Metabolism of aflatoxin B1 in Turkey liver microsomes: The relative roles of cytochromes P450 1A5 and 3A37

The extreme sensitivity of turkeys to aflatoxin B1 (AFB1) is associated with efficient epoxidation by hepatic cytochromes P450 (P450) 1A5 and 3A37 to exo-aflatoxin B1-8,9-epoxide (exo-AFBO). The combined presence of 1A5 and 3A37, which obey different kinetic models, both of which metabolize AFB1 to the exo-AFBO and to detoxification products aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1), respectively, complicates the kinetic analysis of AFB1 in turkey liver microsomes (TLMs). Antisera directed against 1A5 and 3A37, thereby individually removing the catalytic contribution of these enzymes, were used to identify the P450 responsible for epoxidating AFB1 in TLMs. In control TLMs, AFB1 was converted to exo-AFBO in addition to AFM1 and AFQ1 confirming the presence of functional 1A5 and 3A37. Pretreatment with anti-1A5 inhibited exo-AFBO formation, especially at low, submicromolar (∼ 0.1 μM), while anti-3A37, resulted in inhibition of exo-AFBO formation, but at higher (> 50 μM) AFB1 concentrations. Metabolism in immunoinhibited TLMs resembled that of individual enzymes: 1A5 produced exo-AFBO and AFM1, conforming to Michaelis-Menten, while 3A37 produced exo-AFBO and AFQ1 following the kinetic Hill equation. At 0.1 μM AFB1, close to concentrations in livers of exposed animals, 1A5 contributed to 98% of the total exo-AFBO formation. At this concentration, 1A5 accounted for a higher activation:detoxification (50:1, exo-AFBO: AFM1) compared to 3A37 (0.15: 1, exo-AFBO: AFQ1), suggesting that 1A5 is high, while 3A4 is the low affinity enzyme in turkey liver. The data support the conclusion that P450 1A5 is the dominant enzyme responsible for AFB1 bioactivation and metabolism at environmentally-relevant AFB1 concentrations in turkey liver. - Graphical abstract: Display Omitted Highlights: → Efficient bioactivation by P450s 1A5 and 3A4 associated with extreme aflatoxin B1 sensitivity in turkeys. → These P450s exhibit different metabolite profiles and enzyme kinetic models toward AFB1. → Study conducted to determine which P450 is primary bioactivator in turkey liver. → Immunoinhibition studies show 1A5 predominates at low, environmentally-relevant AFB1 concentrations. → 3A37 predominates at only at very high AFB1 concentrations, not relevant to liver in vivo.