Comparative study of cytochromes P450 in three avian species: induction of cytochrome P450-dependent monooxygenase activities and immunochemical relatedness of the avian proteins to rat cytochromes P450
Creators: name: Li, Hong
Publisher: name: University of British Columbia
Year: 2009

description: The overall goal of the present study was to investigate the effects of three prototype inducers, phenobarbital (PB), 3-methyicholanthrene (MC) and Aroclor 1254 (AR), on hepatic cytochromes P450 in three avian species by means of enzymatic assays, immunoblot analysis and immunoinhibition of enzymatic activities. Accordingly, female chickens, female quail as well as male and female pigeons were treated with corn oil (2 mi/kg body weight i.p once daily for 3 to 4 days), PB (80 mg/kg body weight, i.p., once daily for 3 to 4 days), MC (25 mg/kg body weight, i.p., once daily for 3 to 4 days) or AR (300 mg/kg body weight, one i.p. injection). Male Long Evans rats receiving similar treatments were included for the purpose of comparison. Pooled hepatic microsomes were prepared from control and treated animals. Total hepatic cytochrome P450 content and four cytochrome P450-dependent monooxygenase activities, namely 7-ethoxyresorufin 0-deethylase (EROD), benzo[a]pyrene hydroxylase (AHH), 7-pentoxyresorufin 0-depentylase (PROD) and benzphetamine N-demethylase (BPND), were examined. Treatment with PB, MC or AR resulted in 2.2-, 1.5- and 3.6- fold induction of total cytochrome P450 content, respectively, in the rats. It was found that total hepatic cytochrome P450 contents in the three avian species were increased 1.3- to 4-fold by PB, approximately 5- to 6-fold by MC, and 2- to 4-fold by AR. In rats, treatment with PB or AR increased EROD, AHH, PROD and BPND activities (PB: 3.6-, 2.1-, 101- and 3.1-fold, respectively; AR: 74-, 4.3-, 42- and 3.0-fold, respectively), while treatment with MC induced EROD, AHH and PROD activities (56-, 6.9- and 1.2- fold, respectively) but suppressed BPND activity (33%). In the three avian species, treatment with PB appeared to increase AHH (1.3- to 3.9-fold) and BPND activities (1.3- to 4.3-fold) but suppress EROD (22% in chickens and 57% in quail) and PROD (24% to 75%), treatment with MC induced EROD, AHH, PROD and BPND activities (72-, 13-, 16- and 3.6-fold, respectively, in chickens; 40-, 14-, 13- and 8.3-fold, respectively, in quail; 1.2- to 5.7-fold in pigeons), and treatment with AR also increased EROD, AHH, PROD and BPND activities (30-, 4.7-, 7.6- and 1.8-fold, respectively, in chickens; 36-, 7.0-, 26- and 7.7-fold, respectively, in quail; 1.3- to 5.7-fold in pigeons except for a 29% decrease in AHH). Flepatic cytochromes P450 are inducible in the avian species by PB, MC and AR. Species differences exist in terms of the inducibility of cytochrome P450- dependent monooxygenase activity. Slight decreases in EROD activity and moderate induction of AHH activity in the birds treated with PB imply that hepatic EROD and AHH activities may be mediated by different forms of cytochrome P450. Similarly, PBtreatment suppressed hepatic PROD activity and induced BPND activity, suggesting that hepatic PROD and BPND activities should be mediated by different forms of cytochrome P450 in PB-treated birds. Immunoblot analysis was undertaken to determine the presence of different forms of hepatic cytochrome P450 in the three avian species and to compare the immunochemical relatedness of avian and rat cytochromes P450. Immunoblots containing hepatic microsomes from control and treated birds were probed with antibodies generated against several purified rat cytochrome P450 enzymes. Analysis of the immunoblots demonstrates that (1) the three avian species contain hepatic cytochromes P450 that are immunorelated to rat CYP1A1 and are inducible by MC and AR, (2) chickens have a cytochrome P450 that is immunorelated to rat CYP1A2 and is also inducible by MC and AR, (3) weak cross-reactions between polyclonal anti-rat CYP2B1 IgG and the avian microsomes indicate the presence of avian cytochromes P450 that are weakly immunologically related to rat CYP2B isozymes, (4) avian cytochromes P450 immunorelated to CYP2C 11 are present in both male and female chickens and quail but not pigeons, (5) these avian species contain hepatic cytochromes P450 that are immunorelated to other rat CYP2C isozymes (but not to CYP2C7 and CYP2C13), and (5) the three avian species contain hepatic cytochromes P450 immunorelated to rat CYP3A2 but not CYP3A1 and they are inducible by PB. Immunoinhibition of EROD, PROD and BPND activities were conducted in an attempt to find out which forms of avian cytochrome P450 contribute to these monooxygenase activities. It was found that avian cytochrome P450 enzymes immunorelated to rat CYP1A1 mediate not only hepatic EROD activity but also PROD activity in the MC- or AR-treated birds. Avian cytochrome P450 enzymes that are immunorelated to rat CYP2B1, CYP2C11 or CYP3A2 contribute to PROD and BPND activity to varying extents. The present study provides enzymatic and immunological evidence to demonstrate that the three avian species contain many forms of hepatic cytochrome P450 and treatment of the birds with PB, MC or AR results in either induction or reduction in expression of avian cytochromes P450 and their catalytic activities.