Purification and immunochemical detection of β‐naphthoflavone‐and phenobarbital‐induced avian cytochrome P450 enzymes

Abstract

Livers from mallards (Anas platyrhynchos) were treated with either β‐naphthoflavone (50 mg/kg) or phenobarbital (70 mg/kg). Purification of induced hepatic cytochrome P450 was accomplished using both DEAE and hydroxyapatite columns, as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis separation. Polyclonal antibodies to these proteins were then produced in young male New Zealand White rabbits. β‐Naphthoflavone (βNF)‐ and phenobarbital (PB)‐treated red‐winged blackbird, screech owl, European starling, and lesser scaup liver microsomes were analyzed in western blots for species cross‐reactivity. Although all four of these avian species exhibited cross‐reactivity with antibodies to βNF‐induced mallard P450, all but the lesser scaup revealed a protein of higher molecular weight than that of the βNF‐induced mallard. In addition, only the lesser scaup exhibited cross‐reactivity with the anti‐PB‐induced mallard P450 antibodies.

Evaluating cytochrome P450 in lesser scaup (Aythya affinis) and tree swallow (Tachycineta bicolor) by monooxygenase activity and immunohistochemistry: Possible nonlethal assessment by skin immunohistochemistry

“…The CYP1A that is most similar to mammalian CYP1A1 has been named CYP1A4. Based on the characteristics of PAH-type induction in a number of other avian species [26,31,32], it is possible that a CYP1A4 orthologue is common to all avian species. Limited sequence identity exists between chicken CYP1A4 and mammalian CYP1A1 (60-63%) [28].…”

Blarina brevicauda as a biological monitor of polychlorinated biphenyls: Evaluation of hepatic cytochrome P450 induction

“…The nitrocellulose was blocked for 1 h with a 1.0% (w/v) solution of nonfat dry milk in Tris buffered saline with 0.05% tween ([TBST], Sigma Chemicals) for CYP1A blots or 5.0% (w/v) solution for CYP2B blots. After blocking, nitrocellulose blots were rinsed three times with TBST for 5 min, then incubated for 2 h with a 1:1,000 dilution of antimallard βNF‐induced CYP produced in rabbit (preparation was described by Brown et al [21], or a 1:500 dilution of antimouse PB‐induced CYP antibody produced in rat [Daiichi Pure Chemicals, Tokyo, Japan]). After rinsing with TBST, blots were incubated for 1 h in an antirabbit immunoglobulin G (IgG) conjugated to alkaline phosphatase (1:3,000 in TBST), or an antigoat IgG alkaline phosphatase conjugate (1:5,000 in TBST).…”