Relationship: 402



Metabolism of AFB1, Production of Reactive Electrophiles leads to Formation, Pro-mutagenic DNA Adducts

Upstream event


Metabolism of AFB1, Production of Reactive Electrophiles

Downstream event


Formation, Pro-mutagenic DNA Adducts

Key Event Relationship Overview


AOPs Referencing Relationship


AOP Name Directness Weight of Evidence Quantitative Understanding
AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC) directly leads to Strong

Taxonomic Applicability


Sex Applicability


Life Stage Applicability


How Does This Key Event Relationship Work


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.

Weight of Evidence


Biological Plausibility


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 Linkage


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 or 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).

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.

Evidence Supporting Taxonomic 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.



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