USE OF 7-ALKOXYPHENOXAZONES, 7-ALKOXYCOUMARINS AND 7-ALKOXYQUINOLINESAS FLUORESCENT SUBSTRATES FOR RAINBOW-TROUT HEPATIC MICROSOMES AFTER TREATMENT WITH VARIOUS INDUCERS

Various fluorescent substrates have been used as specific indicators o f induction or activity of different cytochrome P450 isozymes in both fish and mammalian species. In an attempt to identify additional defin itive fluorescent substrates for use in fish, we examined a series of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as subs trates in O-dealkylation assays with hepatic microsomes from rainbow t rout (Oncorhynchus mykiss). Microsomes were prepared after 48 hr of tr eatment with beta-naphthoflavone (beta-NF), pregnenolone-16 alpha-carb onitrile (PCN), phenobarbital (PB), isosafrole (ISF), or dexamethasone (DEX). Total P450 spectra were obtained, and spectral binding studies were performed. Microsomal O-dealkylation rates were greater after IS F treatment than after beta-NF treatment for 7-methoxy-, 7-ethoxy-, 7- propoxy- and 7-benzyloxyphenoxazones but not for 7-butoxyphenoxazone. DEX treatment resulted in a significant elevation of pentoxyphenoxazon e metabolism (about a 144-fold increase) compared with microsomes indu ced by beta-NF (11-fold) and ISF (37-fold). The rates of dealkylation of the alkoxyphenoxazones by ISF-treated microsomes occurred in the fo llowing order: methoxy > ethoxy > propoxy > benzxyloxy > butoxy > pent oxy. When beta-NF-treated microsomes were used, the 7-alkoxyphenoxazon es were metabolized as follows: methoxy > ethoxy > propoxy > butoxy > benzyloxy approximate to pentoxy, while the order of metabolism of the 7-alkoxycoumarins was: ethoxy much greater than butoxy > propoxy appr oximate to methoxy > benzyloxy > pentoxy. None of the other treatments significantly increased the rate of metabolism of any of the alkoxyco umarins. Treatment with beta-NF did not significantly elevate the rate of metabolism of any of the alkoxyquinolines. DEX treatment produced significant elevations in the rate of metabolism of benzyloxy-, ethoxy -, and butoxy- approximate to pentoxy- approximate to propoxyquinoline , in that order. ISF treatment significantly elevated the rate of meta bolism of benzyloxy-, methoxy- and butoxyquinoline, in that order. The se results suggest that some of these new fluorescent substrates can b e used to characterize induction of rainbow trout hepatic microsomal m onooxygenase activity by ISF and DEX, in addition to the commonly used ethoxyphenoxazone and ethoxycoumarin for the characterization of indu ction by beta-NF or other 3-methylcholanthrene-type P450 inducers. Dis tinction between ISF-type and beta-NF-type inducers in rainbow trout h epatic microsomes may best be made using 7-methoxycoumarin as a substr ate. Distinction between ISF-type and DEX-type inducers and between be ta-NF-type and DEX-type inducers may best be made using 7-methoxypheno xazone as a substrate. With beta-NF induction 7-methoxycoumarin, with ISF induction 7-methoxy- phenoxazone, and with DEX induction 7-ethoxyq uinoline were metabolized to the greatest extent compared with control s and all other substrates tested.