Peroxide-supported in-vitro cytochrome P450 activities in Haemonchus contortus.
The potential for cytochrome P450 from Haemonchus contortus to operate in the oxygen-poor intestinal environment was investigated by examining the ability of the cytochrome to act in vitro as a peroxygenase in utilising cumene hydroperoxide for substrate oxidations not requiring molecular oxygen. Peroxygenase and NADPH-supported monooxygenase activities were measured in microsomes prepared from L3 and adult nematodes. Both cumene hydroperoxide- and NADPH-supported ethoxycoumarin O-deethylase and aldrin epoxidase activities were detected in larval microsomes. Adult microsomes showed low levels of cumene hydroperoxide-supported ethoxycoumarin O-deethylase, as well as NADPH- and cumene hydroperoxide-supported aldrin epoxidase activities. The use of inhibitors in ethoxycoumarin O-deethylase assays with larval microsomes indicated that the peroxygenase pathway does not proceed via ferrous cytochrome P450 (no inhibition by carbon monoxide), did not require molecular oxygen, and did not depend on electron flow from cytochrome P450 reductase. Larval activity was inhibited by typical cytochrome P450 inhibitors (piperonyl butoxide, SKF-525A, chloramphenicol, metyrapone, n-octylamine) and was unaffected by the peroxidase inhibitor salicylhydroxamic acid. In contrast, adult microsomal cumene hydroperoxide-supported ethoxycoumarin O-deethylase activity was significantly inhibited by both cytochrome P450 inhibitors and salicylhydroxamic acid. Adult microsomes also contained potassium ferrocyanide peroxidase activity utilising cumene hydroperoxide. This activity showed a similar pattern of inhibition by both cytochrome P450 and peroxidase inhibitors. Whilst the ability of larval H. contortus cytochrome P450 to act as a peroxygenase in vitro was demonstrated, the inhibition results with adult microsomes showing both cytochrome P450 and peroxidase activities require further investigation to clarify the nature of the adult microsomal cumene hydroperoxide-supported O-deethylase activity.
Kotze
A C
AC
CSIRO Division of Animal Production, McMaster Laboratory, Blacktown, NSW, Australia. A.Kotze@anprod.csiro.au
D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000818:Animals; D001555:Benzene Derivatives / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006188:Haemonchiasis / Q000469:parasitology; D006189:Haemonchus / Q000201:enzymology* / Q000254:growth & development; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007814:Larva / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D012756:Sheep / Q000469:parasitology
D001555:Benzene Derivatives; D065607:Cytochrome P-450 Enzyme Inhibitors; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; C019647:aldrin epoxidase; D009195:Peroxidase; D015660:7-Alkoxycoumarin O-Dealkylase; C007164:cumene hydroperoxide