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

Abstract

The extreme sensitivity of turkeys to aflatoxin B 1 (AFB 1) is associated with efficient epoxidation by hepatic cytochromes P450 (P450) 1A5 and 3A37 to exo-aflatoxin B 1-8,9-epoxide ( exo-AFBO). The combined presence of 1A5 and 3A37, which obey different kinetic models, both of which metabolize AFB 1 to the exo-AFBO and to detoxification products aflatoxin M 1 (AFM 1) and aflatoxin Q 1 (AFQ 1), respectively, complicates the kinetic analysis of AFB 1 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 AFB 1 in TLMs. In control TLMs, AFB 1 was converted to exo-AFBO in addition to AFM 1 and AFQ 1 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) AFB 1 concentrations. Metabolism in immunoinhibited TLMs resembled that of individual enzymes: 1A5 produced exo-AFBO and AFM 1, conforming to Michaelis–Menten, while 3A37 produced exo-AFBO and AFQ 1 following the kinetic Hill equation. At 0.1 μM AFB 1, 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: AFM 1) compared to 3A37 (0.15: 1, exo-AFBO: AFQ 1), 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 AFB 1 bioactivation and metabolism at environmentally-relevant AFB 1 concentrations in turkey liver.