Identification of furan metabolites derived from cysteine-cis-2-butene-1,4-dial-lysine cross-links

Abstract

Furan is a rodent hepatotoxicant and carcinogen. Because this compound is an important industrial intermediate and has been detected in heat-processed foods and smoke, humans are likely exposed to this toxic compound. Characterization of urinary metabolites of furan will lead to the development of biomarkers to assess human health risks associated with furan exposure. Previous studies indicate that furan is oxidized to a reactive alpha,beta-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA), in a reaction catalyzed by cytochrome P450. Five previously characterized metabolites are derived from the reaction of BDA with cellular nucleophiles such as glutathione and protein. They include the monoglutathione reaction product, N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-l-cysteinylglycine cyclic sulfide, and its downstream metabolite, S-[1-(1,3-dicarboxypropyl)-1H-pyrrol-3-yl]methylthiol, as well as (R)-2-acetylamino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid and N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine and its sulfoxide. The last two compounds are downstream metabolites of a BDA-derived cysteine-lysine cross-link, S-[1-(5-amino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine. In this report, we present the characterization of seven additional urinary furan metabolites, all of which are derived from this cross-link. The cysteinyl residue is subject to several biotransformation reactions, including N-acetylation and S-oxidation. Alternatively, it can undergo beta-elimination followed by S-methylation to a methylthiol intermediate that is further oxidized to a sulfoxide. The lysine portion of the cross-link either is N-acetylated or undergoes a transamination reaction to generate an alpha-ketoacid metabolite that undergoes oxidative decarboxylation. Some of these metabolites are among the most abundant furan metabolites present in urine as judged by LC-MS/MS analysis, indicating that the oxidation of furan to BDA and BDA's subsequent reaction with cellular cysteine and lysine residues may represent a significant in vivo pathway of furan biotransformation. Because they are derived from cellular BDA reaction products, these metabolites are markers of furan exposure and bioactivation and could be explored as potential biomarkers in human studies.

Figure 1

Extracted mass chromatograms obtained for urine from [¹²C₄]furan-treated rats, [¹³C₄]furan-treated rats or untreated controls. A. m/z 301 and 305; B. m/z 373 and 377; C. m/z 402 and 406; D. m/z 345 and 349; E. m/z 386 and 390; F. m/z 329 and 333.

Figure 2

Representative extracted mass chromatograms for the incubation of 12 with RLH. Incubation mixtures were either analyzed directly or following reaction with methoxyamine.

Figure 3

Extracted mass chromatograms obtained metabolite 12 in urine from [¹²C₄]furan-treated rats, [¹³C₄]furan-treated rats or untreated controls obtained through LC-MS/MS analysis of rat urine samples using neutral loss of 147 Da scanning.

Figure 4

Extracted mass chromatograms obtained metabolite 14 in urine from [¹²C₄]furan-treated rats, [¹³C₄]furan-treated rats or untreated controls obtained through LC-MS/MS analysis of rat urine samples using neutral loss of 129 Da scanning.

Figure 5

Extracted ion current for all identified metabolites in urine from furan-treated rats. Top trace: urine from untreated rats. Bottom trace: urine from [¹²C₄]furan-treated rats. * denote the retention times of detected but as yet unidentified furan metabolites. The peaks present at 41.9 and 43.5 min in the urine from [¹²C₄]furan-treated rats were not observed in urine from [¹³C₄]furan-treated rats (data not shown) so these two peaks are not likely metabolites of furan.

Scheme 1

Identified pathways of furan metabolism.

Scheme 2

Proposed cysteine biotransformation pathway in rats. It is not known whether N-acetylation precedes or follows β-elimination. Only one possibility is shown for ease of presentation.

Scheme 3

Proposed lysine biotransformation pathway in rats.

Scheme 4

The chemical and biochemical synthesis of metabolites 9 and 15 and their subsequent chemical transformations.