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Metabolism of AFB1, Production of Reactive Electrophiles leads to Formation, Pro-mutagenic DNA Adducts
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding||Point of Contact||Author Status||OECD Status|
|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)||adjacent||High||Ted Simon (send email)||Open for citation & comment||EAGMST Under Review|
Life Stage Applicability
Key Event Relationship Description
AFB1 must be metabolized via Cytochromes P450 to a specific highly reactive form of AFB1, the exo-epoxide AFB1-8,9-epoxide, in order for DNA binding and formation of a pro-mutagenic DNA adduct to occur. CYP3A4 forms only the exo-form of this reactive epoxide. CYP1A2, inducible in liver, forms both the exo- and the endo-epoxides apparently with a lower Vmax and higher Km than CYP3A4 in human liver (Degen and Neumann,1981; Groopman and Kensler, 2005; Guengerich et al., 1996; Ueng et al., 1995).). Figure X, taken from Pottenger et al., 2014, depicts the metabolism of AFB1. The activated metabolite, exo-epoxide, must then travel from the endoplasmic reticulum, (site of CYP450 enzyme and exo-epoxide of formation) to the nucleus, in order to bind to DNA to form the pro-mutagenic N7-AFB1-G adduct. This can further react to form the AFB1 FAPy adduct.
Evidence Collection Strategy
Evidence Supporting this KER
The biological plausibility of the KER pre-MIE to MIE is strong; without the specific, CYP-450-mediated metabolic activation of AFB1 to the exo-epoxide, the necessary pro-mutagenic N7-AFB1-G adduct will not form, and the sequence of key events will not continue further.
Empirical support for this pre-MIE to MIE KER is indirect but strong. For example, competitive metabolism with other hepatic and extra-hepatic P450 isozymes may result in formation of a decreased proportion of the specific reactive metabolite, AFB1-8,9-epoxide. Alternatively, induction of glutathione-S-transferase (GST) activity, either hepatic or extra-hepatic, can reduce the levels of AFB1-epoxide available for reaction with DNA by increasing conjugation of the epoxide with glutathione. This conjugation renders the epoxide inactive and directs it towards eventual elimination (Guengerich et al., 1996).
Chemoprevention studies with agents that affect AFB1 metabolism, such as oltipraz, can induce detoxifying GST and thus decrease available reactive epoxide. This causes shifts in proportions of different pathways, and also affects formation of downstream events that must go through the pre-MIE to MIE KER (e.g., mutations or altered hepatic foci); thus by correlation the MIE, formation of pro-mutagenic N7-AFB1-G, must be affected (Roebuck et al., 1991; Yates et al., 2006; Johnson et al., 2014).
Uncertainties and Inconsistencies
The available data do not include dose-response data for activation of AFB1 to the key metabolite, exo-8,9-epoxide, which precludes presenting a quantitatively defined relationship between activation and formation of the pro-mutagenic N7-AFB1-G adducts. However, this does not diminish the certainty in the essentiality of this KER.
There are some data to inform the persistence of N7-AFB1-G and its transformation to AFB1-FAPy (Brown et al., 2006; Croy and Wogan, 1991a), but more detailed data, including dose-response data, would be useful.
No inconsistencies were identified vis-à-vis this KER; the conundrum of the high AFB1 metabolic capacity of the mouse and its resistance to the adverse outcome has been investigated and demonstrated to be due to the high rate of detoxication of the exo-epoxide by mouse GSTs (Degen and Neumann, 1981; Monroe and Eaton, 1987, 1988).
Known modulating factors
Quantitative Understanding of the Linkage
Data from a thesis were summarized with limited detail in Lutz (1987) and described levels of tritiated DNA measured in liver following p.o. administration of tritiated AFB1 to male F344 rats. The dose-response, encompassing 4-5 orders of magnitude (1 ng/kg bw to 105 ng/kg bw) was described as linear, although only limited experimental detail was provided. More sophisticated and reliable techniques are available now for structural identification and quantitation of the adducts presumably represented by the tritiated DNA, e.g., mass spectrometric techniques for confirmed specificity. Such specific quantitative data were not identified for AFB1 DNA adducts in rats.
The same chemoprevention studies with agents that affect AFB1 metabolism, such as oltipraz or CDDO-Im, have been shown to decrease the number of pro-mutagenic N7-AFB1-G adducts formed in liver (and eliminated in urine) (Roebuck et al., 1991; Yates et al., 2006; Johnson et al., 2014). These decreases in adduct formation inform a more quantitative understanding of the impact of demonstrated shifts in proportions of different metabolic pathways, typically an increase in detoxication pathways that results in reduced levels of the key reactive metabolite, AFB1 exo-epoxide.
Work conducted in mice, which are less sensitive to AFB1-induced hepatic tumors, provides additional quantitative information on the activation and DNA binding of AFB1 (Monroe and Eaton, 1987, 1988). These studies support the conclusion that the very high (and inducible) GST activity in mouse liver accounts for the resistance of this species to AFB1-induced liver tumors.
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
The requirement for metabolism of AFB1 to a specific reactive form is applicable to all mammalian systems evaluated; it is also applicable to certain birds (turkeys, etc.) (Gregory et al., 1983; IARC, 1993). Humans, non-human primates, rats, mice, poultry, and fish have all demonstrated susceptibility to AFB1-induced liver tumors (Asplin and Canaghan, 1961; Eaton and Gallagher, 1994; Guengerich et al., 1996). Species that preferentially metabolize AFB1 to the exo-8,9-epoxide are more susceptible to AFB1 carcinogenicity.
F.D. Asplin, R.B.A. Carnaghan, (1961). The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Rec. 73:1215– 1219.
Brown KL, Deng JZ, Iyer RS, Iyer LG, Voehler MW, Stone MP, Harris CM, Harris TM (2006). Unraveling the aflatoxin-FAPY conundrum: Structural basis of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J Am Chem Soc 128:15188-15199.
Croy RG, Wogan GN (1981a). Temporal patterns of covalent DNA adducts in rat liver after single and multiple doses of aflatoxin B1. Cancer Res 41:197-203.
Degen GH, Neumann HG (1981). Differences in aflatoxin B1-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin B1-epoxide. Carcinogenesis 2:299–306.
Eaton DL, and Gallagher EP (1994). Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 34:135-172.
Gregory 3rd JF, Goldstein SL, Edds GT. (1983). Metabolite distribution and rate of residue clearance in turkeys fed a diet containing aflatoxin B1. Food Chem Toxicol, 21, 463–7.
Groopman JD, Kensler TW (2005). Role of metabolism and viruses in aflatoxin-induced liver cancer. Toxicol Appl Pharmacol 206:131-137.
Guengerich FP, Johnson WW, Ueng Y-F, Yamazaki H, Shimada T (1996). Involvement of Cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ Health Perspect 104(Suppl 3):557-562.
IARC (1993). Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 56, 245-395.
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.
Lutz, W. (1987). Quantitative evaluation of DNA-binding data in vivo for low-dose extrapolations. Arch.Toxicol, Suppl. 11: 66-74.
Monroe DH, Eaton DL. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and the mouse. Toxicol Appl Pharmacol, 90, 401–409.
Monroe DH, Eaton DL. (1988). Effects of modulation of hepatic glutathione on biotransformation and covalent binding of aflatoxin B1 to DNA in the mouse. Toxicol Appl Pharmacol. 94(1):118-127.
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.
Ueng Y-F, Shimada T, Yamazaki H, Guengerich FP (1995). Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes. Chem Res Toxiol 8:218-225.
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.