Association of cytochrome P450 induction with oxidative stress in vivo as evidenced by 3-hydroxylation of salicylate.
1. Previous studies have shown that formation of 2,3-dihydroxybenzoate (2,3-DHB) from salicylate in vivo is a sensitive and specific marker of *OH radical generation, since 2,3-DHB is formed exclusively by *OH radicals, whereas both *OH radicals and cytochrome P450 (CYP) contribute to the production of 2,5-DHB. In the present study the salicylate-hydroxylation assay was used to examine whether CYP induction by the administration of dexamethasone, phenobarbital or beta-naphthoflavone to the male rat led to oxidative stress in vivo. 2. Dexamethasone was used under conditions that induced an approximately 50-fold induction of CYP P4503A expression in liver microsomal protein. Treatment with dexamethasone caused a 17.2-fold increase in 2,3-DHB plasma concentration compared with control animals. An increase in total hydroxylated salicylate (2,3-DHB plus 2,5-DHB) of 133.5 micromol/l plasma was produced, of which--assuming that the attack by *OH in position 3 or 5 of salicylate occurs at a similar rate--10.9 micromol/l were due to *OH radical attack and 122.6 micromol/l due to metabolism by CYP. 3. Phenobarbital led to a 4.7-fold increase in 2,3-DHB plasma concentration under conditions that induced CYP P4502B and 3A. An increase in total hydroxylated salicylate of 34.3 micromol/l plasma was observed, 2.0 micromol/l due to *OH radical attack and 32.3 micromol/l due to metabolism by cytochrome P450. 4. In contrast to dexamethasone and phenobarbital, beta-naphthoflavone did not cause a significant increase in 2,3-DHB plasma concentrations. 5. SKF 525A, a mixed-function oxidase inhibitor, caused a significant reduction of mean 2,5-DHB plasma concentration by 35% (p < 0.001), whereas 2,3-DHB was not significantly reduced, indicating that in contrast to the situation after induction by dexamethasone or phenobarbital, *OH radical generation by constitutive CYP contributes only to a minor degree to total in vivo *OH radical generation. 6. This study shows for the first time, to the authors' knowledge, that induction of some (but not all) P450s is associated with the production of hydroxyl radicals in vivo.
Strolin-Benedetti
M
M
Zambon Group SpA, Bresso, Milan, Italy.
Brogin
G
G
Bani
M
M
Oesch
F
F
Hengstler
J G
JG
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis; D019388:Cytochrome P-450 CYP1A2 / Q000096:biosynthesis; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D003907:Dexamethasone / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000032:analysis / Q000378:metabolism; D005841:Gentisates; D062385:Hydroxybenzoates / Q000032:analysis / Q000378:metabolism; D006900:Hydroxylation; D008297:Male; D018384:Oxidative Stress / Q000502:physiology*; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis; D010634:Phenobarbital / Q000494:pharmacology; D011335:Proadifen / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012459:Salicylates / Q000378:metabolism*; D019324:beta-Naphthoflavone / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005841:Gentisates; D062385:Hydroxybenzoates; D012459:Salicylates; D019324:beta-Naphthoflavone; C009135:2,3-dihydroxybenzoic acid; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D011335:Proadifen; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; C010925:2,5-dihydroxybenzoic acid; D010634:Phenobarbital