Furan in food: a dietary risk factor for human cholangiocarcinoma? Characterization of functional changes an protein adducts in the hepatobliliary tract of rats exposed to turan
Final Report Abstract
Furan, a potent hepatotoxin and liver carcinogen in rodents, was recently found to be present in a variety of food items that undergo heat treatment. Although data on human intake of furan are limited, there is a relatively narrow margin between human exposure and doses which cause liver tumors in rodents, suggesting that the presence of furan in food may present a potential risk to human health. However, the mechanisms of furan‐induced tumor formation are still poorly understood. Furan is oxidized by cytochrome P450 to yield a chemically reactive α,β‐unsaturated dialdehyde, i.e. cis‐2‐butene‐1,4‐dial, which may covalently modify nucleosides and amino acid residues. Irrespective of its genotoxic potential, it appears that inactivation of protein function through protein binding of cis‐2‐butene‐1,4‐dial may present a key event in the toxicity of furan. However, while it is well established that protein binding by a toxic compound may disrupt cell/tissue homeostasis and cause toxicity, it has long been recognized that adduction of some proteins may be critical to injury, whereas covalent binding to others is not. Thus, the major aim of our study was to identify target proteins of furan reactive metabolites in rat liver to better understand the mechanism of furan toxicity and carcinogenicity in rat liver. Male F344 rats were administered [3,4‐14C]‐furan at doses of 0, 0.1, and 2.0 mg/kg bw and sacrificed after 2h. Identification of target proteins of furan reactive intermediates was achieved by separation of unmodified and furan‐adducted proteins present in liver by two‐dimensional gel electrophoresis, fluorography, in‐gel digestion of separated protein adducts and identification of modified proteins by mass spectrometry and protein sequence database search. In addition, a 28 days oral gavage study was performed in male F344 rats at furan doses of 0, 0.1, 0.5, and 2.0 mg/kg bw to characterize the cellular and functional consequences associated with protein damage at a known carcinogenic dose and doses closer to estimated human exposure. Our results demonstrate that furan binds to a large number of proteins, some of which also represent targets of other compounds thought to cause toxicity via reactive metabolite formation. Target proteins represent ‐ among others ‐ enzymes, transport proteins, structural proteins and chaperones which predominantly localize to the cytosol and mitochondria and participate in various cellular processes, including lipid, amino acid and carbohydrate metabolism, redox homeostasis and protein folding. Among the most adducted furan targets are structural proteins and transport proteins. While their contribution to the mechanism of furan toxicity is not apparent, it is conceivable that binding to these high abundance proteins may function as a mechanism of detoxification/inactivation of reactive furan metabolites to prevent damage and loss of function of proteins which may be less abundant but perhaps more important for cell survival. In contrast, our finding that furan binds to a range of mitochondrial enzymes involved in glucose metabolism, mitochondrial β‐oxidation and ATP synthesis suggests that the mechanism of furan toxicity may involve impaired mitochondrial energy production and oxidative stress through mitochondrial uncoupling. This is in line with data showing that uncoupling of hepatic oxidative phosphorylation is an early event in furan‐mediated cell death. In addition, adduct formation with proteins which participate in the maintenance of redox homeostasis and protein folding may result in reduced ability to cope with cellular/oxidative stress, including accumulation of misfolded proteins. Although functional (altered bile acid homeostasis) and proliferative changes developing from the subcapsular region of furan target lobes were seen in rat liver following repeated administration of furan, furan did not appear to trigger the unfolded protein response, a homeostatic control mechanism which serves to increase the cells capacity to recognize misfolded proteins and target them for degradation by the proteasome. Interestingly, however, GRP78, the primary sensor of ER stress, was also identified as a target of furan, suggesting that inactivation of GRP78 function through covalent binding may in fact contribute to furan toxicity by preventing activation of the unfolded protein response. In summary, our data suggest that functional loss of several individual proteins and pathways, most notably mitochondrial energy production, redox regulation and protein folding, may combine to disrupt cell homeostasis and cause hepatocyte cell death and subsequent regenerative cell proliferation. However, further work is needed to establish if adduction by furan reactive metabolites indeed results in loss of individual protein function.
Publications
- Furan in Food: 28 Day Oral Toxicity and Cell proliferation in Male F344/N Rats. Society of Toxicology 48th Annual Meeting, Baltimore, 2009. The Toxicologist, Supplement to Toxicological Sciences
Mally, A., Graff, C., Moro, S., Hamberger, C., Schauer, U.M., Brück, J., Özden, S., Sieber, M., Steger, U., Hard, G.C., Chipman, J.K., Schrenk, D., and Dekant, W.
- Identification of target proteins of furan in rat liver. 46th Congress of the European Society of Toxicology, Dresden, 2009. Toxicology Letters, Vol. 189S
Moro, S., Hamberger, C., Dekant, W., Chipman, K. and Mally, A.
- Modulation of hepatic gene expression independent of DNA methylation in furan treated F344/N Rats. Society of Toxicology 48th Annual Meeting, Baltimore, 2009. The Toxicologist, Supplement to Toxicological Sciences
Chen, T., Mally, A., Ozden, S., Dekant,W., Chipman, J.K.
- Role of genetic and non‐genetic mechanisms in furan risk. 10th International Conference on Environmental Mutagens, 20.‐25.8.2009, Florence, Italy
Mally, A.
- Role of genotoxic and non‐genotoxic mechanisms in furan carcinogenicity. 46th Congress of the European Society of Toxicology, Dresden, 2009. Toxicology Letters, Vol. 189S
Chipman, K., Chen, T., Hickling, K., Dekant, W., and Mally, A.
- (2010). Low Doses of the Carcinogen Furan Alter Cell Cycle and Apoptosis Gene Expression in Rat Liver Independent of DNA Methylation. Environ Health Perspect. 2010 Jun 18 [Epub ahead of print]
Chen, T., Mally, A., Ozden, S. and Chipman, J. K.
- Furan in Food: 28 Day Oral Toxicity and Cell proliferation in Male F344/N Rats. Mol. Nutr. Food Res., 2010 Jun 10 [Epub ahead of print]
Mally, A., Graff, C., Moro, S., Hamberger, C., Schauer, U.M., Brück, J., Özden, S., Sieber, M., Steger, U., Hard, G.C., Chipman, J.K., Schrenk, D., and Dekant, W.
- Heat‐treatment induced toxicant in food: Needs for health risk assessment. Chipman, K. Role of genetic and epigenetic mechanisms in furan toxicity. XII International Congress of Toxicology, 19‐23 July 2010, Barcelona, Spain
Dekant, W.
- Identification of target proteins of furan in rat liver. (Poster) Society of Toxicology 49th Annual Meeting, Salt Lake City, 2010. The Toxicologist, Supplement to Toxicological Sciences
Moro, S., Hamberger, C., Dekant, W., Chipman, K. and Mally, A.
- Role of genetic and epigenetic mechanisms in furan toxicity. XII International Congress of Toxicology, 19‐23 July 2010, Barcelona, Spain
Chipman, K.
