data dependent scanning experiments, a full MS scan (100 – 1800 m/z ) was obtained, followed by five data dependent scans (100 – 1800 m/z ). Identification of peptides and sequence coverage were facilitated by MyriMatch using a 32-protein subset database containing mouse and human albumin peptide sequences.…”

“…21,22,26,35 The adducted peptides 31 LQQC *[desaminoPhIP] PF 36 and 411 Y *[desaminoPhIP] TK 413 , resulted from the ex vivo treatment of transgenic human albumin with NO 2 -PhIP, were detected. The desamino adduct of NO 2 -PhIP was also identified at 411 Y *[desaminoPhIP] QTK 414 - of mouse albumin; however, the formation of the NO 2 -PhIP adduct at the Cys 34 of mouse albumin was not detected.…”

Acid-labile protein-adducted heterocyclic aromatic amines in human blood are not viable biomarkers of dietary exposure: A systematic study

“…However, these are almost exclusively studies of in vitro HCA-adducts created using liver microsome systems (Lynch et al, 1991) or conjugation using reactive intermediates (Peng and Turesky, 2011;Wang et al, 2015). Furthermore, some studies are applied to cell cultures (Crosbie et al, 2000) or extraction procedures from plasma (Lezamiz et al, 2008) or proteins (Busquets et al, 2006) fortified with free HCAs but not to incurred samples containing known HCA-protein adducts.…”