Characterization of the activation of hepatic microsomal hydroxylation by betamethasone and α naphthoflavone

Biphenyl 2-hydroxylation is selectively activated in vitro by incubation of betamethasone or α naphthoflavone with control male rat liver microsomes. Biphenyl 3- and 4-hydroxylation activities are unchanged or marginally inhibited. The nature of the enzymes involved in the activation has been investigated. Metyrapone (1 mM) completely inhibited the expression of the activation but had a lesser effect on the basal 2-, 3- and 4-hydroxylation activities. SKF525A (1 mM) 2 inhibited both basal and betamethasone-activated enzyme activities by 25–35 per cent. Of other drug metabolizing enzymes investigated, only benzo[a]pyrene hydroxylation activity was increased by betamethasone and α naphthoflavone. Acetone (0.6M) caused a small activation (40 per cent) of biphenyl 2-hydroxylation but inhibited 4-hydroxylation. The non-ionic detergent Brij 35 inhibited biphenyl 2-, 3- and 4-hydroxylation. It was concluded that activation of biphenyl 2-hydroxylation differs from activation of aromatic amine hydroxylation and glucuronyl transferase but may be related to activation of benzo[a]pyrene hydroxylation by naphthoflavones.     

Tissue and sex differences in the activation of aromatic hydrocarbon hydroxylases in rats

Betamethasone and α-naphthoflavone produced similar activation of biphenyl 2-hydroxylase and benzo[a]pyrene 3-hydroxylase in control male rat liver microsomes. In small intestinal epithelial microsomes, betamethasone had no effect whereas α-naphthoflavone caused a pronounced activation of benzo[a]pyrene hydroxylation and a lesser activation of biphenyl 2-hydroxylation. In lung microsomes, betamethasone had no effect on either enzyme activity whereas α-naphthoflavone had no effect on biphenyl 2-hydroxylase but inhibited benzo[a]pyrene hydroxylase. In kidney cortex microsomes from male rats both compounds caused inhibition or had no effect whereas in kidney cortex microsomes from female rats betamethasone activated whereas α-naphthoflavone had no effect.Activation also occurred in isolated viable hepatocytes from male rats. The response of biphenyl 2-hydroxylase was very similar to that found in male rat liver microsomes but benzo[a]pyrene hydroxylase was more sensitive to activation and less sensitive to inhibition than in microsomes. The findings are interpreted as demonstrating the presence of more than one ‘latent’ aromatic hydrocarbon hydroxylase in rodents.     

Biphenyl hydroxylations and spectrally apparent interactions with liver microsomes from hamsters pre-treated with phenobarbitone and 3-methylcholanthrene.

1. Metabolism of [14-C]biphenyl by hamster liver microsomes has been studied by t.l.c., quantitative fluorimetry and difference absorption spectrophotometry. 2. 4-Hydroxybiphenyl (major metabolite) and 2-hydroxybiphenyl (minor) accounted for at least 83% of total biphenyl metabolism. Small quantities of 2,2'- and 4,4'-dihydroxybiphenyl metabolites were also tentatively identified. 3. Biphenyl 2- and 4-hydroxylations exhibited different NADPH-NADH specificities and pH profiles. 4. Phenobarbitone preferentially induced formation of 4-hydroxybiphenyl, while 3-methylcholanthrene induced 2- and 4-hydroxylation almost equally by affecting both production and further metabolism of 2- and 4-hydroxybiphenyl. 5. Biphenyl, 2- and 4-hydroxy- and 2,2'-dihydroxybiphenyl gave both high- and low-affinity type I spectrally apparent microsomal interactions, whereas 4,4'-dihydroxybiphenyl promoted a reverse type I spectral change. There was an inverse correlation between the spectral dissociation constants (Ks) and lipid solubilities for the low-affinity type I interactions and a possible direct correlation for the high-affinity type I interactions. 6. Phenobarbitone and 3-methylcholanthrene induced cytochrome P-450 and cytochrome P-448 respectively and produced complex changes in the biphenyl type I interaction kinetics. No direct relationship was found in 'control' or 'induced' microsomes between biphenyl 2- or 4-hydroxylation, the type I interaction and cytochrome P-450 concentration. The results are discussed in terms of a 3-methylcholanthrene-inducible biphenyl 2- and 4-hydroxylase and a phenobarbitone-inducible biphenyl 4-hydroxylase.     

Cholesterol 7α-hydroxylase—Evidence for a distinct hepatic microsomal enzyme system

The interrelationship of microsomal 7α-hydroxylation and drug oxidation reactions was studied in liver microsomes obtained from the male Wistar rat. When several compounds were administered to rats, a variety of changes ensued concerning the rate of cholesterol 7α-hydroxylation, ethylmorphine N-demethylase activity, and alterations in electron transport components. Cholestyramine and d-thyroxine administration resulted in significant increases in cholesterol 7α-hydroxylase activity. Both phenobarbital (PB) and spironolactone pretreatment did not produce an elevation of cholesterol 7α-hydroxylation, but significant increases in ethylmorphine N-demethylation over controls were realized. There was a lack of congruence with the rate of cholesterol 7α-hydroxylation and the content or activity of electron transport components whereas there was a demonstrable dependency of ethylmorphine N-demethylation on the monitored electron transport components for PB- and spironolactone-treated rats. Along with an elevation of cytochrome P448 content, 3-methylcholanthrene (3-MC) pretreatment reduced cholesterol 7α-hydroxylase activity and the rate of ethylmorphine N-demethylation. Concomitant treatment with 3-MC and cholestyramine caused no alteration of cholesterol 7α-hydroxylase activity. The inhibitory action of 3-MC on cholesterol 7α-hydroxylase activity was not due to competition for reducing equivalents in liver microsomes. The inhibitory phenomenon caused by NADH on microsomal cholesterol 7α-hydroxylase activity was reproducible and was dose-dependent at both subsaturating and saturating levels of NADPH. In conclusion, the results of this study indicate that the cholesterol 7α-hydroxylase enzyme system is distinctly different from that which catalyzes drug oxidations.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced changes in the hydroxylation of biphenyl by rat liver microsomes

Biphenyl 2- and 4-hydroxylase activities and cytochrome P-450 concentrations in microsomes were increased by oral doses of less than 1 μg TCDD/kg. Female rats were more sensitive than male rats to the inductive effects of TCDD. since highly significant increases in biphenyl-hydroxylating activities were observed at the dose level of 0.2 μg TCDD/kg in female but not in male rats. The inductive effect was very persistent: biphenyl 2- and 4-hydroxylases remained stimulated even after 73 days following a single oral dose of 25 μg TCDD/kg. The levels to which the hydroxylases were stimulated in female rats were the same as in male rats. Rats of all ages from 10 to 335 days responded to hepatic microsomal effects of TCDD to approximately the same degree. The enzyme inductive effect was diminished by the simultaneous administration of actinomycin D. The K_m of biphenyl 2-hydroxylase (1.42 mM) was not altered significantly by TCDD treatment, but the K_m of biphenyl 4-hydroxylase (0.62 mM) was increased to approximately the same value (1.6 mM) as that of the 2-hydroxylase. The V_max of biphenyl 4-hydroxylase was increased 4.5-fold but that of biphenyl 2-hydroxylase was increased 16.5-fold. Rates of 2β- and 16α-hydroxylation of testosterone were suppressed by TCDD but rates of 7α- and 6β-hydroxylation were unaffected. It would appear that the hepatic microsomal mixed-function oxidases responsible for the hydroxylation of biphenyl and testosterone are different.     

The alkaloid rutaecarpine is a selective inhibitor of cytochrome P450 1A in mouse and human liver microsomes.

Rutaecarpine, evodiamine, and dehydroevodiamine are quinazolinocarboline alkaloids isolated from a traditional Chinese medicine, Evodia rutaecarpa. The in vitro effects of these alkaloids on cytochrome P450 (P450)-catalyzed oxidations were studied using mouse and human liver microsomes. Among these alkaloids, rutaecarpine showed the most potent and selective inhibitory effect on CYP1A-catalyzed 7-methoxyresorufin O-demethylation (MROD) and 7-ethoxyresorufin O-deethylation (EROD) activities in untreated mouse liver microsomes. The IC(50) ratio of EROD to MROD was 6. For MROD activity, rutaecarpine was a noncompetitive inhibitor with a K(i) value of 39 +/- 2 nM. In contrast, rutaecarpine had no effects on benzo[a]pyrene hydroxylation (AHH), aniline hydroxylation, and nifedipine oxidation (NFO) activities. In human liver microsomes, 1 microM rutaecarpine caused 98, 91, and 77% decreases of EROD, MROD, and phenacetin O-deethylation activities, respectively. In contrast, less than 15% inhibition of AHH, tolbutamide hydroxylation, chlorzoxazone hydroxylation, and NFO activities were observed in the presence of 1 microM rutaecarpine. To understand the selectivity of inhibition of CYP1A1 and CYP1A2, inhibitory effects of rutaecarpine were studied using liver microsomes of 3-methylcholanthrene (3-MC)-treated mice and Escherichia coli membrane expressing bicistronic human CYP1A1 and CYP1A2. Similar to the CYP1A2 inhibitor furafylline, rutaecarpine preferentially inhibited MROD more than EROD and had no effect on AHH in 3-MC-treated mouse liver microsomes. For bicistronic human P450s, the IC(50) value of rutaecarpine for EROD activity of CYP1A1 was 15 times higher than the value of CYP1A2. These results indicated that rutaecarpine was a potent inhibitor of CYP1A2 in both mouse and human liver microsomes.     

Stereochemical biotransformation of warfarin as a probe of the homogeneity and mechanism of microsomal hydroxylases

The hepatic microsomal metabolism of R and S warfarin by normal Wistar and Sprague-Dawley rats was compared with that of phenobarbital (PB) and 3-methylcholanthrene (3-MC)-pretreated animals. In all the microsomal systems examined, R warfarin was metabolized faster than the S enantiomer. Induction of microsomal mixed-function oxidase activity with PB, and especially 3-MC, caused significant alterations in the normal stereochemical pattern of R and S warfarin hydroxylation which were independent of the method of microsomal preparation and the technique employed in the quantitation of hydroxylated products. PB pretreatment of Sprague-Dawley rats resulted in an increase in all hydroxylated products but to differing extents. Similar results were obtained from Wistar rats except for the processes of 4' and benzylic hydroxylation of (S) warfarin. 3-MC pretreatment resulted in the selective induction of 6- and 8-hydroxylation in both species. These results suggest that liver microsomes from normal animals contain at least two major, A and B, and two minor, C and D, mono-oxygenases which differ in their stereoselectivity (as measured by the rates for the formation of two enantiomeric products), regioselectivity (as measured by the ratio of any two isomeric products from the same substrate), and inducibility. In this model, normal animals have hydroxylase activity enzyme A which is not inducible by PB or 3-MC and which is stereoselective for the R enantiomer of warfarin in 7- and benzylic hydroxylation and for the S enantiomer in 4'-hydroxylation. Microsomes from normal and PB-induced animals contain additional hydroxylase activity, enzyme B, which catalyzes both the 6- and 8-hydroxylation of warfarin and which has low stereoselectivity but is regioselective for 6-hydroxylation. Enzyme B may also be responsible for some 4'-hydroxylation. PB-induced animals have additional mono-oxygenase activity, enzyme C, which displays the opposite stereoselectivity compared to enzyme A for benzylic and less stereoselectivity for 7-hydroxylation. 3-MC-induced animals have greatly enhanced levels of 6- and 8-hydroxylase activity, enzyme D, which is stereoselective for the R enantiomer and regioselective for 8-hydroxylation of R warfarin and 6-hydroxylation of S warfarin.     

Activation of hepatic microsomal biphenyl 2-hydroxylation by corticosteroids

1. 4-Hydroxylation was a major route of biphenyl metabolism in liver microsomes from control and phenobarbitone-pretreated rats, with 2- and 3-hydroxybiphenyl as lesser metabolites.

2. Many corticosteroids, when added to the microsomal incubation mixture, selectively increased 2-hydroxylation with little or no effect on 3- and 4-hydroxylation. Betamethasone caused the greatest activation (400%).

3. In liver microsomes from control hamsters and 3-methylcholanthrene-pretreated rats, the basal hydroxylase activity, especially 2-hydroxylation, was much higher, but the quantitative increase following betamethasone addition was similar to that in liver microsomes from control and phenobarbitone-pretreated rats.

4. Pretreatment of rats with betamethasone also resulted in a small increase in biphenyl 2-hydroxylation activity after 4?h, returning to control values after 6?h.

5. In vitro addition of estradiol or testosterone had no effect on either basal or betamethasone-activated biphenyl 2-hydroxylation.     

Lauric acid hydroxylation in human liver and kidney cortex microsomes

The ω- and (ω- 1 )-hydroxylation of the medium-chain fatty acid, dodecanoic or lauric acid, was studied in liver and kidney cortex microsomes from seven human cadavers. The rates of laurate hydroxylation in human liver microsomes were found to exceed the rates recorded in human kidney cortex microsomes by 4-to 30-fold. The mean specific activity of laurate hydroxylation from the seven human kidneys was six to fourteen times lower than the specific activities found in pig, rat or hamster kidney microsomes. The effects of several known inhibitors of the liver microsomal cytochrome P-450-dependent mono-oxygenase system were also studied. Metyrapone preferentially inhibited the (ω- 1)-hydroxylase activity of human liver microsomes, but did not affect the ω-hydroxylation reaction. In the presence of 7,8-benzoflavone, the human liver microsomal (ω- 1 )-hydroxylase activity was stimulated, but an inhibitory effect was observed on the ω-hydroxylation reaction. 2-Diethylaminoethyl-2,2-diphenylvaIerate (SKF 525A) inhibited both hydroxylase activities in human liver microsomes. Neither metyrapone nor SKF 525A inhibited the laurate hydroxylation reactions catalyzed by human kidney microsomes. These studies indicate that the cytochrome P-450-mediated hydroxylations of medium chain fatty acids in human kidney cortex microsomes are much less active than in kidneys of other species investigated. The effects of the inhibitors, metyrapone and SKF 525A, on ω- and (ω- 1)-hydroxylation of laurate in human liver and kidney microsomes were similar to the effects reported in other mammalian species.     

In vivo and in vitro effects of 3-methylcholanthrene on the microsome-mediated in vitro mutagenicity of 2-acetylaminofluorene

Pretreatment of rat, hamster or mouse by 3-methylcholanthrene (3-MC) largely induces the liver microsomal N-hydroxylase activity. The same pretreatment given simultaneously with 2-acetylaminofluorene (2-AAF) inhibits the hepatocarcinogenicity in the rat but not in the hamster.The present report compares the in vivo and in vitro effects of 3-MC on liver microsomal N-hydroxylation and liver microsome-mediated mutagenicity of 2-AAF in hamster, rat and mouse. The induction of hamster or mouse liver microsomal N-hydroxylase activity correlates well with the increase in the microsome-mediated mutagenicity of 2-AAF. With rat, however, even though the N-hydroxylase activity is largely enhanced, microsome-mediated mutagenicity is significantly reduced after pretreatment with 3-MC. Such a reduction parallels a decrease in enzyme affinity.Added in vitro to the incubation medium, 3-MC (μM concentration) inhibits both the N-hydroxylase activity and the microsome-mediated mutagenicity of 2-AAF. Those data are discussed in relationship with the biological interactions between 3-MC and 2-AAF.     

Induction of aryl hydrocarbon hydroxylase by 3-methylcholanthrene in liver,lung and kidney of gonadectomized and sham-operated wistar rats

Detailed dose-response curves have been obtained for the induction of aryl hydrocarbon hydroxylase (AHH) m liver, lung and kidney by intraperitoneally administered 3-methylcholanthrene (3-MC) in Wistar rats. The effects of gonadectomy also were studied. Lung was the tissue most sensitive to induction, followed by liver, and then kidney in all cases. Although liver exhibited the greatest overall activity, the per cent increase over control AHH levels was much higher in the two extrahepatic tissues. Gonadectomy did not affect either control or induced AHH activity in any of the three organs in the female, or in the male lung. However, castration of the males decreased control liver enzyme levels and increased these levels in kidney. The AHH levels reached after maximal 3-MC induction were the same in castrated and sham-operated male rat livers. A different pattern was seen in male kidney enzyme levels where significantly increased maximal induction was observed in castrated animals.     

Effects of betamethasone on the in vitro and in vivo 2-hydroxylation of biphenyl in the rat

The in vitro addition of betamethasone to rat liver microsomes caused a concentration-dependent stimulation of biphenyl 2-hydroxylation. At a 100 microM concentration of betamethasone, the formation of 2-hydroxybiphenyl was increased by approximately 4-fold in microsomes from 28-day-old rats and 10-fold in liver microsomes from 5-day-old rats. The steroid had little or no effect on the hydroxylation of biphenyl in the 3- or 4-position except at the highest concentration tested (1 mM), where a 20-30% inhibition was observed. The ip injection of 0.01 to 10 mumol of betamethasone to 5-day-old rats had little or no effect on the total body metabolism of 0.01 to 3.5 mumol of biphenyl-2-3H to 2-hydroxybiphenyl as measured by 3H2O formation. Although betamethasone had no effect on the 2-hydroxylation of biphenyl in the intact rat, this reaction was stimulated 4- to 9-fold by the addition of 100 microM betamethasone to hepatocyte monolayer cultures.     

Betamethasone-mediated activation of biphenyl 2-hydroxylation in rat liver microsomes. Studies on possible mechanisms

The addition of the steroid betamethasone to intact or detergent-solubilized rat liver microsomes caused a concentration-dependent increase in the rate of biphenyl 2-hydroxylation. Betamethasone (100 microM) increased the apparent Vmax for 2-hydroxybiphenyl formation 2- to 4-fold but had no effect on the apparent Km when either the biphenyl or NADPH concentration was varied. Betamethasone had little or no effect on the apparent Vmax or apparent Km of the 3- and 4-hydroxylations of biphenyl. The steroid did not enhance biphenyl 2-hydroxylation through a peroxidative mechanism. Betamethasone had little or no effect on the rate of the NADPH-dependent reduction of cytochrome c or total microsomal cytochrome P-450. The addition of purified NADPH-cytochrome P-450 reductase to cholate-solubilized liver microsomes increased the rate of hydroxylation of biphenyl in positions 2 and 4. Betamethasone (100 microM) decreased the apparent Km for purified cytochrome P-450 reductase by 48% and increased the apparent Vmax of 2-hydroxybiphenyl formation by 2-fold when the concentration of cytochrome P-450 reductase was varied. The steroid did not alter the Km or Vmax values for the 4-hydroxylation of biphenyl. The data suggest that betamethasone enhances the interaction between the reductase and the form(s) of cytochrome P-450 responsible for the 2-hydroxylation of biphenyl.     

Aryl hydrocarbon hydroxylase activity induced by injected diesel particulate extract vs inhalation of diluted diesel exhaust

The effects of long-term inhalation of diluted diesel exhaust on aryl hydrocarbon hydroxylase activity (AHH) and cytochrome P-450 content in lung and liver microsomes were investigated in male Fischer-344 rats and compared with repeated parenteral administration of organic solvent extracts of hydrocarbons adsorbed on the diesel particulate surface during the combustion process. The animals were exposed to concentrations of 750 micrograms m-3 or 1500 micrograms m-3 of diesel particulates from a 5.7L GM diesel engine 20 h per day, 5 1/2 days per week for up to 9 months or treated by repeated IP injections of diesel particulate extract (dissolved in corn oil) from the same engine at several dose levels for 4 days. No significant effects of long-term inhalation exposure were observed in liver microsomal AHH activity. A slight decrease in lung microsomal AHH activity was found in rats following 6 months of exposure to diesel exhaust at the particulate concentration of 1500 micrograms m-3. The total mass of particles deposited in the lung during the inhalation exposure was estimated and an equivalent dose of extractable hydrocarbons was administered intraperitoneally; no increase in AHH activity was observed in the lung or liver microsomes. In contrast, 1.4- to 9-fold increases in AHH activity were observed in liver and lung microsomes of rats pretreated by intraperitoneal doses 10-50 times larger than the most conservative estimate of the deposited lung burden. No changes in cytochrome P-450 content were observed in the microsomes of rat liver after inhalation or injection treatment studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interaction between two forms of cytochrome P-450 during drug oxidation in the reconstituted system containing limited amount of NADPH-cytochrome P-450 reductase

The activities of drug oxidation in a reconstituted system which contains two forms of cytochrome P-450 and a limiting amount of NADPH-cytochrome P-450 reductase were determined. Cytochrome P-450 (termed MC P-4481 and MC P-4482) purified from liver microsomes of 3-methyl-cholanthrene-treated rats was active in both 2- and 4-hydroxylation of biphenyl but cytochrome P-450 (termed PB P-450) purified from liver microsomes of phenobarbital-treated rats was active in 4-hydroxylation of biphenyl only. PB P-450, MC P-4481 and MC P-4482 were most active toward benzphetamine N-demethylation, aniline hydroxylation and 7-ethoxycoumarin O-deethylation, respectively. PB P-450 inhibited the activity of biphenyl 2-hydroxylation supported by MC P-4481 or MC P-4482. On the contrary, no inhibition of PB P-450 supported benzphetamine N-demethylation was observed when MC P-4481 or MC P-4482 was added to the system containing PB P-450 and limited amount of the reductase. The apparent Km of PB P-450 for the reductase obtained from double reciprocal plot of the reductase concentration and the activity of biphenyl hydroxylase or benzphetamine N-demethylation was lower than that of MC P-4481 or MC P-4482. These and other results suggest that there is a certain hierarchy among the cytochrome P-450 species for receiving electrons from reductase.     

Role of CYP2C19 in stereoselective hydroxylation of mephobarbital by human liver microsomes.

The 4-hydroxylation of mephobarbital enantiomers was investigated by using human liver microsomes from the extensive metabolizers (EM) and poor metabolizers of CYP2C19. The 4-hydroxylase activity of R-mephobarbital in the EM microsomes was >10 times higher than that of S-mephobarbital. In the poor metabolizer microsomes, the 4-hydroxylase activity of R-mephobarbital was much lower than that in the EM microsomes, and the ratio of 4-hydroxylase activity of R-mephobarbital to that of S-mephobarbital was also lower than that in the EM microsomes. Moreover, the 4-hydroxylase activity of R-mephobarbital showed a high correlation (r = 0.985, p<0.001) with the 4'-hydroxylase activity of S-mephenytoin in a panel of nine human liver microsomes. Anti-CYP2C antibody inhibited R-mephobarbital 4-hydroxylase activity by 85% of the control activity. R-Mephobarbital competitively inhibited S-mephenytoin 4'-hydroxylase activity (K(i) = 34 microM), while S-mephenytoin inhibited R-mephobarbital 4-hydroxylase activity (K(i) = 103 microM). Among the seven cDNA-expressed CYPs studied, only CYP2C19 catalyzed R-mephobarbital 4-hydroxylation. These findings suggest that the 4-hydroxylation of mephobarbital catalyzed by CYP2C19 is preferential for R-enantiomer in human liver microsomes.     

Effect of asbestos fibers on aryl hydrocarbon hydroxylase and aminopyrine N-demethylase activities of rat liver microsomes

Chrysotile asbestos fibers impair the activities of rat liver microsomal aryl hydrocarbon hydroxylase (AHH), aminopyrine (AP) N-demethylase and dimethylnitrosamine (DMN) demethylase in vitro. This inhibition is concentration-dependent. Preincubation of 3-methylcholanthrene (3-MC)-pretreated rat liver microsomes with chrysotile depresses the overall metabolism of [G-3H]benzo[a]pyrene (BaP). Various forms of asbestos employed inhibit AHH activity to the same extent. However, other types of asbestos are not as effective as chrysotile in diminishing AP demethylase activity. Chrysotile and crocidolite fibers are not found to significantly change the apparent Km of AHH activity, from 3-MC-pretreated rat liver microsomes, for BaP. Increasing the microsomal protein concentration partially abolishes the inhibition of AHH activity caused by chrysotile fibers. Inhibition of AP demethylase and AHH activities is attenuated by bovine serum albumin (BSA) or ferritin. Depression of AHH activity by crocidolite is significantly reversed by ferritin. Since polymers such as ferritin override enzyme inhibition by chrysotile as well as crocidolite, surface chemical groups of the fibers may be involved in enzyme modification.     

Studies of the mechanisms of metabolism of diethyl p-nitrophenyl phosphorothionate (parathion) by rabbit liver microsomes

Based on the protein content of microsomes, the administration of 3-methylcholanthrene (3-MC) and phenobarbital (PB) to adult rabbits leads to an increased rate of metabolism of parathion (diethyl 4-nitrophenyl phosphorothionate) by rough-surfaced and whole microsomes but not by smooth-surfaced microsomes. Although prior administration of both PB and 3-MC increased the cytochrome P-450 content of the microsomes, when the rate of metabolism of parathion was calculated on the basis of the concentration of cytochrome P-450 in these microsomes, there is no difference in the rate of metabolism of parathion by rough-surfaced and smooth-surfaced microsomes from the untreated, 3-MC-treated and PB-treated animals. However, based on the cytochrome P-450 concentration, the rate of metabolism of parathion by whole microsomes from 3-MC and PB-treated animals is less than the rate with whole microsomes from untreated animals. Further studies have shown there is no correlation between the concentration of high spin or low spin cytochrome P-450 in any of the microsomal fractions or subfractions and the rate of metabolism of parathion to paraoxon or diethyl phosphorothionate.     

Differential metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate by rat and chicken hepatic microsomes

The in vitro hepatic metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) was investigated in the hen (a species that is sensitive to EPN delayed neurotoxicity) and the rat (an insensitive species). EPN, which produced a Type I binding spectrum on incubation with cytochrome P-450, was converted by liver microsomes from both species to its oxygen analog, O-ethyl O-4-nitrophenyl phenylphosphonate (EPNO), and to p-nitrophenol (PNP). The formation of EPNO and PNP was dependent on the presence of NADPH in the reaction mixture and could be inhibited by either SKF-525A or by anaerobic conditions. The rates of EPNO and PNP formation by rat liver microsomes were, however, 3- and 20-fold higher, respectively, than the rates of formation by chicken liver microsomes. There was also a 4-fold difference in the cytochrome P-450 contents of the liver microsomes. The EPNO-hydrolyzing activity of rat liver microsomes was much greater than that of chicken liver microsomes. EPNO metabolism, in contrast to EPN metabolism, did not require NAPDH nor was it inhibited by SKF-525A or by anaerobic conditions. Prior exposure of rats to phenobarbital (PB) or Arochlor 1254 resulted in an increase in hepatic microsomal EPN metabolism and cytochrome P-450 content. On the other hand, 3-methylcholanthrene (3-MC) treatment elevated microsomal cytochrome P-450 but did not increase EPNO or PNP formation. Pretreatment with EPN did not alter either microsomal EPN metabolism or cytochrome P-450 levels. In chickens, prior exposure to PB, 3-MC or 100 mg/kg EPN increased EPNO and PNP formation by liver microsomes as well as cytochrome P-450 levels; prior exposure of chickens to 15 mg/kg EPN did not alter these variables. The λ_max Soret bands of the reduced hepatic cytochrome P-450 complexes from these animals differed as follows (rat then chicken): untreated, 450 vs 452 nm; PB-treated, 450 vs 451 nm; and 3-MC-treated, 448 vs 449 nm. None of the above treatments had an effect on EPNO metabolism by liver microsomes.     

Metabolism of phenanthrene by brown bullhead liver microsomes

We have investigated the regio- and stereoselective metabolism of phenanthrene by the liver microsomes of brown bullhead (Ameriurus nebulosus), a bottom dwelling fish species. The liver microsomes from untreated and 3-methylcholanthrene (3-MC)-treated brown bullheads metabolized phenanthrene at a rate of 14.1 and 20.7 pmol/mg protein/min, respectively, indicating that the hydrocarbon is a rather poor substrate for bullhead liver microsomes contrary to what has been reported for rat liver microsomes. The major phenanthrene metabolites formed by liver microsomes from untreated and 3-MC-treated bullheads included benzo-ring 1,2-dihydrodiol (25.3 and 11.6%), K-region 9,10-dihydrodiol (9.6 and 9.6%), and phenols (40.5 and 54.5%). The 3,4-dihydrodiol represented a minor proportion of the total phenanthrene metabolites. The low proportion of the 9,10-dihydrodiol formed by both control and 3-MC-treated bullhead microsomes sharply contrasts the previous data reported for the corresponding rat liver microsomes which metabolized phenanthrene predominantly to its 9,10-dihydrodiol representing 76.6 and 67.1%, respectively of the total metabolites. Liver microsomes from 3-MC-treated bullheads, like rat liver microsomes, were more selective in their attack at the 1,2-position of the benzo-ring than at the 3,4-position of the benzo-ring. Phenanthrene 1,2-dihydrodiol and 3,4-dihydrodiol formed by liver microsomes from both control and 3-MC-treated bullheads consisted predominantly of their R,R enantiomer. Phenanthrene, compared with benzo[a]pyrene and chrysene, is metabolized by bullhead liver microsomal enzymes to its benzo-ring dihydrodiols with a relatively low degree of stereoselectivity.