Validation of putative biomarkers of furan exposure through quantitative analysis of furan metabolites in urine of F344 rats exposed to stable isotope labeled furan

Abstract

Humans are chronically exposed to furan, a potent liver toxicant and carcinogen that occurs in a variety of heat-processed foods. Assessment of human exposure based on the furan content in foods is, however, subject to some uncertainty due to the high volatility of furan. Biomarker monitoring is thus considered an alternative or complementary approach to furan exposure assessment. Previous work suggested that urinary furan metabolites derived from the reaction of cis-2-butene-1,4-dial (BDA), the reactive intermediate of furan, with glutathione (GSH) or amino acids may serve as potential biomarkers of furan exposure. However, some metabolites were also reported to occur in urine of untreated animals, indicating either background contamination via animal feed or endogenous sources, which may limit their suitability as biomarkers of exposure. The overall aim of the present study was to accurately establish the correlation between external dose and concentration of furan metabolites in urine over time and to discriminate against endogenous formation and furan intake via feed. To this end, the furan metabolites GSH-BDA (N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine), NAcLys-BDA (R-2-(acetylamino)-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid), NAcCys-BDA-NAcLys (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine) and NAcCys-BDA-NAcLys sulfoxide (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine sulfoxide) were simultaneously analyzed by stable isotope dilution ESI-LC-MS/MS as unlabeled and [¹³C₄]-furan dependent metabolites following oral administration of a single oral dose of isotopically labelled [¹³C₄]-furan (0.1, 1, 10, 100 and 1000 µg/kg bw) to male and female F344/DuCrl rats. Although a linear correlation between urinary excretion of [¹³C₄]-furan-dependent metabolites was observed, analysis of unlabeled NAcLys-BDA, NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide revealed substantial, fairly constant urinary background levels throughout the course of the study. Analysis of furan in animal feed excluded feed as a source for these background levels. GSH-BDA was identified as the only furan metabolite without background occurrence, suggesting that it may present a specific biomarker to monitor external furan exposure. Studies in humans are now needed to establish if analysis of urinary GSH-BDA may provide reliable exposure estimates.

Keywords: Biomarker of exposure; Biomonitoring; Furan; Metabolism; Process-related food contaminant; Urinary metabolite excretion.

Fig. 1

Metabolic pathways of furan and potential urinary biomarkers of furan exposure. Furan is predominantly metabolized by CYP 2E1, leading to the formation of the reactive dialdehyde cis-2-butene-1,4-dial (BDA). The reactivity of BDA towards cellular nucleophiles results in a broad spectrum of urinary metabolites: GSH-BDA (1), the product of the conjugation with glutathione and a subsequent intramolecular reaction; NAcLys-BDA (2), the adduct of BDA and lysine, followed by acetylation of the ⍺-amino group of lysine; NAcCys-BDA-NAcLys (3) and its corresponding sulfoxide (4), which derive from crosslinks of cysteine and lysine by BDA and subsequent N-acetylation and oxidation. Note that conjugation with GSH and cysteine can also occur in position 2 instead of 3 in the pyrrol ring

Fig. 2

Extracted ion chromatograms of GSH-BDA (t_R 11.3 min), NAcLys-BDA (t_R 11.5 min), NAcCys-BDA-NAcLys (t_R 13.4 min) and NAcCys-BDA-NAcLys sulfoxide (11.7 min) as [¹³C₄]-furan metabolites, [¹²C]-furan metabolites and matching internal standards obtained from a standard mix solution and a 16 h urine sample from a male F344/DuCrl rat treated with 1000 µg/kg bw [¹³C₄]-furan

Fig. 3

Urinary excretion of GSH-BDA, NAcLys-BDA, NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide as [¹³C₄]-furan-derived metabolites (filled symbols) and corresponding unlabeled compounds (open symbols) in urine of male (A) and female (B) F344/DuCrl rats. Inserts present zoomed plots of the highlighted areas of the diagrams, showing metabolite excretion in the lower dose groups (100–1 µg/kg bw [¹³C₄]-furan). Data are presented as mean ± standard deviation (n = 5) of the amount of metabolite excreted within 8 h

Fig. 4

Linear correlation between external [¹³C₄]-furan dose and 24 h urinary excretion of furan metabolites in male (A) and female (B) rats. Data are presented as mean ± standard deviation (n = 5)