Degraded protein adducts of cis-2-butene-1,4-dial are urinary and hepatocyte metabolites of furan

Abstract

Furan is a liver toxicant and carcinogen in rodents. On the basis of these observations and the large potential for human exposure, furan has been classified as a possible human carcinogen. The mechanism of tumor induction by furan is unknown. However, the toxicity requires cytochrome P450-catalyzed oxidation of furan. The product of this oxidation, cis-2-butene-1,4-dial (BDA), reacts readily with glutathione, amino acids, and DNA and is a bacterial mutagen in Ames assay strain TA104. Characterization of the urinary metabolites of furan is expected to provide information regarding the structure(s) of the reactive metabolite(s). Recently, several urinary metabolites have been identified. We reported the presence of a monoglutathione-BDA reaction product, N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-l-cysteinylglycine cyclic sulfide. Three additional urinary metabolites of furan were also characterized as follows: R-2-acetylamino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid, N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine, and its sulfoxide. It was postulated that these three metabolites are derived from degraded protein adducts. However, the possibility that these metabolites result from the reaction of BDA with free lysine and/or cysteine was not ruled out. In this latter case, one might predict that the reaction of thiol-BDA with free lysine would not occur exclusively on the epsilon-amino group. Reaction of BDA with N-acetylcysteine or GSH in the presence of lysine indicated that both the alpha- and the epsilon-amino groups of lysine can be modified by thiol-BDA. The N-acetylcysteine-BDA-N-acetyllysine urinary metabolites were solely linked through the epsilon-amino group of lysine. A GSH-BDA-lysine cross-link was a significant hepatocyte metabolite of furan. In this case, the major product resulted from reaction with the epsilon-amino group of lysine; however, small amounts of the alpha-amino reaction product were also observed. Western analysis of liver and hepatocyte protein extracts using anti-GSH antibody indicated that GSH was covalently linked to proteins in tissues or cells exposed to furan. Our data support the hypothesis that GSH-BDA can react with either free lysine or protein lysine groups. These data suggest that there are multiple pathways by which furan can modify cellular nucleophiles. In one pathway, BDA reacts directly with proteins to form cysteine-lysine reaction products. In another, BDA reacts with GSH to form GSH-BDA conjugates, which then react with cellular nucleophiles like free lysine or lysine moieties in proteins. Both pathways will give rise to N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine. Given the abundance of these metabolites in urine of furan-treated rats, these pathways appear to be major pathways of furan biotransformation in vivo.

Figure 1

HPLC trace of lysine incubation with BDA and N-acetylcysteine (A) or GSH (B), monitoring at 254 nm. GS = glutathione.

Figure 2

Extracted chromatograms at 400 amu for the standards 4a and 4b as well as for urine from [¹²C₄]furan-treated rats.

Figure 3

Extracted chromatograms at 416 amu for the standards 5a and 5b as well as for urine from [¹²C₄]furan-treated rats.

Figure 4

Extracted chromatograms at 398/402 and 414/418 amu in urine from [¹³C₄]furantreated rats (8 mg/kg).

Figure 5

Extracted chromatographs for furan metabolites formed in hepatocytes. A) 356 amu; B) 400 amu; C) 358 amu; D) 502 amu and E) 544 amu.

Figure 6

Western analysis of A) S9 from rat liver or B) hepatocyte extracts with rabbit anti- GSH antibody. A) Lane 1: liver S9 from furan-treated rats, lane 2: liver S9 from furantreated rats plus 75 mM DTT; lane 3: liver S9 from furan-treated rats plus 135 mM DTT; lane 4: liver S9 from untreated rats; lane 5, liver S9 from untreated rats plus 75 mM DTT; lane 6: liver S9 from untreated rats plus 135 mM DTT. B) Lane 1: extracts from control hepatocytes; Lane 2: extracts from control hepatocytes plus 75 mM DTT; Lane 3: extracts from furan-treated hepatocytes; Lane 4: extracts from furan-treated hepatocytes plus 75 mM DTT; Lane 5: extracts from SKF525A- and furan-treated hepatocytes; Lane 6: extracts from SKF525A- and furan-treated hepatocytes plus 75 mM DTT; Lane 7: extracts from 1-phenylimidazole- and furan-treated hepatocytes; Lane 8: extracts from 1- phenylimidazole- and furan-treated hepatocytes plus 75 mM DTT.

Scheme 1

Identified pathways of furan metabolism.

Scheme 2

Proposed mechanism for the addition of methanol to the 2-pyrrole position of 5a.

Scheme 3

Proposed pathways of furan metabolism.

Chart 1

Structure of GSH-BDA-glutamine and GSH-BDA-N^α-acetyllysine reaction products. GS = glutathione