D. E. Williams

Rabbit lung flavin-containing monooxygenase is immunochemically and catalytically distinct from the liver enzyme

Flavin-containing monooxygenase has been purified to homogeneity from lung microsomes of pregnant rabbits. Antibody to rabbit lung flavin-containing monooxygenase was raised in guinea pig. Ouchterlony double diffusion analysis with this antibody produced precipitin lines of identity with the purified rabbit lung enzyme and lung microsomes from pregnant and non-pregnant rabbits. No cross-reaction was seen with liver microsomes from rabbit or with purified pig liver enzyme. The tricyclic antidepressant drugs, imipramine and chlorpromazine, are not substrates for the rabbit lung enzyme, whereas they are rapidly oxidized by the pig liver enzyme. These results indicate that rabbit lung and liver flavin-containing monooxygenases differ in substrate specificity, are immunochemically distinct proteins, and may be different gene products.

Sex differences in cytochrome P-450 isozyme composition and activity in kidney microsomes of mature rainbow trout

Kidney microsomes from sexually mature male, as opposed to female, rainbow trout displayed an approximately 20-fold higher cytochrome P-450 specific content, NADPH-cytochrome c reductase activity, and rates of hydroxylation of lauric acid, testosterone, progesterone and aflatoxin B1. Little or no sex difference in metabolism was observed with benzo[a]pyrene or benzphetamine as substrates. A similar pattern was observed in hepatic microsomes from these fish, but the difference was much less striking (approximately 2-fold higher activity in males). Juvenile trout (both sexes) possessed activities intermediate between mature males and females. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of kidney and liver microsomes of juvenile and sexually mature male and female trout suggested that the striking sex difference in kidney could be due to the high amount of trout P-450 isozyme LM2 in sexually mature males. Immunoquantitation of LM2, performed by Western Blotting and immunostaining with rabbit anti-trout LM2-IgG, confirmed that mature male kidney contained much higher levels of P-450 LM2 than juvenile or female kidney, or even of liver microsomes of all three groups. The amount of P-450 LM2 in mature female kidney microsomes was barely detectable. The high amount of LM2 in male trout kidney is consistent with the high activity of these microsomes towards lauric acid and aflatoxin B1, which have been shown previously to be preferentially metabolized by trout P-450 LM2. It is suggested that rainbow trout may serve as an alternative to the rat as an animal model for the study of sex-dependent differences in cytochromes P-450.

Hexachlorobenzene‐induced porphyria in Japanese quail: Changes in microsomal enzymes

Hexachlorobenzene (HCB) was administered orally (500 mg/kg d) for 1, 2, 5, or 10d) to sexually mature Japanese quail to compare altered hepatic porphyrin levels with changes that occur in hepatic xenobiotic metabolizing enzymes. Porphyrin levels rapidly increased following the administration of HCB (three times control levels after a single dose of HCB), and birds began to develop porphyria (i.e., porphyrin levels were at least 10 times higher than controls) following 5 d of treatment. Following 10 d of HCB treatment, 3 of 4 treated quail were porphyric. Coincident with the HCB-induced disruption of the heme biosynthetic pathway were increases in various hepatic constituents. Changes included elevation of microsomal protein concentrations and increases in the specific content of cytochrome P-450, in the activities of aryl hydrocarbon hydroxylase (AHH), biphenyl hydroxylase (BPH), ethoxyresorufin-O-deethylase (EROD), and ethoxycoumarin-O-deethylase (ECOD), and in cytosolic and microsomal glutathione S-transferase (GSH-t) levels. In addition, the lambda max of the CO versus CO-reduced absorption spectra of hepatic microsomes from HCB-dosed birds showed a hypsochromic shift of 450 to 448 nm. The activity of NADPH-cytochrome P-450 reductase was increased following 10 d of HCB, and the activity of epoxide hydrolase was increased following 5 d of HCB. Most of these changes occurred with a single HCB treatment, and no further alterations developed in the nature of the response with repetitive dosing. Only weight loss, increased cytochrome P-450 content, and increases in GSH-t activity occurred simultaneously with the induction of porphyria.

Hexachlorobenzene‐induced porphyria in japanese quail: An in vitro study of changes in cytochrome P‐450 and monooxygenases

The ability of hexachlorobenzene (HCB) to cause changes in the isozymic composition of hepatic monooxygenases of Japanese quail was assessed. HCB-induced changes in the relative concentrations of benzo[a]pyrene metabolites produced in vitro were apparent. HCB treatment also resulted in changes in the responsiveness of ethoxycoumarin O-deethylase (ECOD) and ethoxyresorufin O-deethylase (EROD) to the in vitro cytochrome P-450 inhibitors metyrapone, SKF-525A, and alpha-naphthoflavone. These changes may be indicative of alterations in the major cytochrome P-450 isozymes present following HCB treatment. Of these changes, only an increased responsiveness of ECOD to SKF-525A correlated with the onset of porphyria. The response of Japanese quail to the porphyrogenic action of HCB is more rapid than that found with more commonly used mammalian models. This rapid response is probably due either to the ability of quail to produce greater amounts of porphyrogenic metabolites of HCB than mammals or to a greater sensitivity of the heme pathway in quail to metabolites produced. In either case, this rapid response makes Japanese quail a good model for studying the biochemical mechanism for HCB-induced porphyria. The work presented here extends previous in vivo studies by using in vitro techniques to address the possibility that changes in the proportions of the major cytochrome P-450 isozymes occur in response to HCB and these changes, rather than changes in the total concentration of cytochrome P-450, are important to the development of porphyria.