ToxSci Advance Access originally published online on March 16, 2009
Toxicological Sciences 2009 109(1):1-3; doi:10.1093/toxsci/kfp052
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Protection against Aflatoxin B1 in Rat—A New Look at the Link between Toxicity, Carcinogenicity, and Metabolism
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G1 1XW, UK
1 For correspondence via Fax: +44 141 553 4124. E-mail: Elizabeth.ellis{at}strath.ac.uk.
Received March 3, 2009; accepted March 4, 2009
The ability of aflatoxin B1 (AFB1) to cause liver cancer has a profound impact on the health of many people living in certain regions of the developing world. A clearer understanding of the metabolism of this toxin in model systems may ultimately lead to effective strategies for preventing its harmful effects. To this end, the work described by Roebuck et al. in this issue of Toxicological Sciences illustrates how transgenic animals have been used to determine not only which routes of metabolism prevail in vivo, but also whether these contribute to the prevention of toxicity and carcinogenicity. By overexpressing AKR7A1, an aldo–keto reductase known to be capable of metabolizing AFB1, they also show that in the rat, the link between metabolism, toxicity and carcinogenicity is not as clear-cut as previously thought. Although the AKR7A1 enzyme is shown to be involved in the metabolism of AFB1 in vivo, its effect in reducing toxicity is surprisingly muted. This work makes a significant contribution to our understanding of AFB1 metabolism and toxicity and the relative roles of specific enzymes in mediating protection.
Aflatoxin B1 (AFB1) is a natural toxin produced as a secondary metabolite by the mould Aspergillus flavus and one of the most potent human hepatocarcinogens known. It presents a particular problem in areas of the world where temperature and humidity are conducive to growth of the mould, such as Africa and Southeast Asia. The carcinogenicity, mutagenicity, toxicity, and metabolism of AFB1 in many different species has been investigated over the past 40 years by several groups using a variety of in vivo and in vitro approaches, yet the interrelatedness of these processes is by no means completely understood.
In terms of carcinogenicity, AFB1 is thought to initiate cancer through the formation of adducts with DNA, observed both in vitro and in rat liver (Croy et al., 1978
; Essigmann et al., 1977). These adducts are derived from highly reactive exo-epoxide metabolites of AFB1, the result of oxidation reactions within the liver (Swenson et al., 1974). Several cytochromes P450 have been implicated in this activation, and in human these were identified as CYP1A2 and CYP3A4 (Gallagher et al., 1994; Shimada et al., 1994). The formation of adducts between the AFB1-exo-epoxide and the N7 of guanine bases in DNA leads to mutations, and is strongly associated with the presence of preneoplastic lesions, characterized as GST-P–positive foci, providing a direct link between DNA damage, mutagenesis, and carcinogenesis. However, AFB1 may also contribute to the development of cancer through other metabolites or through indirect effects.
AFB1 causes acute toxicity as well as carcinogenicity, and this was observed in early studies in the rat (Butler, 1964; Wogan and Newberne, 1967). Acute toxicity was initially attributed to mainly genotoxic effects of the epoxide, dependent on the formation of DNA adducts which at high levels lead to cell death. However, a dialdehyde metabolite of AFB1 that rapidly forms from the epoxide, can form adducts with proteins, and these were proposed to contribute to the acute toxicity (Neal et al., 1981). In addition, such cellular necrotic damage caused by AFB1 dialdehyde may lead to compensatory liver hyperplasia and by so doing may promote the incorporation of mutations into the DNA of dividing cells and hence contribute towards carcinogenicity initiated by the AFB1-exo-epoxide (Roebuck, 2004).
Examining AFB1 metabolites in the bile of rats indicated that a major product of metabolism was the glutathione conjugate of the AFB1-8,9-epoxide (Degen and Neumann, 1978). These conjugates are formed by the action of a glutathione S-transferase identified as GSTA5 (Hayes et al., 1991). Other metabolic pathways were also demonstrated, including the reduction of the cytotoxic AFB1 dialdehyde to AFB1-dialcohol catalyzed by AKR7A1 (Ellis et al., 1993). As both the glutathione conjugate and the dialcohol products were less toxic than the parental compounds, these were deemed to represent detoxication steps, and the GSTA5 and AKR7A1 enzymes were seen to be pivotal in protecting cells against AFB1 toxicity.
Establishing the identity of the individual enzymes that are capable of metabolizing AFB1 was an important development in understanding AFB1-induced toxicity and carcinogenesis. However it was also important to investigate the effect these enzymes had in toto. Studying the kinetics of the various competing reactions led to the conclusion that the route of metabolism followed by AFB1 could be directed by the levels and activities of the major enzymes involved, and this could significantly affect the overall toxicity and carcinogenicity (Guengerich et al., 1998). Variance in enzyme levels and activities may well account for some of the differences in sensitivity observed between species, but another more compelling reason for investigating these enzymes was apparent. Treating rats with compounds such as ethoxyquin, tert-butyl hydroquinone, oltipraz, and coumarin could reduce the toxicity as well as carcinogenicity of AFB1 (Cabral and Neal, 1983; Liu et al., 1988). A series of experiments designed to understand this protective effect pinpointed an elevation in the levels of several detoxication enzymes, notably AKR7A1 and GSTA5 (Hayes et al., 1991, 1993). Both enzymes appeared to be regulated via an antioxidant response element which responds to dietary chemoprotectors (Ellis et al., 2003; Pulford and Hayes, 1996). This correlation between elevation of enzyme levels and protection highlighted that one or both enzymes might be important for the detoxication of AFB1, and may be responsible for the observed reduction in toxicity and carcinogenicity in chemoprotector-treated animals. In support of this hypothesis, extracts prepared from the livers of treated animals were capable of increased metabolism of AFB1 through both detoxication routes.
A direct role for AKR7A1 in protecting against the cytotoxicity of AFB1 in cells was not addressed until recently when experiments were carried out using cell lines in which recombinant AKR7A1 was overexpressed (Bodreddigari et al., 2008). This expression lead to increased protection against the cytotoxicity of the dialdehyde metabolite of AFB1 as well as a reduction in protein-adduct formation, and indicated that AKR7A1 could be responsible for protecting against toxicity in vivo (Bodreddigari et al., 2008). However, the overexpression of AKR7A1 in cell lines had no significant effect on the level of DNA adducts formed following AFB1 exposure and activation, indicating that AKR7A1 is not able to protect against DNA damage and genotoxicity.
The work described by Roebuck et al. (2009) in this issue is an extension of the cell-based work, and was designed to establish the role of AKR7A1 not only in protecting against cytotoxicity and protein-adduct formation, but also against toxicity and carcinogenicity in vivo. A key element of the experimental design of this study is that unlike the situation following treatment with chemoprotective compounds that induce expression of both AKR7A1 and GSTA5, in the transgenic animals only the expression of AKR7A1 is elevated, thereby enabling the role of this enzyme to be studied in isolation. The experimental results show that levels of AFB1-dialdehyde reductase activity are significantly increased in the transgenic animals, leading to an increase in AFB1 dialcohol levels in the urine, and a decrease in AFB1-protein adducts. However, surprisingly this increase in activity does not protect the liver against AFB1 toxicity, as measured by bile duct proliferation, nor does it protect against carcinogenesis as judged by the formation of GST-P–positive foci.
A general point that emerges from this work and from the previous study by Bodreddigari et al. (2008) is that the mode of AFB1 toxicity appears to vary dependent on the concentration of AFB1 and the level of enzyme expressed. At low concentrations in cell lines, AFB1 is cytotoxic, and AKR7A1 can protect against cytotoxicity and protein-adduct formation. This suggests that this cytotoxicity is being mediated via the dialdehyde metabolite. At higher AFB1 concentrations, there is a dose-dependent decrease in cell viability, suggesting that either there is insufficient AKR7A1 to remove the dialdehyde; or that the observed cytotoxicity at these concentrations is now dependent on other metabolites such as the epoxide. In vivo, however, there does not appear to be any protective effect from AKR7A1 against AFB1 toxicity at the concentration used, indicating that AKR7A1 expression alone is not sufficient for significant protection in vivo. As with many detoxication pathways, it is possible that the sequential or concerted action of enzymes is required; that is, the product of the AKR7A1 reduction reaction, the dialcohol, may need to be removed to enable the reductive step to proceed at a useful rate. Hence overexpression of enzymes catalyzing subsequent or accessory steps may also be necessary to see the full benefit of AKR7A1 overexpression. These enzymes may be part of the chemoprotector-induced response and may include glutathione S-transferases.
A counterpoint to the question addressed in the current study is "What would happen if AKR7A1 is not present?". In other words, is AKR7A1 necessary for protection against AFB1 carcinogenesis? This could be pursued in cell lines and ultimately in animals using gene knockout or siRNA approaches. Similarly the role of other aflatoxin-metabolizing enzymes, for example, the glutathione S-transferase GSTA5, in ameliorating toxicity and carcinogenicity in the rat is awaiting investigation.
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