Capturing Labile Sulfenamide and Sulfinamide Serum Albumin Adducts of Carcinogenic Arylamines by Chemical Oxidation

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-<i>b</i>]­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 Cys³⁴ residue. Oxidation of the carcinogen-modified SA with <i>m</i>-chloroperoxybenzoic acid (<i>m</i>-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

Integrated Process of Coke-Oven Gas Tri-Reforming and Coal Gasification to Methanol with High Carbon Utilization and Energy Efficiency

The hydrogen to carbon (H/C) ratio of coal gasified gas in the range 0.2–1.0, far less than the desired value for the coal to methanol process. Therefore, a water gas shift unit is needed to raise the H/C ratio, which results in a great deal of CO₂ emission and carbon resource waste. At the same time, there is 7 × 10¹⁰ m³ coke-oven gas (COG) produced in coke plants annually in China. The hydrogen-rich COG consists of 60% hydrogen and 26% methane. However, a massive amount of COG is utilized as fuel or discharged directly into the air, which makes a waste of precious hydrogen resources and causes serious environmental pollution. This paper proposes an integrated process of coke-oven gas and coal gasification to methanol, in which a tri-reforming reaction is used to convert methane and CO₂ to syngas. The carbon utilization and energy efficiency of the new process increase about 25% and 10%, whereas CO₂ emission declines by 44% in comparison to the conventional coal to methanol process

Mapping Serum Albumin Adducts of the Food-Borne Carcinogen 2‑Amino-1-methyl-6-phenylimidazo[4,5‑<i>b</i>]pyridine by Data-Dependent Tandem Mass Spectrometry

2-Amino-1-methyl-6-phenylimidazo­[4,5-<i>b</i>]­pyridine (PhIP) is a heterocyclic aromatic amine that is formed during the cooking of meats. PhIP is a potential human carcinogen: it undergoes metabolic activation to form electrophilic metabolites that bind to DNA and proteins, including serum albumin (SA). The structures of PhIP-SA adducts formed in vivo are unknown and require elucidation before PhIP protein adducts can be implemented as biomarkers in human studies. We previously examined the reaction of genotoxic N-oxidized metabolites of PhIP with human SA in vitro and identified covalent adducts formed at cysteine³⁴ (Cys³⁴); however, other adduction products were thought to occur. We have now identified adducts of PhIP formed at multiple sites of SA reacted with isotopic mixtures of electrophilic metabolites of PhIP and 2-amino-1-methyl-6-[²H₅]-phenylimidazo­[4,5-<i>b</i>]­pyridine ([²H₅]-PhIP). The metabolites used for study were 2-nitro-1-methyl-6-phenylimidazo­[4,5-<i>b</i>]­pyridine (NO₂-PhIP), 2-hydroxyamino-1-methyl-6-phenylimidazo­[4,5-<i>b</i>]­pyridine (HONH-PhIP), or <i>N</i>-acetyloxy-2-amino-1-methyl-6-phenylimidazo­[4,5-<i>b</i>]­pyridine (<i>N</i>-acetoxy-PhIP). Following proteolytic digestion, PhIP-adducted peptides were separated by ultra performance liquid chromatography and characterized by ion trap mass spectrometry, employing isotopic data-dependent scanning. Analysis of the tryptic or tryptic/chymotryptic digests of SA modified with NO₂-PhIP revealed that adduction occurred at Cys³⁴, Lys¹⁹⁵, Lys¹⁹⁹, Lys³⁵¹, Lys⁵⁴¹, Tyr¹³⁸, Tyr¹⁵⁰, Tyr⁴⁰¹, and Tyr⁴¹¹, whereas the only site of HONH-PhIP adduction was detected at Cys³⁴. <i>N</i>-Acetoxy-PhIP, a penultimate metabolite of PhIP that reacts with DNA to form covalent adducts, did not appear to form stable adducts with SA; instead, PhIP and 2-amino-1-methyl-6-(5-hydroxy)-phenylimidazo­[4,5-<i>b</i>]­pyridine, an aqueous reaction product of the proposed nitrenium ion of PhIP, were recovered during the proteolysis of <i>N</i>-acetoxy-PhIP-modified SA. Some of these SA adduction products of PhIP may be implemented in molecular epidemiology studies to assess the role of well-done cooked meat, PhIP, and the risk of cancer

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

2-Amino-1-methyl-6-phenylimidazo­[4,5-<i>b</i>]­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-<i>b</i>]­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 <i>N</i>-sulfooxy-2-amino-1-methyl-6-phenylimidazo­[4,5-<i>b</i>]­pyridine (<i>N</i>-sulfooxy-PhIP), which is thought to be a major genotoxic metabolite of PhIP formed <i>in vivo</i>. Targeted and data-dependent scanning methods showed that <i>N</i>-sulfooxy-PhIP adducted to the Cys³⁴ of albumin in human plasma to form LQ­QC*^[SO₂PhIP]PF­E­D­H­VK at levels that were 8–10-fold greater than the adduct levels formed with <i>N</i>-(acetyloxy)-2-amino-1-methyl-6-phenylimidazo­[4,5-<i>b</i>]­pyridine (<i>N</i>-acetoxy-PhIP) or HONH-PhIP. We also discovered that <i>N</i>-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-<i>b</i>]­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

Mass Spectrometric Characterization of an Acid-Labile Adduct Formed with 2‑Amino-1-methyl-6-phenylimidazo[4,5‑<i>b</i>]pyridine and Albumin in Humans

2-Amino-1-methyl-6-phenylimidazo­[4,5-<i>b</i>]­pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine formed during the high-temperature cooking of meats. The cytochrome P450-mediated N-hydroxylation of the exocyclic amine group of PhIP produces 2-hydroxyamino-1-methyl-6-phenylimidazo­[4,5-<i>b</i>]­pyridine, an electrophilic metabolite that forms adducts with DNA and proteins. Previous studies conducted by our laboratory showed that the reaction of N-oxidized PhIP metabolites with human albumin <i>in vitro</i> primarily occurs at the Cys³⁴ residue, to produce an acid-labile linked sulfinamide adduct. On the basis of these findings, we developed a sensitive ultraperformance liquid chromatography–mass spectrometry method to measure acid-labile albumin–PhIP adducts in human volunteers administered a dietary-relevant dose of ¹⁴C-labeled PhIP [Dingley, K. H., et al. (1999) <i>Cancer Epidemiol., Biomarkers Prev. 8</i>, 507–512]. Mild acid treatment of albumin (0.1 N HCl, 37 °C for 1 h) or proteolytic digestion with Pronase [50 mM ammonium bicarbonate buffer (pH 8.5) at 37 °C for 18 h] released similar amounts of covalently bound PhIP, which was characterized by multistage scanning and quantified by Orbitrap mass spectrometry. The amount of [¹⁴C]­PhIP recovered by acid treatment of albumin 24 h following dosing accounted for 7.2–21.3% of the [¹⁴C]­PhIP bound to albumin based on accelerator mass spectrometry measurements. 2-Amino-1-methyl-6-(5-hydroxy)­phenylimidazo­[4,5-<i>b</i>]­pyridine, a hydrolysis product of the Cys³⁴ S–N linked sulfenamide adduct of PhIP, was not detected in either acid-treated or protease-treated samples. These findings suggest that a portion of the PhIP bound to albumin <i>in vivo</i> probably occurs as an acid-labile sulfinamide adduct formed at the Cys³⁴ residue