A study was conducted to identify the cytochrome P450 (CYP, CYP450) enzyme orthologs involved in the bioactivation of aflatoxin B1 (AFB1) into the hi

A study was conducted to determine the cytochrome (CYP) P450 enzymes responsible for the bioactivation of aflatoxin B1 (AFB1) into its epoxide form (AFBO) in duck liver microsomes. Six male and six female 6-week-old Pekin ducks were used. The biochemical toxicology strategies applied included the use of selective inhibitors, prototype substrate activity for specific human P450s, correlation between aflatoxin bioactivation and enzymatic activity of prototype substrates, and the expression of specific CYP450 enzymes using antibodies against human CYP450s. Enzymatic activity was detected for the duck orthologues CYP1A1/2, CYP2A6 and CYP3A4 but not for the CYP2D6 orthologue. Immunoreactive proteins for CYP1A1, CYP2A6 and CYP3A4 were also detected. Inhibition studies suggested that the duck turkey CYP2A6 orthologue and, to a lesser extent, the CYP1A1 orthologue are involved in the bioactivation of AFB1. Correlation studies, however, suggest that CYP3A4, CYP2A6 and CYP1A1/2 are all involved in AFBO formation. The finding that four CYP enzymes may be involved in AFB1 bioactivation in ducks could explain the high sensitivity of this species to AFB1. Further studies are needed to fully elucidate the phase I hepatic metabolism of AFB1 in ducks, the only poultry species that develops hepatic cancer from AFB1 exposure.

1. A study was conducted to determine the cytochrome P450 enzymes responsible for the bioactivation of aflatoxin B1 into its epoxide form (AFBO) in turkey liver microsomes. 2. The strategies used included the measurement of prototype substrate activity for specific human P450s, use of selective inhibitors, determination of correlation between aflatoxin bioactivation and enzymatic activity of prototype substrates and the determination of immunoreactive proteins using antibodies against human P450s. 3. Enzymatic activity and immunoreactive proteins corresponding to the turkey orthologs CYP1A1, CYP1A2, CYP2A6 and CYP3A4 were detected, but not for the CYP2D6 ortholog. 4. The results of the inhibition and correlation studies strongly suggest that the turkey CYP2A6 ortholog and, to a lesser extent, the CYP1A1 ortholog, are involved in the bioactivation of aflatoxin B1 in turkey liver microsomes. 5. This is the first study reporting the role of CYP2A6 in the bioactivation of AFB1 in an avian species and the role of CYP1A1 in any species.

A study was conducted to determine the enzymatic kinetic parameters V_max, K_M, and intrinsic clearance (CL_int) for the hepatic in vitro production of aflatoxin B₁-dihydrodiol (AFB₁-dhd) from aflatoxin B₁ (AFB₁) in four commercial poultry species, ranging in sensitivity to AFB₁ from highest (ducks) to lowest (chickens). Significant but small differences were seen for V_max, while large significant differences were observed for K_M. However, the largest inter-species differences were observed for the CL_int parameter, with ducks being extraordinarily efficient in converting AFB₁ into AFB₁-dhd. Since AFB₁-dhd is considered the metabolite responsible for the acute toxic effects of AFB₁, the high hepatic production of AFB₁-dhd from AFB₁ in ducks is the possible biochemical explanation for the extraordinary high sensitivity of this poultry species to the adverse effects of AFB₁.

Comparative studies designed to investigate the role of glutathione S-transferase (GST) activity on the enzyme catalyzed trapping of aflatoxin B-1-8,9-epoxide (AFBO) with glutathione, and the relationship with aflatoxin B-1 (AFB(1)) resistance have not been conducted in poultry. Hepatic cytosolic fractions of chickens, quail, turkeys and ducks were used to measure in vitro the enzymatic parameters maximal velocity (V-max), Michaelis-Menten constant (K-m) and intrinsic clearance (CLint) for GST activity. AFB(1) used ranged from 2.0 to 157.5 mu M and the AFB(1)-GSH produced was identified and quantitated by HPLC. Significant differences were found in GST V-max values, being the highest in chickens, followed by quail, ducks and turkeys. The K-m values were also significantly different, with chickens < ducks < turkeys < quail. Chickens had the higher CLmt value in contrast to ducks. Differences by sex showed that duck females had a higher CLmt value than the turkey and quail, whereas duck males had a CLmt close to that of turkey. The ratio "AFBO production/AFB(1)-GSH production" follows the order duck>turkey>quail>chicken, in agreement with the known poultry sensitivity. The extremely high "AFB(1) epoxidation activity/GST activity" ratio observed in ducks might be the explanation for the development of hepatocellular carcinoma in this species.

Cytochrome P450 enzymes (CYP) are a group of monooxygenases able to biotransform several kinds of xenobiotics including aflatoxin B1 (AFB1), a highly toxic mycotoxin. These enzymes have been widely studied in humans and others mammals, but there is not enough information in commercial poultry species about their biochemical characteristics or substrate specificity. The aim of the present study was to identify CYPs from avian liver microsomes with the use of prototype substrates specific for human CYP enzymes and AFB1. Biochemical characterization was carried out in vitro and biotransformation products were detected by high-performance liquid chromatography (HPLC). Enzymatic constants were calculated and comparisons between turkey, duck, quail and chicken activities were done. The results demonstrate the presence of four avian ortholog enzyme activities possibly related with a CYP1A1, CYP1A2, CYP2A6 (activity not previously identified) and CYP3A4 poultry orthologs, respectively. Large differences in enzyme kinetics specific for prototype substrates were found among the poultry species studied. Turkey liver microsomes had the highest affinity and catalytic rate for AFB1 whereas chicken enzymes had the lowest affinity and catalytic rate for the same substrate. Quail and duck microsomes showed intermediate values. These results correlate well with the known in vivo sensitivity for AFB1 except for the duck. A high correlation coefficient between 7-ethoxyresorufin-Odeethylase (EROD) and 7-methoxyresorufin- O-deethylase (MROD) activities was found in the four poultry species, suggesting that these two enzymatic activities might be carried out by the same enzyme. The results of the present study indicate that four prototype enzyme activities are present in poultry liver microsomes, possibly related with the presence of three CYP avian orthologs. More studies are needed in order to further characterize these enzymes.