Effects of acetylenic and olefinic pyrenes upon cytochrome P-450 dependent benzo[a]pyrene hydroxylase activity in liver microsomes

1-Ethynylpyrene, trans-, & cis-1-(2-bromovinyl)pyrene, methyl 1-pyrenyl acetylene, and phenyl 1-pyrenyl acetylene are substrates for cytochrome P-450 dependent monooxygenases and also inhibitors of cytochrome P-450 dependent benzo[a]pyrene hydroxylase activities in liver microsomes from 5,6-benzoflavone or phenobarbital pretreated rats. 1-Ethynylpyrene, trans-1-(2-bromovinyl)pyrene, and methyl 1-pyrenyl acetylene cause a mechanism based inhibition (suicide inhibition) of the benzo[a]pyrene hydroxylase activities in microsomes from 5,6-benzoflavone or phenobarbital pretreated rats, while cis-1-(2-bromovinyl)pyrene only causes suicide inhibition of the hydroxylse activities in the 5,6-benzoflavone induced microsomes and phenyl 1-pyrenyl acetylene does not cause a detectable suicide inhibition of these activities in either type of microsome. Incubation with NADPH and 1-ethynylpyrene, trans-, or cis-1-(2-bromovinyl)pyrene causes a loss of the P-450 content in the microsomes from 5,6-benzoflavone or phenobarbital pretreated rats, but incubations with methyl 1-pyrenyl acetylene or phenyl 1-pyrenyl acetylene did not cause a loss of the P-450 content of either microsomal preparation.     

Induction of microsomal aryl hydrocarbon (3,4-benzo(alpha)pyrene) hydroxylase and cytochrome P-450K in rat kidney cortex. II. Interactions with the induced hemoprotein

Benzo(α)pyrene treatment resulted in stimulation of only cytochrome P-450_K and benzo(α)pyrene hydroxylase activity in rat kidney cortex microsomes. Spectral properties of cytochrome P-450_K showed that the 452 nm peak of the reduced hemoprotein CO-complex was not shifted in benzo(α)pyrene-treated rats. The off-balance absolute spectrum of oxidized cytochrome P-450_K displayed an absorption maximum at 414 nm, another band at 385 nm, and a distinct shoulder at 398 nm. Addition of benzo(α)pyrene to kidney microsomes resulted in a type I spectral change seen only in benzo(α)pyrene-treated rats. The addition of ethyl isocyanide to dithionitetreated microsomes from control rats gave rise to two Soret peaks, 432 nm and 458 nm. These peaks were proportionately increased in benzo(α)pyrene-treated rats; furthermore, the 458 nm peak was not shifted. The relative heights of the two peaks were in a pH-dependent equilibrium similar to that observed in liver; however, in contrast to liver, the pH, at which the ratio of the peak heights equals one, was the same for both benzo(α)pyrene-treated and control microsomes. These data indicate that the newly induced hemoprotein has spectral properties markedly different from those of the benzo(α)pyrene-induced liver hemoprotein, yet similar to those of the “noninduced” kidney hemoprotein. α-Naphthoflavone, an inhibitor of the aryl hydroxylase system, induced a type I spectral change, suggesting the mode of action of α-naphthoflavone to be its interaction with cytochrome P-450_K probably at or near the active site. Finally, the rate of reduction of cytochrome P-450_K was not affected by the presence of benzo(α)pyrene.     

Effect of intratracheally instilled benzo(a)pyrene on the pulmonary and hepatic drug-metabolizing enzymes in normal and vitamin A deficient rats

The effect of intratracheal instillation of different doses of benzo(a)pyrene (0.1, 1.0 and 2.0 mg) on the drug metabolizing enzymes of lung and liver was analysed in rats fed diet with or without vitamin A for 5-6 weeks. Benzo(a)pyrene exposure at 2.0 mg dose only elevated the level of cytochrome P-450 and b5, and activity of benzopyrene hydroxylase in liver, and extent of increase was similar in normal and vitamin A deficient groups. Contrary to this, pulmonary contents of cytochrome P-450 and b5, and benzopyrene hydroxylase activity increased over control values in both the groups even at lower doses of benzo(a)pyrene. Moreover, their values were higher in vitamin A deficient-treated groups compared to normal-treated controls. Increase in these parameters was greater in lung as compared to increase in liver. NADPH cytochrome C-reductase in lung and liver was not affected either by inducing vitamin A deficiency or exposing these rats further to benzo(a)pyrene. Uridine-diphospho-glucuronosyl-transferase (UDP-GT) activity in normal and vitamin A deficient groups was enhanced following exposure to benzo(a)pyrene both in lung and liver. However, activity of this enzyme remained impaired in vitamin A deficient groups, benzo(a)pyrene exposed or not exposed when compared to respective normal controls. Glutathione S-transferase activity remained unchanged following exposure to benzo(a)pyrene both in lung and liver. The apparent increase in hepatic glutathione S-transferase and decrease in pulmonary glutathione S-transferase activity in vitamin A deficiency was only due to vitamin A deficient status of rats with no further effect of benzo(a)pyrene.     

Effect of vitamin A deficiency on the pulmonary and hepatic drug-metabolizing enzymes in rat

The effect of vitamin A deficiency on the drug-metabolizing enzyme system of the lung and liver was analyzed in rats fed diets with or without vitamin A for 5-6 weeks. The hepatic level of vitamin A was significantly reduced in vitamin A deficient animals. The hepatic cytochrome P-450 and b5 contents and activity of benzo(a)pyrene hydroxylase was significantly reduced in deficient animals. Contrary to this, pulmonary cytochrome P-450 and b5 contents were above the control values. No alteration in pulmonary benzo(a)pyrene hydroxylase was noted. The uridine diphosphate-glucuronosyltransferase activity of digitonin-treated microsomal membranes was below the control values both in lung and liver. However, the native uridine diphosphate-glucuronosyltransferase activity remained unchanged in the liver and was below control values in the lung.     

Effect of chronic ethanol ingestion on intestinal metabolism and mutagenicity of benzo(α)pyrene

Chronic ethanol ingestion in male rats causes an increase in cytochrome P-450 content and in the activity of microsomal benzo(α)pyrene hydroxylase in the upper intestinal mucosa. Intestinal microsomes from ethanol fed rats also exhibit an enhanced capacity to activate the ubiquitous procarcinogen benzo(α)pyrene to a mutagen. These findings could be of relevance with respect to the increased incidence of cancer in the alcoholic.     

Effect of vitamin A deficiency on pulmonary and hepatic in vitro metabolism of benzo(a)pyrene in rat

The metabolism of (3H)-benzo(a)pyrene and the activities of enzymes involved in its metabolism were studied in rat lung and liver in vitamin A deficiency. Deficiency of vitamin A resulted a significant decrease in the overall metabolism of benzo(a)pyrene in the liver in vitro, whereas no significant difference was evident in the lung. The ethyl acetate-soluble metabolites of benzo(a)pyrene formed by lung and liver preparations were unaltered qualitatively by vitamin A deficiency. However, quantitative analysis revealed that vitamin A deficiency decreased the yield of dihydrodiols, quinones and phenols in liver, and dihydrodiols in lung. The hepatic cytochrome P-450 content, arylhydrocarbon hydroxylase and uridine diphosphate-glucuronosyl transferase activities were reduced, whereas glutathione S-transferase activity was increased in the vitamin A deficient animals. Contrary to this, pulmonary cytochrome P-450 content was above the control values (p less than 0.01) and no alteration in pulmonary arylhydrocarbon hydroxylase activity was observed in vitamin A deficient rats. Uridine diphosphate-glucuronosyltransferase and glutathione S-transferase activities were impaired in lung by inducing vitamin A deficiency. However, no significant difference was evident in the overall metabolism of benzo(a)pyrene by lung supernatants from the two groups.     

Temperature compensation in the hepatic mixed-function oxidase system of bluegill

Bluegill (Lepomis macrochirus R.) were acclimated to 12, 22 or 32 degrees C for 5 or 14 days. Liver weight to body weight ratio and the rate of metabolism of benzo[alpha]pyrene by liver microsomes varied inversely with the acclimation temperature of the fish. Concentration of microsomal cytochrome P-450, as determined by CO-difference binding spectra, was not significantly affected by acclimation temperature. There were no qualitative or quantitative differences in the electrophoretic patterns of proteins with molecular weights similar to those reported for cytochrome P-450. There were no shifts in the temperature optima of the microsomal benzo[alpha]pyrene hydroxylase activity.     

Effects of phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls on sex-specific forms of cytochrome P-450 in liver microsomes of rats

The effects of treatment with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls (PCB) on the amounts of sex-specific forms of cytochrome P-450, namely P-450-male and P-450-female, in male and female rats were studied. Although treatment with phenobarbital, 3-methylcholanthrene or PCB markedly increased the total amount of hepatic cytochrome P-450, P-450-male and P-450-female were rather decreased or not significantly changed. Thus, the percentages of P-450-male and P-450-female in the total cytochrome P-450 were decreased in liver microsomes from the treated rats. The increases in specific cytochrome P-450, such as P-448-H, P-448-L, and P-450I-c accounted for the increase in the total amount of cytochrome P-450 in the treated rats. The treatment with phenobarbital or PCB increased the activities of testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase and aminopyrine N-demethylase more markedly in female rats than in male rats. Similarly, the treatment with 3-methylcholanthrene increased benzo(a)pyrene hydroxylase more markedly in female rats. Therefore, the sex-differences in testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase, and aminopyrine N-demethylase activities became smaller after the drug treatment. These results indicate that sex-specific P-450-male and P-450-female were unaffected, or even depressed by the agents in some cases.     

Nonadditive effects of combined in vivo hepatic microsomal enzyme inducers in the Wistar rat

Compounds that are known to increase the hepatic microsomal cytochrome P-450 dependent monooxygenases were administered to adult female rats, alone or in combination, to determine whether their effects on certain substrate oxidations were additive. 3-Methylcholanthrene (3-MC) and pregnenolone-16 alpha-carbonitrile (PCN), known to induce different forms of cytochrome P-450, when administered together increased benzo[a]pyrene oxidation to the same level as observed following 3-MC treatment alone. Phenobarbital (Pb) and PCN when administered concomitantly increased benzo[a]pyrene, amino-pyrine, and ethylmorphine metabolism to the same extent as seen following PCN administration alone. Both compounds are known to induce different forms of cytochrome P-450. Nonadditive effects were also observed with Pb and spironolactone, as well as with Pb and trans-stilbene oxide. Treatment of adult male rats with either PCN or 3-MC resulted in significantly smaller increases in benzo[a]pyrene oxidation than observed in adult female rats. These results suggest that oxidative metabolism in hepatic microsomes is not the sum of activities of a number of cytochrome P-450s, but may represent the activity of a single predominant hemeprotein. In addition, it appears that the oxidation of substrate by a particular cytochrome P-450, in intact microsomes, is greatly influenced by the presence of another form.     

Effects of dietary benzo(a) pyrene on intestinal phase I and phase II drug metabolizing systems in normal and vitamin A-deficient rats

Feeding of vitamin A-deficient diet to male weanling rats for 10 weeks caused significant increase in the activities of Phase I enzyme system, i.e., cytochrome P-450, cytochrome b5 and arylhydrocarbon hydroxylase in the proximal, middle and distal segments of the intestine. Of the Phase II enzymes studied, UDP-glucuronyltransferase showed significant decrease whereas glutathione S-transferase showed significant increase. Treatment with benzo(a)pyrene caused greater induction in the levels of Phase I enzymes in deficient animals as compared to controls. In contrast to this, benzo(a)pyrene treatment induced the level of UDP-glucuronyltransferase in control rats more than in deficient rats. Intestinal NADPH cytochrome C-reductase and glutathione S-transferase remained insensitive to benzo(a)pyrene induction.     

Purification and properties of cytochrome P-450 generally acting as a catalyst on benzo[a]pyrene hydroxylation from liver microsomes of untreated rats

A form of cytochrome P-450 generally catalyzing benzo[a]pyrene (B[a]P) hydroxylation was purified from liver microsomes of untreated rats on the basis of the catalytic activity. The purification procedures consisted of cholate solubilization and chromatography in 3 steps, on DEAE-Toyopearl (at room temperature), hydroxylapatite, and CM-Toyopearl columns. Cytochrome P-450 purified in this way (named P-450/B[a]P) was homogeneous on SDS-polyacrylamide gel electrophoresis, and the molecular weight was estimated to be 51,000. The absorption spectra of the oxidized form of P-450/B[a]P showed a Soret peak at 417 nm, characteristic of low-spin hemoprotein, and the Soret peak of the reduced cytochrome P-450-CO complex was at 451 nm. Immunochemical analysis of P-450/B[a]P indicated that P-450/B[a]P is immunologically distinct from P-450b (a major phenobarbital-inducible form of P-450) and P-450c (a major 3-methylcholanthrene-inducible form of P-450, which highly catalyzes the hydroxylation of B[a]P). B[a]P hydroxylase activity in liver microsomes of untreated rats was inhibited to about 20% by the P-450/B[a]P antibody. These results demonstrate that P-450/B[a]P is a different form of P-450 from P-450b and P-450c, and generally catalyzes B[a]P hydroxylation in liver microsomes of untreated rats.     

Induction of microsomal aryl hydrocarbon (3,4-benzo(α)pyrene) hydroxylase and cytochrome P-450k in rat kidney cortex: I. Characteristics of the hydroxylase system

Both the rat kidney cortex aryl hydrocarbon hydroxylase activity and cytochrome P-450_K are induced by benzo(α)pyrene treatment. Following a single injection of benzo(α)pyrene, maximal hydroxylase activity and cytochrome P-450_K content occur at 24 hr, returning to control levels within 72–96 hr. Induction of both the enzyme activity and hemoprotein is inhibited by cycloheximide. The enzyme system is localized in the microsomal fraction, has an absolute requirement for NADPH and molecular oxygen, and a pH optimum at 7.4; the induced activity is linear with microsomal protein concentration up to 0.8 mg and with time up to 20 min. Both the hydroxylase activity and cytochrome P-450_K follow the same pattern of inactivation with increasing temperature. The apparent K_m for the induced hydroxylase was 7.7 μm and V was increased fourfold above control value. In the presence of laurate, a substrate for the kidney microsomal cytochrome P-450_K-dependent monooxygenase system, the amount of inhibition of hydroxylase activity corresponded to the level of activity present in untreated kidney cortex microsomes. α-Naphthoflavone (10^?5m), a type I inducer (36) produced a greater inhibitory effect on the induced hydroxylase activity than on the control (55% vs 20%). The presence of cytochrome c or carbon monoxide markedly decreased hydroxylase activity. This evidence in addition to aforementioned characteristics of the enzyme suggests a cytochrome P-450_K-dependent aryl hydroxylase activity which differs from that present in the control rat.     

Cytochrome P-450 isozymes from the marine teleost Stenotomus chrysops: their roles in steroid hydroxylation and the influence of cytochrome b5

Two new cytochrome P-450 forms were purified from liver microsomes of the marine fish Stenotomus chrysops (scup). Cytochrome P-450A (Mr = 52.5K) had a CO-ligated, reduced difference spectrum lambda max at 447.5 nm, and reconstituted modest benzo[a]pyrene hydroxylase activity (0.16 nmol/min/nmol P-450) and ethoxycoumarin O-deethylase activity (0.42 nmol/min/nmol P-450). Cytochrome P-450A reconstituted under optimal conditions catalyzed hydroxylation of testosterone almost exclusively at the 6 beta position (0.8 nmol/min/nmol P-450) and also catalyzed 2-hydroxylation of estradiol. Cytochrome P-450A is active toward steroid substrates and we propose that it is a major contributor to microsomal testosterone 6 beta-hydroxylase activity. Cytochrome P-450A had a requirement for conspecific (scup) NADPH-cytochrome P-450 reductase and all reconstituted activities examined were stimulated by the addition of purified scup cytochrome b5. Cytochrome P-450B (Mr = 45.9K) had a CO-ligated, reduced difference spectrum lambda max at 449.5 nm and displayed low rates of reconstituted catalytic activities. However, cytochrome P-450B oxidized testosterone at several different sites including the 15 alpha position (0.07 nmol/min/nmol P-450). Both cytochromes P-450A and P-450B were distinct from the major benzo[a]pyrene hydroxylating form, cytochrome P-450E, by the criteria of spectroscopic properties, substrate profiles, minimum molecular weights on NaDodSO4-polyacrylamide gels, peptide mapping and lack of cross-reaction with antibody raised against cytochrome P-450E. Cytochrome P-450E shares epitopes with rat cytochrome P-450c indicating it is the equivalent enzyme, but possible homology between scup cytochromes P-450A or P-450B and known P-450 isozymes in other vertebrate groups is uncertain, although functional analogs exist.     

Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.     

Interaction of 9-hydroxyellipticine with two forms of partially purified rabbit lung cytochrome P-450. Inhibition of lung microsomal benzo[a]pyrene hydroxylase.

9-Hydroxyellipticine (9-OHE), a potent inhibitor of rat liver monooxygenase activities, binds to the various forms of partially purified lung cytochromes P-450 from untreated and 3-methylcholanthrene (3-MC)-treated rabbits. The spectral data (lambda max: 428 nm (ox.), 447 nm (red.), Ks: 10 microM and 5 muM for cytochrome I and cytochrome II from 3-MC-treated rabbits respectively) resemble those obtained with cytochrome P-450 purified from liver of Aroclor 1254-pretreated rats (lambda max: 428 nm (ox.), 445 nm (red.), Ks: 8 microM). 9-OHE has been shown to inhibit the benzo[a]pyrene hydroxylase activity of rat and rabbit lung microsomes. The inhibitory effect was higher towards the 3-MC-induced lung microsomes than with the control microsomes. However, the lung microsomes, as well as the liver microsomes of rabbits were less sensitive to inhibition by 9-OHE than the corresponding microsomes from rats. These results suggest that rabbit and rat cytochromes P-450 have subtle structural differences.     

Aryl hydrocarbon hydroxylase in larvae of the southern armyworm (Spodoptera eridania)

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity in midgut microsomes of southern armyworm (Spodoptera eridania) larvae was induced 11-fold and 5.6-fold respectively following three days of feeding on diets containing pentamethylbenzene or naphthalene (both 0.2% w/v). β-Naphthoflavone and Aroclor 1254 were less effective inducers of AHH activity, phenobarbital was only slightly active and 3-methylcholanthrene caused a decrease in enzyme activity. AHH activity in microsomes from untreated and induced larvae was susceptible to inhibition by α-naphthoflavone, 1-phenylimidazole and piperonyl butoxide. Equilibrium dialysis studies with 1-(4′-³H-phenyl)imidazole showed that control and induced armyworm midgut microsomes contained a class of cytochrome(s) P-450 with a uniformly high affinity for phenylimidazole. It is concluded that AHH activity in the armyworm is catalyzed by a class of cytochrome(s) P-450 with characteristics intermediate between mammalian cytochrome(s) P-450 and P-448.     

Xenobiotic-metabolizing enzymes and benzo[a]pyrene metabolism in the benzo[a]pyrene-sensitive mutant strain of Drosophila simulans.

Basal levels of aryl hydrocarbon hydroxylase, epoxide hydrolase and glutathione S-transferase enzyme activities, cytochrome P-450 content and inducibility of enzymes with phenobarbital were found to be similar in the microsomes of D. simulans mutant strain 364yv, which is sensitive to the toxic and mutagenic effects of benzo[a]pyrene (BP), and of the wild resistant Turku strain. In contrast, increases in the rate of BP turnover per molecule of cytochrome P-450, intensity of the hemoprotein band with apparent molecular weight 56,000 and the yield of BP 7,8-dihydrodiol and 9,10-dihydrodiol occurred only in microsomes of BP-pretreated 364yv flies but not of Turku ones. It is likely that BP induces an aberrant form of cytochrome P-450 in 364yv flies with a rare mutation in one of the P-450 regulating genes.     

Regional and subcellular distribution of cytochrome P-450-dependent drug metabolism in monkey brain: the olfactory bulb and the mitochondrial fraction have high levels of activity

In brain of female monkey (M. fascicularis) the content of cytochrome P-450 and benzo(a)pyrene hydroxylase activity in the mitochondrial fraction exceeded that of the microsomes by more than 4-fold. The mitochondrial drug metabolism activity exhibited substrate specificity and, unlike the microsomes, did not catalyze 7-ethoxyresorufin O-deethylase reaction. Moreover, the rate of benzo(a)pyrene hydroxylase activity by both the mitochondrial and the microsomal fractions displayed regional variation with the olfactory bulb displaying the highest hydroxylase activity. In contrast, the microsomal 7-ethoxyresorufin O-deethylase activity was uniformally distributed in all brain regions.     

Electron paramagnetic resonance studies on hepatic microsomal cytochrome P-450 from a marine teleost fish

Hepatic microsomal cytochrome P-450 from fish (Stenotomus versicolor), untreated or treated with 3-methylcholanthrene, 5, 6-benzoflavone, or tricaine methanesulfonate, exhibited an absorption maximum at 450 nm when reduced and ligated to CO. Microsomes from all groups exhibited EPR spectra with g values near 2.4, 2.24 and 1.9, yielding crystal field parameters similar to those for cytochrome P-450 from a variety of other sources. Treatment with 3-methylcholanthrene or 5, 6-benzoflavone resulted in elevated levels of aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity yet produced no apparent change in the levels or optical properties of CO-ligated cytochrome P-450. Tricaine methanesulfonate, a common fish anaesthetic, caused a decrease in the levels of fish cytochrome P-450.     

Induction of cytochrome P-450 forms in liver microsomes of rats in the early neonatal period after administration of phenobarbital and 3-methylcholanthrene

The activity of cytochrome P-450 dependent monooxygenase system from rat liver microsomes after induction by phenobarbital and 3-methylcholantrene in early neonatal period (3-16 days after birth) was studied. It was found that the total amount of cytochrome P-450 increases after injection of these inducers in neonatal rats of all age groups. In parallel, in the case of 3-methylcholantrene induction the benz(a)pyrene hydroxylase and 7-ethoxyresorufin deethylase activities increase; phenobarbital induction causes a rise in the benzphetamine-N-demethylase and benz(a)pyrene hydroxylase activities. Immunochemical analysis involving the use of antibodies specifically directed against cytochrome P-450 of adult rats revealed that the level of cytochrome P-450 in the case of 3-methylcholantrene induction increases from 5 to 50%, whereas that of cytochrome P-450 upon phenobarbital induction increases from 5 to 40% in liver microsomes of 3- and 16-day-old rats. The mode of inhibition of various substrates metabolism by antibodies in neonatal rat microsomes suggests that the 3-methylcholantrene-induced cytochrome P-448, like in adult rats, participates in the hydroxylation of benz(a)pyrene and O-deethylation of 7-etoxyresorufin. The participation of phenobarbital-induced cytochrome P-450 in the metabolism of benzphetamine and aldrin in neonatal rats is much lower than in the adult ones. The metabolism of benz(a)pyrene in phenobarbital-induced neonatal rat microsomes in all age groups is not inhibited by antibodies. The age-dependent differences in inhibition of metabolism and the increase in the benz(a)pyrene hydroxylase activity in phenobarbital-induced rats suggest that the spectrum of inducible forms of cytochrome P-450 in neonatal rats differ from that in adult animals.