Xenobiotic biotransformation in wild birds: Activity,induction, characterization and comparison with rat and mouse microsomal enzymes

1. Benzo[a]pyrene hydroxylase, aminopyrine demethylase and glutathione S-trans-ferase activities in hepatic and extrahepatic tissues of five wild birds, rat and mouse were compared.

2. Hepatic benzo[a]pyrene hydroxylase of wild pigeon was at least three times higher than that of rat or mouse. Hepatic aminopyrine demethylase and GSH S-transferase activities of wild birds were lower than those of rodents.

3. Renal GSH S-transferase of wild birds was 2-3 times more than in rat and mouse.

4. Avian benzo[a]pyrene hydroxylase activity of hepatic and renal tissue was linear up to 3.0?mg enzyme protein with pH optima of 7.4 and 7.2, at 42°C Apparent K_m values were 11.76 and 3.33 μM respectively.

5. Hepatic enzyme activity was induced four-fold by 3-methylcholanthrene and about two-fold by phenobarbitone administration.     

Sex differences in cytochrome P-450 and mixed-function oxygenase activity in gonadally mature trout

Levels of microsomal cytochrome P-450 and aminopyrine demethylase activity in liver and of cytochrome P-450 in kidney of gonadally mature rainbow and brook trout were markedly greater in males than in females. Similar differences appeared in hepatic microsomal NADH- but not in NADPH-cytochrome c reductase activity or cytochrome b₅ content. When normalized to cytochrome P-450 content, benzo[a]pyrene hydroxylase activity in both liver and kidney was greater in females. In liver, there was a pronounced sex difference in the response of this activity to 7,8-benzoflavone, suggesting cytochromes P-450 of different catalytic function. Electron paramagnetic resonance spectra of hepatic microsomal cytochromes P-450 in mature brook trout were not demonstrably different between males and females, and crystal field parameters indicate that axial ligands to the neme are the same in these as in other cytochromes P-450. Mixed-function oxygenase activities in liver of gonadally immature brook trout differed from those in mature fish, and there was no sex difference. The appearance of seasonally dependent sex differences suggests that fish may provide interesting models for studying regulation of sex-specific forms of cytochromes P-450.     

Kinetics, activation, and induction of aortic mono-oxygenases--biotransformation of benzo[a]pyrene.

Aortic aryl hydrocarbon hydroxylase (EC 1.14.14.2), a cytochrome P-450-dependent mono-oxygenase complex potentially important in the etiology of atherosclerosis, was detected previously in aortic homogenates of humans, monkeys and rabbits [M. R. Juchau, J. A. Bond and E. P. Benditt, Proc. natn. Acad. Sci. U.S.A.73, 3723 (1976)]. The present study more fully characterizes the mono-oxygenase activity in aortas of treated and untreated New Zealand White rabbits. A 2-fold activation was obtained with NADH (7 × 10^?4 M) at saturating concentrations of NADPH. Addition of heme (9 μm) increased the enzymatic activity 2- to 4-fold during a 15-min incubation and over 25-fold during a 2-hr incubation. The results suggest the presence of relatively high concentrations of apoprotein in the aortic tissues. Kinetic studies in the presence of heme yielded an apparent K_m of 1 × 10^?4 M and V_max of 15.24 pmoles/mg of protein/min with respect to NADPH. A sigmoidal curve was obtained with varying benzo[a]pyrene concentrations (0.5 to 80 μM), suggesting the possibility of allosterism. Aroclor 1254,3-methylcholanthrene and 5,6-benzoflavone acted to induce the cytochrome P-450-dependent mono-oxygenase, while pheno-barbital and pregnenolone 16α-carbonitrile demonstrated little, if any, induction capacity. Analyses of metabolites formed in induced aortas with high-pressure liquid chromatography revealed the formation (in each case) of primarily the phenolic metabolites of benzo[a]pyrene.     

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.     

Studies on the properties of highly purified cytochrome P-448 and its dependent activity benzo[a]pyrene hydroxylase,from Saccharomyces cerevisiae

1. The yeast Saccharomyces cerevisiae, produces a cytochrome P-450 enzyme with a Soret peak in the reduced-CO difference spectrum at 448 nm. The enzyme purified to homogeneity (88–97% pure on a specific content basis) has a molecular wt. of 55 500 as determined by SDS-PAGE.

2. Amino acid analysis of yeast cytochrome P-448 revealed 407 amino acid residues per molecule with a 43% complement of hydrophobic residues. Although the number of residues is smaller than cytochrome P-448 enzymes from mammalian sources, the percentage of hydrophobic residues is almost identical. Estimation of the haem content of yeast cytochrome P-448 showed that one haem group was present per molecule. Phospholipid was present at very low levels. The molecular wt. of the polypeptide chain plus an estimated 5–6 units of hexose and of hexosamine is in good agreement with the molecular wt. value obtained from SDS-PAGE.

3. A reconstituted system of purified cytochrome P-448, purified NADPH-cytochrome P-450 (c) reductase and phospholipid showed aryl hydrocarbon hydroxylase activity towards benzo[a]pyrene. Both protein components, NADPH and dilauroyl phosphatidylcholine (or emulgen 911) were necessary for full activity. The NADPH requirement could be replaced by cumene hydroperoxide or H₂O₂ generated in situfrom a glucose oxidase system; in each case V_max is increased, but the apparent affinity for benzo[a]pyrene, as measured by an increased K_m, is lowered.

4. The spin state of purified yeast cytochrome P-448 was 94% low spin (22°C) as determined from the temperature-dependent spin-state equilibrium. The addition of benzo[a]pyrene to this enzyme resulted in a change to higher spin state (18% high spinat 22°C).

5. Equilibrium gel filtration analysis of the number of benzo[a]pyrene binding sites per mole of enzyme monomer showed a value of 1 for purified yeast cytochrome P-448 and 6 for this enzyme in microsomal form. The corresponding values for purified and microsomal cytochrome P-450 from phenobarbital-pretreated rats are 1 and 6, respectively. However, purified cytochrome P-448 from β-naphthoflavone-induced rats gave a value of 6 benzo[a]pyrene binding sites.

6. Type I binding spectra with purified yeast cytochrome P-448 were observed with benzo[a]pyrene, lanosterol, ethylmorphine, dimethylnitrosamine, sodium phenobarbitone and perhydrofluorene. Type II spectral changes were observed with imidazole, aniline and benzphetamine.

7. Cytochrome P-448 from Saccharomyces cerevisiae is identified as a distinct enzyme of the P-450 family. This enzyme however has many properties in common with cytochrome P-448 from mammalian sources.

8. A more specific and efficient form of benzo[a]pyrene hydroxylase is induced by the addition of benzo[a]pyrene to the yeast growth medium at zero time. The efficiency of the enzyme, as indicated by the V_max / K_m ratio, increases progressively with concentration of benzo[a]pyrene. This indicates that multiple forms of yeast cytochrome P-448 occur. Induction of more efficient forms occurs at the expense of less efficient forms as little increase in total enzyme concn. is observed.     

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.     

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.     

In vitro inhibitory action of cadmium on microsomal monooxygenases of rabbit lung

In vitro addition of cadmium (Cd) salts to pulmonary microsomes isolated from male rabbits decreased the cytochrome P-450 levels and the activity of benzo[a]pyrene hydroxylase and aminopyrine N-demethylase but not that of NADPH-cytochrome c reductase. The Cd-induced reduction of pulmonary monooxygenase activity was enhanced when microsomes were preincubated in the presence of Cd and inhibition increased as the time of preincubation progressed, attaining its maximum rate at 20 min of preincubation. When hepatic microsomes were used, this delayed effect of Cd on monooxygenase was less apparent. The presence of NADH and/or NADPH in the preincubation did not markedly enhance the Cd-induced inhibition rate of monooxygenase activity. The addition of Cd-acetate to pulmonary microsomes produced a concentration-dependent inhibition of the monooxygenase activity and the estimated IC50 values of Cd-acetate (i.e. the concentration required to inhibit control enzyme activity by 50%) were 3.8 x 10(-5)M, 6.5 x 10(-6)M and 5.3 x 10(-5)M for cytochrome P-450, benzo[a]pyrene hydroxylase and aminopyrine N-demethylase, respectively. The inhibitory action of Cd-acetate on the monooxygenase activity was also observed with microsomes isolated from the lungs of male guinea-pigs and rats.     

The intestinal metabolism and DNA binding of benzo[a]pyrene in guinea-pigs fed normal,high-fat and high-cholesterol diets

1. Strains of intestinal bacteria were capable of deconjugating benzo[a]pyrene metabolites in vitro. The hydrolysis products, and other primary oxidative metabolites of benzo[a]pyrene, were stable to further degradation by the strains tested.

2. Cytochromes P-450 and b5 were detectable in the mucosa of the guinea-pig small intestine, but not in the mucosae of the colon or rectum. The concentrations were unaltered by administration of benzo[a]pyrene and/or the feeding of high-fat or high-cholesterol diets.

3. Benzo[a]pyrene hydroxylase was measurable in the mucosa of the upper intestine, but was present in the lower gut only at very low levels in some animals. The activity was inducible, by oral administration of benzo[a]pyrene, in the small intestinal mucosa of guinea-pigs fed normal diet but not in those fed high-fat and high-cholesterol diets.

4. Low levels of covalent binding of ³H to DNA of liver and gut mucosa were obtained in guinea-pigs dosed orally with ³H-benzo[a]pyrene. Comparison with data for animals given ³H₂O suggested that approx. one quarter of the binding was probably due to ³H exchange during metabolism. The feeding of high-fat and high-cholesterol diets did not increase this binding. Guinea-pigs fed high-fat and high-cholesterol diets excreted a greater proportion of an oral dose of ³H-benzo[a]pyrene in urine, and less in faeces than animals fed a normal diet.

5. Due to the low, and apparently non-inducible, levels of benzo[a]pyrene hydroxylase activity and of covalent binding in the colonic mucosa, the administration of benzo[a]pyrene to guinea-pigs fed high-fat or high-cholesterol diets appears unlikely to provide a novel animal model for studies on mechanisms of colon carcinogenesis.     

NADPH-cytochrome P-450 reductase: Preferential inhibition by ellipticine and other type II compounds having little effect on NADPH-cytochrome c reductase

Ellipticine (5,11-dimethyl-[6H]-pyrido[4,3b]carbazole) binds with an affinity greater than most other compounds known to interact with P-450. Control and 3-methylcholanthrene-induced aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2) and acetanilide 4-hydroxylase and control and phenobarbital-induced ethylmorphine N-demethylase activities are all markedly inhibited by ellipticine to about the same extent. Ellipticine and other Type II compounds (metyrapone, octylamine-1, pyridine and aniline) preferentially inhibit NADPH-cytochrome P-450 reductase activity, while affecting NADPH-cytochrome c reductase activity very little. Butanol-1, a compound having pure Reverse Type I character, does not block P-450 reductase activity like these Type II compounds. These data suggest that Type II compounds bind to P-450 ferric iron in the sixth coordinate position in such a way as to impede transfer of the first electron from the hydrophobic binding site of the reductase to the P-450-substrate complex, while leaving unencumbered any transfer of electrons from the hydrophilic binding site of the reductase to soluble electron acceptors such as cytochrome c. These data indicate that ellipticine may be very useful in attempting to understand the mechanism by which electrons are transferred from the reductase to the cytochrome(s) P-450.     

Sites of action of cadmium in vitro on hepatic,adrenal, and pulmonary microsomal monooxygenases in guinea pigs

Preincubation of hepatic, adrenal, or pulmonary microsomal preparations with cadmium produced time-dependent decreases in monooxygenase (benzphetamine demethylase, benzo(a)pyrene hydroxylase) activities. Addition of cadmium after the preincubation period had little or no effect on microsomal metabolism. As a result of preincubation with cadmium, hepatic cytochrome P-450 levels declined and the magnitude of the benzphetamine-induced type I spectral change in hepatic microsomes decreased. Cadmium also decreased hepatic NADPH-cytochrome c and NADPH-cytochrome P-450 reductase activities but had no effect on NADH-cytochrome c reductase activity. Cadmium similarly decreased cytochrome P-450 concentrations and NADPH-cytochrome c reductase activity in lung microsomes without affecting NADH-cytochrome c reductase activity. Preincubation of adrenal microsomes with cadmium had no effects on cytochrome P-450 levels, on the benzphetamine-induced type I spectrum, or on NADH-cytochrome c reductase activity. However, decreases in adrenal NADPH-cytochrome c and NADPH-cytochrome P-450 reductase activities resulted which closely paralleled the decline in adrenal monooxygenase activities. EDTA extraction of hepatic, adrenal, or pulmonary microsomes after the preincubation exposure removed about 95% of the cadmium but did not diminish the effects of the metal on microsomal monooxygenases. The results indicate that cadmium has somewhat varying sites of action on hepatic, adrenal, and pulmonary monooxygenases, but in all three tissues electron transfer to cytochrome P-450 is compromised. In addition, the effects of cadmium on microsomal metabolism persist fully even after removal of approximately 95% of the metal.     

Effects of naphthalene on benzo[a]pyrene hydroxylase and cytochrome P-450 in Fundulus heteroclitus

Hepatic benzo[a]pyrene (B[a]P) hydroxylase, cytochrome P-450 and cytochrome b₅ were investigated in the mummichog, Fundulus heteroclitus, following acute exposure to naphthalene dissolved in the water. Control experiments revealed that males had significantly higher levels of BaP hydroxylase and cytochrome P-450 compared to females. Significant variation of B[a]P hydroxylase activity was also observed in control fish during a 6-mth period which may reflect seasonal or reproductive influences.Naphthalene at a concentration of 4 mg/l caused a significant reduction of B[a]P hydroxylase activity in males and females. The naphthalene exposure effected significant decreases of cytochrome P-450 in males, but not in females. Addition of naphthalene directly to reaction mixtures containing liver preparation from control fish caused reduced B[a]P hydroxylase activity in vitro. The concentrations of naphthalene necessary to produce in vitro reductions in B[a]P hydroxylase appeared to be much higher than would be realized by in vivo exposures. These data indicate that metabolite formation or other mechanisms such as generalized physiological stress as a result of in vivo exposures may be important factors in reducing B[a]P hydroxylase activity. The nature of the reduction of B[a]P hydroxylase was not elucidated. Cytochrome b₅ concentrations did not change significantly.     

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.     

On the capacity of human placental enzymes to catalyze the formation of diols from benzo[a]pyrene

Studies were performed on the oxidative biotransformation of benzo[a]pyrene in fortified preparations of human placental microsomes by analysis with high-pressure liquid chromatography. These investigations revealed that the utilization of substrate concentrations (1–2 × 10^?4m) sufficiently high to assure zero-order reaction kinetics (in terms of the generation of phenolic metabolites) produced a marked inhibitory effect on the formation of dihydrodiols in the same reaction mixtures. Relative quantities of dihydrodiols generated increased with decreasing substrate concentrations between 200 and 2.7 μm. Additions of manganese or ferric ions to reaction mixtures altered the ratios of generated phenols to dihydrodiols but did not provide an explanation for the differences observed in the literature. Identical results were obtained with either ¹⁴C- or ³H-labeled benzo[a]pyrene as substrates. The data suggested the possibility that considerable quantities of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, a proximate mutagen/carcinogen, may be generated in vivo by placental tissues of women who smoke.     

Octachloronaphthalene induction of hepatic microsomal aryl hydrocarbon hydroxylase activity in the immature male rat

Administration of octachloronaphthalene to immature male Wistar rats resulted in a dose-dependent increase in several enzymic, electrophoretic and spectral parameters associated with induction of the hepatic microsomal enzymes. Compared to corn-oil (control) treated animals octachloronaphthalene (150 μmol · kg^?1 induced hepatic cytochrome P-450 (1.5-fold), benzo [a]pyrene hydroxylase (18-fold) and 4-chlorobiphenyl hydroxylase (18-fold) enzyme activities. In addition to increases in the relative peak intensities of the reduced microsomal cytochrome P-450 : CO and ethylisocyanide (EIC) difference spectra the peak maxima were observed at 448.5 and 452.2/428.0 nm, respectively. The effects of administering octachloronaphthalene to the rat were similar to those observed after pretreatment with 3-methylcholanthrene (MC) and electrophoresis of the induced microsomal proteins showed that both compounds enhanced heme-staining peptides with comparable electrophoretic mobilities. Moreover coadministration of MC (3 × 10 βmol · kg^?1) and octachloronaphthalene (2 × 150 μmol · kg^?1) indicated that their inductive effects were not additive. It was concluded that octachloronaphthalene was an MC-type inducer of hepatic microsomal enzymes.     

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.     

Inhibition by 17-alpha-ethinyl estradiol of benzo[a]-pyrene metabolism in isolated adult rat hepatocytes

The effects of 17-α-ethinyl estradiol [EE] were studied on benzo[a]pyrene (BP) metabolism and covalent binding to DNA and on some enzymes involved in the activation/inactivation pathway. Suspensions of hepatocytes freshly isolated from adult rats either untreated or pretreated with phenobarbital or methylcholanthrene were used as an experimental model closer to the in vivo situation than subcellular fractions. The formation of water-soluble and of organic metabolites of benzo[a]pyrene was inhibited by EE at 10^?4 M in hepatocytes from untreated rats and at 10^?3 M in those isolated from rats pretreated with phenobarbital. Dihydrodiols were the main class of metabolites affected by EE. The pretreatment of rats with 3-methylcholanthrene rendered hepatocytes insensitive to the effect of EE. The effect of EE on BP metabolism was well correlated to its inhibitory effect on BP metabolite binding to DNA in untreated and phenobarbital-treated rats. These data could not however be related to a quantitative decrease in cytochrome P-450 of hepatocytes incubated in the presence of EE. Epoxide hydratase, UDP-glucuronyl-transferase and glutathione S-transferase activities were lowerea by 36, 27 and 19% respectively in the presence of 10^?4 M EE.The impairment of BP metabolism by EE might be due to a functional alteration specific for cytochrome P-450, or to a non-destructive mechanism and/or to a competition of both chemical. for a number of enzymatic pathways common to their metabolism.     

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.     

Studies in the metabolism of carcinogenic polycyclic heteroaromatic compounds. I. The hepatic microsomal metabolism of 7-methylbenz[c]acridine

An enzyme assay for the metabolism of the carcinogenic aza-aromatic polycyclic compound 7-methylbenz[c]acridine has been developed using a modification of a radiochemical assay described for the polycyclic aromatic hydrocarbon benzo[a]pyrene by DePierre et al. [J. W. DePierre, M. S. Moron, K. A. M. Johannesen and L. Ernster, Analyt. Biochem. 63, 470 (1975)]and Van Cantfort et al. [J. Van Cantfort, J. DeGraeve and J. E. Gielen, Biochem. biophys. Res. Commun. 79, 505 (1977)]. When the activities of control microsomes and microsomes of phenobarbital-, 3-methylcholanthrene-and 7-methylbenz[c]acridine-pretreated animals were compared, strong similarities were displayed toward oxidation of benzo[a]pyrene and 7-methylbenz[c]acridine. These similarities were seen in turnover numbers, Michaelis constants, and inducibility of both enzyme systems. 7-Methylbenz[c]acridine afforded a type I difference spectrum with 3-methylcholanthrene-pretreated microsomes. It is suggested that 7-methylbenz[c]acridine is oxidized by the same or a similar set of enzymes which is responsible for benzo[a]pyrene metabolism.     

Depression of theophylline metabolism and elimination by troleandomycin and erythromycin

Clinical observations have suggested that elevated levels of theophylline may occur during the use of macrolide antibiotics. In the present study, the plasma clearance of theophylline was studied in rabbits treated with troleandomycin or erythromycin (400 mg · kg^?1 · day^?1) over a 10-day period. The elimination of theophylline was impaired significantly after 10 days of antibiotic treatment. No change in theophylline elimination occurred when the antibiotics were given for shorter periods of time. Protein, cytochrome P-450 and cytochrome b₅ levels, and aminopyrine N-demethylase and benzo[α]pyrene hydroxylase activities were unchanged in hepatic microsomes prepared from rabbits treated with antibiotics for 10 days. Pretreatment of rabbits for 10 days with troleandomycin completely abolished production of 1-methyluric acid from theophylline in isolated hepatic microsomes, but production of 1,3-dimethyluric acid was unaffected. Troleandomycin, when added in vitro to microsomes, had no direct effect on theophylline metabolism. It is concluded that long-term treatment with troleandomycin selectively blocks or destroys one pathway of theophylline metabolism. This mechanism may explain the clinically observed interaction between theophylline and the macrolide antibiotics.