Polyamines are traps for reactive intermediates in furan metabolism

Abstract

Furan is toxic and carcinogenic in rodents. Because of the large potential for human exposure, furan is classified as a possible human carcinogen. The detailed mechanism by which furan causes toxicity and cancer is not yet known. Since furan toxicity requires cytochrome P450-catalyzed oxidation of furan, we have characterized the urinary and hepatocyte metabolites of furan to gain insight into the chemical nature of the reactive intermediate. Previous studies in hepatocytes indicated that furan is oxidized to the reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA), which reacts with glutathione (GSH) to form 2-(S-glutathionyl)succinaldehyde (GSH-BDA). This intermediate forms pyrrole cross-links with cellular amines such as lysine and glutamine. In this article, we demonstrate that GSH-BDA also forms cross-links with ornithine, putrescine, and spermidine when furan is incubated with rat hepatocytes. The relative levels of these metabolites are not completely explained by hepatocellular levels of the amines or by their reactivity with GSH-BDA. Mercapturic acid derivatives of the spermidine cross-links were detected in the urine of furan-treated rats, which indicates that this metabolic pathway occurs in vivo. Their detection in furan-treated hepatocytes and in urine from furan-treated rats indicates that polyamines may play an important role in the toxicity of furan.

Figure 1

Mass chromatogram generated upon extraction of the CNL 129 ion current from LC/MS/MS analysis of media from A) 0 or B) 100 μM furan-treated rat hepatocytes (HPLC method 1 with 10 mM ammonium formate, pH 2.8) obtained on an Agilent ion trap mass spectrometer. Previously characterized metabolites are m/z 502 (GSH-BDA-lysine) and m/z 544 (GSH-BDA-N^α-acetyl-lysine). Unknowns are: m/z 488 and m/z 501.

Figure 2

LC/ESI-MS/MS analysis of media from furan- and control-treated hepatocytes for GSH-BDA-putrescine in the absence and presence of synthetic standard. The traces were generated by monitoring for the neutral loss of 129 from the molecular ion (m/z → 444 m/z 315).

Figure 3

LC/ESI-MS/MS analysis of rat urine for NAC-BDA-spermidine cross-links. Animals were treated with either corn oil alone or corn oil containing [¹²C₄]- or [¹³C₄]furan. A. The traces were generated by monitoring for the neutral loss of 71 from the molecular ion ([¹²C₄]: m/z 357 → m/z 286; [¹³C₄]: m/z 361 → m/z 290). B. The traces were generated by monitoring for the neutral loss of 129 from the molecular ion ([¹²C₄]: m/z 357 → m/z 228; [¹³C₄]: m/z 361 → m/z 232).

Figure 4

Representative mass chromatograms of solutions of 5 mM GSH, 100 μM BDA and an equimolar mixture of the amines (100 μM each putrescine, cadaverine, spermine, spermidine, ornithine, and lysine) in either 150 mM sodium phosphate, pH 7.4, (top) in hepatocyte media (RPMI 1640 media containing 10 mM HEPES, pH 7.4, bottom).

Figure 5

Relative distribution of GSH-BDA-amine products in sodium phosphate, pH 7.4, hepatocyte media, and media from furan-treated rat hepatocytes as determined by LC-MS analysis. The amount of GSH-BDA-N^α-lysine (2b) in furan treated-hepatocytes was calculated from the relative amount of 2b/2a analysis using SRM monitoring. *Includes N^α-acetyl-l-lysine derivative of GSH-BDA-lysine.

Scheme 1

Major pathways of furan biotransformation in rats.

Scheme 2

The reaction products formed when GSH-BDA is reacted with a variety of cellular amines.

Scheme 3

Proposed pathways of furan metabolism. GSH: l-Glutathione; NAC: N-Acetyl-l-cysteine