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.     

Some characteristics of hamster liver and lung microsomal aryl hydrocarbon (biphenyl and benzo(a)pyrene) hydroxylation reactions

Aryl hydrocarbon hydroxylation (AHH) reactions were compared using liver and lung microsomes of corn oil- and 3-methylcholanthrene (3-MC)-treated hamsters, employing benzo(a)pyrene (BAP) and biphenyl as substrates. The predominant AHH activity of liver and lung microsomes from corn oil- or 3-MC-treated hamsters was biphenyl 4-hydroxylase. Biphenyl 2-hydroxylase and BAP-hydroxylase activities were approximately 50 per cent as active as biphenyl 4-hydroxylase in liver and approximately 1–3 per cent as active as biphenyl 4-hydroxylase in lung microsomes. Biphenyl 4-hydroxylase activity was 70–80 per cent as active in lung as in liver microsomes. Treatment with 3-MC in vivo induced the biphenyl 4-hydroxylation reaction in liver but not in lung microsomes, the biphenyl 2-hydroxylation reaction both in lung and liver microsomes, and the BAP hydroxylation reaction in lung but not in liver microsomes. Biphenyl 2- and 4-hydroxylase activities of liver microsomes displayed similar sensitivities to inhibition by a number of chemical inhibitors in vitro. Inhibition of biphenyl hydroxylation reactions by metyrapone or carbon monoxide did not distinguish between lung or liver microsomal mono-oxygenases of corn oil- or 3-MC-treated hamsters. While small differences were expressed by inhibition with ethylmorphine, large differences became apparent through inhibition studies with BAP or α-naphthoflavone. It is concluded that the major aromatic hydroxylase activity of lung microsomes from corn oil- or 3-MC-treated hamsters resembles the constitutive (uninduced) AHH of the liver microsomes and that the minor aromatic hydroxylase activity of lung microsomes from corn oil- or 3-MC-treated hamsters resembles the induced AHH of the liver microsomes.     

Effect of isoniazid administration on selected rat and mouse hepatic microsomal mixed- function oxidases and in vitro [14C]acetylhydrazine-derived covalent binding

The effect of isoniazid on selected microsomal mixed-function oxidase and activities and on the microsomal metabolism of its own metabolite, acetylhydrazine, to a highly reactive compound which covalently binds to intracellular macromolecules was characterized in male C57BL6 mice and male Sprague-Dawley rats. In comparison with controls, isoniazid pretreatment of rats significantly increased the sp. act. of acetanilide 4-hydroxylase and the in vitro [¹⁴C]acetylhydrazine-derived covalent binding to hepatic microsomes but significantly decreased the sp. act. benzo[a]pyrene hydroxylase and testosterone 16α-hydroxylase. Isoniazid treatment of mice had no effect on any of these parameters except for a significant reduction is sp. act. of testosterone 7α-hydroxylase. Thus the pathway of isoniazid metabolism leading to the formation of reactive metabolites of acetylhydrazine is enhanced by isoniazid pretreatment in rats but not in mice. The presence of similar routes of isoniazid metabolism in man may account for the 8.7–24% incidence of subclinical hepatocellular damage observed in patients receiving isoniazid alone in the chemoprophlaxis of tuberculosis.     

Induction of hepatic microsomal cytochrome P-448-mediated oxidases by 3,3′-Dichlorobenzidine in the rat

Intraperitoneal administratioin of the hepatocarcinogen 3,3′-dichlorobenzidine (4,4′-diamino, 3,3′-dichlorobiphenyl) to adult male rats caused the induction of hepatic microsomal ethoxycoumarin O-deethylase and p-nitrophenetole O-deethylase activities comparable in magnitudes to those induced by 3-methylcholanthrene; neither anilin hydroxylase nor aminopyrine N-demethylase activity was affected by the pretreatment. The induction was not accompanied by a significant increase in content of hepatic microsomal cytochrome P-450; however, a shift in the absorption maxium of the reduced + CO spectrum of the cytochrome to 448 nm and an increase in the ratio of the 455 nm : 430 nm peaks of the reduced + ethylisocyanide spectrum of the hemoprotein was affected. Arylhydrocarbon hydroxylase activitity was stimulated 5-fold by dichlorobenzidine pretreatment in comparison with a 12-fold stimulation following 3-methylcholanthrene pretreatment. However, enzymatically mediated covalent binding of benzo[a]pyrene to microsomal protein was greater in microsomes from dichlorobenzidine-pretreated rats than in those from methylcholanthrene-pretreated rats. All of the dichlorobenzidine-induced enzymic activities were inhibited by α-naphthoflavone but not by SKF-525A. Hepatic microsomes from dichlorobenzidine than those from untreated animalsl both sets of microsomes elicited the Type II spectral change on combination with the compound, albeit with different binding affinities and capacities. The results show that dichlorobenzidine, although only a dihalogenated biphenyl derivative, is a potent inducer of cytochrome P-448.     

Enhanced aryl hydrocarbon hydroxylase activity after interaction between solubilized cytochrome P-448 and microsomes low in endogenous cytochrome P-450

The effects of cumene hydroperoxide on microsomal mixed-function oxidase components and enzyme activities were determined. In vitro cumene hydroperoxide treatment decreased cytochrome P-450 content, benzphetamine N-demethylase activity and aryl hydrocarbon hydroxylase activity of hepatic and renal microsomes from adult male and female rats, and of hepatic microsomes from fetal rats. Cumene hydroperoxide-treated microsomes, as well as fetal liver and adult renal microsomes, which are naturally low in cytochrome P-450 and mixed-function oxidase activity, were used to incorporate partially purified hepatic cytochrome P-448 isolated from 2,3,7,8-tetrachlorodibenzo-p-dioxin-pretreated immature male rats. This resulted in an enhanced rate of benzo[a]pyrene hydroxylation. Aryl hydrocarbon hydroxylase activity was increased 12-, 26-. 31- and 53-fold when 1.0 nmole of partially purified cytochrome P-448 was incubated with fetal liver microsomes, microsomes from kidney cortex of female rats, and cumene hydroperoxide-pretreated hepatic microsomes from female and male rats, respectively. The increased rate of benzo[a]pyrene hydroxylation was linear with cytochrome P-448 over the range 0.25 to 1.0 nmole. Because cumene hydroperoxide-pretreated microsomes from male rat liver and the hepatic and renal microsomes from female rats have a combination of high NADPH-cytochrome c reductase activity and low mixed-function oxidase activity, they are an attractive choice for catalytic studies of the interaction between cytochrome P-448 and microsomes.     

Differential effects of disulfiram and diethyldithiocarbamate on small intestinal and liver microsomal benzo[a]pyrene metabolism

Microsomes isolated from rat small intestinal mucosa and liver were used to study the effects of disulfiram and diethyldithiocarbamate on benzo[a]pyrene monooxygenase activity. This activity was decreased in the intestinal microsomes to 25 per cent of control 24 hr after a single oral dose of disulfiram. In contrast, daily administration of disulfiram for 5 days produced a dose related increase of benzo[a]pyrene monooxygenase activity, above control level. The elevated activities were accompanied by a concomitant increase in the concentration of cytochrome P-450. This benzo[a]pyrene monooxygenase activity was further stimulated by addition of α-naphthoflavone to the incubation medium. Furthermore, the absorption maximum of this cytochrome was at 450 nm in the CO bound reduced difference spectrum. These observations indicate that the disulfiram induced cytochrome P-450 was of the control type. Daily pretreatment with diethyldithiocarbamate impaired both intestinal and liver microsomes at benzo[a]pyrene monooxygenase activities. Pretreatment with a single dose of 3-methylcholanthrene resulted in a more than 10-fold increase of intestinal benzo[a]pyrene monooxygenase activity after 24 hr. Administration of disulfiram 24 hr before treatment appeared to potentiate the 3-methylcholanthrene induced increase of intestinal benzo[a]pyrene monooxygenase activity. In vitro addition of disulfiram and diethyldithiocarbamate to incubates of intestinal or liver microsomes inhibited benzo[a]pyrene metabolism to various extents; the liver being more sensitive. Disulfiram was approximately 50-fold more potent as an inhibitor than diethyldithiocarbamate. The in vitro inhibition of intestinal benzo[a]pyrene monooxygenase activity obtained with disulfiram appeared to be caused both by direct interaction with the monooxygenase system and through NADPH dependent metabolic activation of disulfiram, while the inhibition of diethyldithiocarbamate may be a result of the latter process only.     

Hydroxylation of benzo[a]pyrene and binding of (−)trans 5-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to deoxyribonucleic acid catalyzed by purified forms of rabbit liver microsomal cytochrome P-450: Effect of 7,8-benzoflavone,butylated hydroxytoluene and ascorbic acid

The catalytic activities of hepatic microsornes from untreated, phenobarbital-treated and 3-methylcholanthrene-treated adult rabbits with respect to benzo[a]pyrene hydroxylation and the activation of (?)(rflw-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene[(?)trans-7,8-diol] to DNA-binding metabolites were determined in the absence and presence of mixed-function oxidase inhibitors and compared to the corresponding activities of the individual enzyme systems. Treatment of rabbits with phnobarbital led to induction of P-450LM₂ and a concomitant 3-fold enhancement in microsomal benzo[a]pyrene hydroxylase activity, whereas the conversion of (?)trans-7,8-diol to DNA-binding products was unaffected. Homogeneous phenobarbital-inducible P-450LM₂ exhibited the highest activity and specificity toward benzo[a]pyrene and the lowest activity toward (?)trans-7,8-diol. Conversely, P-450LM₄ was the major form of cytochrome P-450 induced in rabbit liver by 3-methylcholanthrene or β-naphthoflavone, and this was associated in microsomes with an increase in the metabolism of (?)trans-7, 8-diol but not of benzo[a]pyrene. Homogeneous P-450LM₄ preferentially Catalyzed the oxygénation of (?)trans-7,8-diol, but was largely ineffective with benzo[a]pyrene. Partially purified P-450LM₇ lacked substrate specificity, for it metabolized both benzo[a]pyrene and (?)trans-7, S-diol at comparable rates. Additionally, 7,8-benzoflavone strongly inhibited benzo[a]pyrene hydroxylation by P-450LM₄ and phenobarbital-induced microsomes, as well as (?)trans-7,8-diol metabolism by P-450LM₄ and 3-methyl-cholanthrene-induced microsomes; in contrast, the activity of control microsomes with either substrate, and the activities of P-450LM₄ and LM₂ with benzo[a]pyrene and (?)trans-7 ,8-diol, respectively, were only partially or slightly decreased by 7,8-benzoflavone. Unlike 7,8-benzoflavone, butylated hydroxytoluene inhibited benzo[a]pyrene hydroxylation only. Thus, different forms of rabbit liver microsomal cytochrome P-450 were involved in the metabolism of benzo[a]pyrene and its 7,8-dihydrodiol. The results also demonstrate that the changes in substrate specificity and inhibitor sensitivity seen in phenobarbital- and 3-methylcholanthrene-induced microsomes relative to control rabbit liver microsomes can be accounted for by the catalytic properties of a specific form of cytochrome P-450 that prevails in these preparations, P-450LM₂ and LM₄, respectively.     

Species differences in the substrate specificity of hepatic cytochrome P-448 from polycyclic hydrocarbon-treated animals.

The in vitro effects of α-naphthoflavone on four hepatic mono-oxygenase activities associated with aromatic hydrocarbon responsiveness in the mouse (aryl hydrocarbon hydroxylase, 2-acetylamino-fluorene N-hydroxylase, biphenyl 2-hydroxylase, and biphenyl 4-hydroxylase) were investigated before and after methylcholanthrene treatment of C57BL/6N and DBA/2N mice, rats, hamsters, guinea pigs and rabbits. The electrophoretic pattern of cytochrome P-450 subunits and reduced CO-hemoprotein difference spectra of the microsomal fractions were also studied. Pretreatment of animals with methylcholanthrene caused: (1) a 1.5 to 2 mm hypsochromic shift in the Soret peak of the reduced hemoprotein-CO complexes in liver microsomes from a C57BL/6N mouse, rat, hamster and rabbit; a 0.5-nm hypsochromic shift in the guinea pig and no shift in the DBA/2N mouse; and (2) an increase in cytochrome P-450 apoproteins of the following molecular weights on sodium dodecyl sulfate-polyaerylamide gel electrophoresis: 54,000 and 55,000 in the C57BL/6N mouse; 48,000, 54,000 and 55,000 in the rat; 49,000 and 54,000 in the hamster; and 54,000 and 57,000 in the rabbit; a small increase in the 54,000 band was seen in the DBA/2N mouse and no increase in the guinea pig. In vitro addition of α-naphthoflavone selectively inhibited all four mono-oxygenase activities from the methylcholanthrene-treated C57BL/6N mouse, rat and hamster; 2-acetylaminofluorene N-hydroxylase and biphenyl 4-hydroxylase activities in the rabbit; and aryl hydrocarbon hydroxylase, 2-acetylaminofluorene N-hydroxylase and biphenyl 4-hydroxylase activities in the guinea pig. The addition of α-naphthoflavone enhanced the activities of aryl hydrocarbon hydroxylase and biphenyl 2-hydroxylase in liver microsomes from both control and methylcholanthrenetreated rabbits, but only biphenyl 2-hydroxylase activity was increased in the guinea pig: the activitity of 2-acetylaminofluorene N-hydroxylase was increased in both control and methylcholan-threne-treated DBA/2M mice, but only in the control C57BL/6N mouse. These data indicate that hepatic cytochrome P-448 is composed of multiple cytochromes, which differ among animal species, each catalyzing different mono-oxygenase activities.     

Dose-dependent effects of medroxyprogesterone acetate on the hepatic drug-metabolizing enzyme system in rats

The activity of the hepatic microsomal drug metabolism was examined in vitro in rats pretreated with 10–600 mg/kg medroxyprogesterone acetate intraperitoneally daily for seven days. In both sexes there was a significant increase in the liver weight, amount of cytochrome P-450, activity of NADPH-cytochrome c reductase, benzo[a]pyrene hydroxylase and 2,5-diphenyloxazole hydroxylase. The increase in 7-ethoxycoumarin-O-deethylase activity was also significant in female rats, but not in male rats. In the female rats pretreated with medroxyprogesterone acetate, the ability of α-naphtho-flavone and SKF 525A to inhibit benzo[a]pyrene hydroxylase was decreased and slightly increased, respectively. The results show that medroxyprogesterone acetate has a dose-dependent inducing effect on the hepatic drug metabolism in rats. Female rats seem to be more sensitive to the inducing effect of medroxyprogesterone acetate than the males. The characteristics of medroxyprogesterone acetate induction resemble mostly those caused by phenobarbital and pregnenolone-16α-carbonitrile.     

Inhibition of cytochrome P-450c-mediated benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by dihydrosafrole

1. Inhibitory activity of dihydrosafrole towards benzo[a]pyrene (BP) hydroxylase activity in hepatic microsomes from β-naphthoflavone (BNF)-induced rats, and in reconstituted systems containing cytochrome P-450c, increased dramatically on preincubation of the inhibitor with NADPH; no inhibition occurred without preincubation. The level of BP hydroxylase inhibition was associated with the progressive formation of the 456 nm dihydrosafrole metabolite-cytochrome P-450c spectral complex during preincubation.

2. Inhibition of BP hydroxylase by dihydrosafrole in control microsomes, and inhibition of ethoxyresorufin O-deethylase (EROD) in microsomes (control or BNF-induced) and in reconstituted systems with cytochrome P-450c, did not require preincubation and apparently was not dependent on prior formation of the dihydrosafrole metabolite-cytochrome P-450 complex.

3. Kinetic studies established that, following preincubation with NADPH, dihydrosafrole was a noncompetitive inhibitor of both BP hydroxylase and EROD activities. In the absence of preincubation, dihydrosafrole was an effective competitive inhibitor of EROD in BNF-induced microsomes and in reconstituted systems with cytochrome P-450c.

4. Both ethoxyresorufin and benzo[α]pyrene inhibited the development of the type 1 optical difference spectrum of dihydrosafrole in reconstituted systems containing cytochrome P-450c. Inhibition by ethoxyresorufin was competitive while that caused by benzo[α]pyrene was noncompetitive in nature.

5. The type II ligand phenylimidazole was an effective noncompetitive inhibitor of EROD activity but failed to exert any inhibitory effect on cytochrome P-450c-mediated BP hydroxylase activity. Phenylimidazole inhibited formation of the dihydrosafrole type 1 optical difference spectrum non-competitively.

6. The results indicate that ethoxyresorufin and benzo[α]pyrene may occupy different binding sites on cytochrome P-450c and that dihydrosafrole binds primarily to the site utilized by ethoxyresorufin.     

Effects of steroid hormones in vitro on adrenal xenobiotic metabolism in the guinea pig

Studies were carried out to determine the effects of steroid hormones in vitro on adrenal and hepatic microsomal benzphetamine demethylation and benzo[a] pyrene hydroxylation. Testosterone inhibited adrenal drug metabolism but had no effect on hepatic enzymes, whereas 6β-hydroxytestosterone had no effect in either tissue. All of the corticosteroids tested (cortisol, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, progesterone, and 17-hydroxyprogesterone) produced a concentration-dependent inhibition of adrenal drug metabolism, but had little or no effect on hepatic metabolism. The 17-deoxy-steroids were more potent inhibitors of adrenal metabolism than were their 17-hydroxylated counterparts. Cortisol was a potent inhibitor of adrenal benzphetamine and benzo[a]pyrene metabolism, produced a type I difference spectrum in adrenal microsomes, and diminished the magnitude of the benzphetamine-induced spectrum; 6 β-hydroxycortisol had none of these effects. Prior addition of benzphetamine to adrenal microsomes reduced the size of cortisol-induced spectral change. The results demonstrate that the effects of corticosteroids in vitro are relatively specific for adrenal enzymes and established a close association between the 6 β-hydroxylase and some drug-metabolizing enzymes. Adrenal steroids may have an important role in the regulation of adrenal xenobiotic metabolism.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) effects on hepatic microsomal cytochrome P-448-mediated enzyme activities.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatic microsomal mixed function oxidase (MFO) enzyme systems were examined in female rats. Although TCDD had little effect on NADPH-cytochrome c reductase activity and cytochrome P-450 content, the activities of the cytochrome P-448-mediated enzymes benzo[α]pyrene hydroxylase, ethoxyresorufin O-deethylase, and biphenyl 2-hydroxylase were greatly increased. Three months after a single oral dose of 2 μg/kg TCDD, the cytochrome P-450 content and benzo[α]pyrene hydroxylase and ethoxyresorufin O-deethylase activities were still significantly increased. In addition, the microsomal metabolism of the novel substrate 4,4′-dimethylbiphenyl was greatly increased by TCDD pretreatment. Low dose studies revealed that the ED50 of TCDD induction of benzo[α]pyrene hydroxylase was 0.63 μg/kg and the lowest dose of TCDD which caused a significant increase in enzyme activity was 0.002 μg/kg. Studies in which [1,6-³H]TCDD was used to determine the extent of hepatic uptake of orally administered TCDD at the lowest effective dose of 0.002 μg/kg lead to the estimate that only 65 molecules of TCDD per hepatocyte were required to produce a measurable increase in benzo[α]pyrene hydroxylation. These results attest to the specificity and persistence of TCDD in the induction of cytochrome P-448-mediated enzyme activities in rat liver. The small number of molecules required to induce benzo[α]pyrene hydroxylase suggests that TCDD is among the most potent MFO-inducing agents yet demonstrated in mammalian liver.     

Benzo[a]pyrene metabolism in the Mongolian gerbil: influence of chlordecone and mirex induction

1. The metabolism of benzo[a]pyrene (BP) by gerbil hepatic microsomes is increased following induction by phenobarbital (PB), chlordecone, mirex and 3-methylcholanthrene (3-MC).

2. By several criteria including the influence of α-naphthoflavone (α-NF) on BP-hydroxylase activity and BP-metabolite profiles, the cytochromes P-450 responsible for benzo[a]pyrene metabolism appear to be similar in microsomes isolated from PB-, chlordecone-, or mirex-treated gerbils. The cytochromes P-450 present in microsomes isolated from control animals and those treated with 3-MC are different from each other and from those present in PB, chlordecone, or mirex microsomes by the same criteria.

3. Of the inducers used, only PB induced microsomal epoxide hydrolase activity.     

Mutagenic activation and detoxification of benzo[a]pyrene in vitro by hepatic cytochrome P450 1A1 and phase II enzymes in three meat-producing animals

The mutagenic activation activity of hepatic microsomes from three meat-producing animals (cattle, deer and horses) was compared with those of rats as a reference species. In the Ames Salmonella typhimurium TA98 assay, the liver microsomes of all examined animals mutagenically activated benzo[a]pyrene, an ideal promutagens, in terms of production of histidine-independent revertant colonies. The microsomes of horses had the highest ability to produce revertant colonies of the examined animals under both low and high substrate concentrations. Inhibition of this mutagenic activity using α-naphthoflavone, anti-rat CYP1A1, CYP3A2 and CYP2E1 antibodies suggests that this activity was mainly because of CYP1A1 in these animals as well as in rats. The addition of co-factors for two phase II enzymes, microsomal UDP glucoronosyl transferase and cytosolic glutathione-S-transferase, reduced the production of the revertant colonies in a concentration-dependent manner. Interestingly, horses had the highest reduction rate among the examined animals, suggesting that phase II enzymes play a great role in producing a state of balance between the bioactivation and detoxification of xenobiotics in these meat-producing animals. This report is the first to investigate the mutagenic activation activity of the hepatic microsomes and the role of phase II enzymes against this activity in meat-producing animals.     

Hepatic and extrahepatic microsomal electron transport components and mixed-function oxygenases in the marine fish Stenotomus versicolor.

NADPH-cytochrome c reductase, benzo[a]pyrene hydroxylase and aminopyrine demethylase activities in hepatic microsomes from the marine fish scup (Stenotomus versicolor) were characterized according to dependence of Ph, temperature, ionic strength and Mg²⁺. The kinetic properties of benzo[a] pyrene hydroxylase were variable, depending on protein and substrate concentration, with measured K_m values for benzo[a]pyrene between 4 × 10^?7 M and 4 × 10^?5 M. The K_m for aminopyrine was 7 × 10^?4 M, and NADPH-cytochrome c reductase had K_m values of 2.1 × 10^?5 M and 1.3 × 10^?5 M for cytochrome c and NADPH. respectively. NADH supported benzo[a]pyrene hydroxylation at 10 per cent of the rate seen with NADPH, and no synergism was observed. Aminopyrine demethylation proceeded at least as well with NADH as with NADPH, and there was synergism when combined. NADPH- and NADH-cytochrome c reductases were detected in “microsomes” from fourteen extrahepatic tissues, including kidney, testis, foregut, gill, heart, red muscle, hindgut, buccal epidermis, pyloric caecum, spleen, brain, lens, ovary and white muscle. Benzo[a]pyrene hydroxylase was detected in all but white muscle, while cytochrome P-450 and aminopyrine demethylase were detectable in fewer tissues. Reduced, CO-ligated absorption maxima in the Soret region were 450 nm for all those but liver (occasionally 449 nm) and heart (about 447 nm). The estimated turnover numbers for benzo[a]pyrene hydroxylase and aminopyrine demethylase, and the influence of 7,8-benzoflavone in vitro on benzo[a]pyrene hydroxylase indicate that the cytochromes P-450 in different fish tissues are not catalytically equivalent.     

Organic solvent extraction of liver microsomal lipid: Effect on the kinetic parameters of benzo[a]pyrene hydroxylase and benzphetamine n-demethylase activity

Extraction of lyophilized hepatic microsomes from untreated rats, once with l-butanol and twice with acetone, increased benzo[a]pyrene hvdroxylase and benzphetamine N-demethylase activities by 25 per cent: these activities were increased further by the addition of dilauroylglyceryl-3-phosphorycholine (di-12-GPC). More extensive extraction with 1-butanol decreased the activities by 50 per cent; addition of di-12-GPC restored activity to control levels. Kinetic analysis indicated that a single extraction with 1-butanol decreased the apparent K_m for benzo[a]pyrene 6-fold, with no change in V_max; addition of di-12-GPC had no effect on the apparent K_m or V_max. In contrast, a single extraction with l-butanol of microsomes from 3-methycholanthrene (3-MC)-treated rats had no effect on the apparent K_m or V_max for benzo[a]pyrene. Lineweaver-Burk plots of benzphetamine 4N-demethylase activity in extracted microsomes from untreated rats and in both unextracted and extracted microsomes from 3-MC-treated rats were non-linear with a marked increase in activity at higher benzphetamine concentrations.     

Effect of caffeine on the hepatic microsomal mixed function oxidase system during phenobarbital and benzo[a]pyrene treatment in rats

Simultaneous administration of caffeine (100 mg/kg, i.p., 3 days) and phenobarbital (80 mg/kg, i.p., 3 days) to adult male rats resulted in a significant decrease in hepatic cytochrome P-450 and acetanilide hydroxylase activity, compared to phenobarbital administration alone. While simultaneous administration of caffeine and benzo[a]pyrene (20 mg/kg, i.p., 2 days) increased acetanilide hydroxylase, compared to benzo[a]pyrene administration, no change was seen in the cytochrome P-450 concentration. In vitro addition of 2.5 mM caffeine to microsomal incubations from untreated, phenobarbital- and benzo[a]pyrene-treated rats inhibited aminopyrine N-demethylase activity. No significant difference was seen in the extent of aminopyrine N-demethylase inhibition due to the in vitro addition of caffeine to microsomes from untreated or phenobarbital-treated rats, whereas inhibition in microsomes from benzo[a]pyrene-treated rats was greater.     

Modifications of carcinogen metabolism in hepatic microsomes of suckling young by 3-methylcholanthrene or β-naphthoflavone administered to lactating rats

The effects of treating lactating rats with 3-methylcholanthrene (3-MC) or β-naphthoflavone (β-NF) (three i.p. injections of 20 or 40 mg compound/kg of body weight) on hepatic microsomal enzymes of their suckling young were examined. This treatment had no apparent effect on the contents of cytochromes P-450 and b₅ or on the activities of NADH- and NADPH-cytochrome c reductases in hepatic microsomes of the pups. However, these microsomes had 8- and 6-fold increased capacities for hydroxylations of benzo[a]pyrene (B[a]P) and N-2-fluorenylacetamide (2-FAA) respectively. These increases were about 5-fold greater in the hepatic microsomes of the dams, in which they were inhibited by 0.1 mM α-naphthoflavone (α-NF) invitro 72–81 and 89–95% and by 0.1 mM β-NF in vitro 12–41 and 60–76% respectively. In the pups, the induced activities were also inhibited, whereas the basal hydroxylations of B[a]P and 2-FAA were stimulated by α-NF 2.7- and 5.0-fold and by β-NF 1.4- and 2.4-fold respectively. The inhibition of the induced hydroxylations by α-NF and β-NF may be explained by their higher affinities (K_s, 0.14 and 0.28 μM, respectively) than those of B[a]P and 2-FAA (K_s 4.4 to 8.8 and 2.4 to 3.1 μM, respectively) for cytochrome P-450. Whereas β-NF gave a type I binding spectrum, α-NF gave a spectrum composed of type I and reverse-type I elements. Analysis of metabolites of 2-FAA showed differences in their type and amounts formed by hepatic microsomes of β-NF-treated lactating rats and their pups. Thus, in the dams the formation of 1-, 3-, 5-, 7-, 9- and N-hydroxy-2-FAA was increased by 9-, 30-, 40-, 5-, 20- and 5-fold respectively. In the pups, the formation of 1-, 3-, 5-, 7- and N-hydroxy-2-FAA was increased by 2-, 30-, 18-, 4- and 27-fold respectively. All these increased hydroxylations were inhibited by 0.1 mM α-NF in vitro. In the hepatic microsomes of pups from the corn oil-treated dams, α-NF stimulated all ring-hydroxylations, but not N-hydroxylation of 2-FAA. The results support earlier findings that microsomal enzymes differ in immature and mature rat liver and suggest that N-hydroxylation of 2-FAA, the activation required for carcinogenesis, and specific ring-hydroxylations are catalyzed by different cytochrome P-450 isozymes. Our studies showed that 3-MC and β-NF and/or their metabolites were transferred with milk of dams to their suckling pups in which they modified metabolism of carcinogens.     

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.