The activities of aminopyrine N-demethylase, aniline 4-hydroxylase and UDP-glucuronyltransferase in tissues of camels, desert sheep and Nubian goats

1. The activities of the drug-metabolizing enzymes, aminopyrine N-demethylase, aniline 4-hydroxylase and UDP-glucuronyltransferase have been measured in vitro in the liver, kidney and duodenal mucosa of camels, sheep, goats and rats. 2. Enzyme activities were generally higher in the liver, followed by the duodenal mucosa, then the kidney in all species. 3. Male kids had much lower enzyme activity in the liver when compared to adult goats, and in the former animal, no measurable activity could be detected in the duodenal mucosa or kidney. 4. In general, goats seemed to have the highest and camels the lowest enzyme activity when compared to the other species. 5. Some sex differences were noted in the three enzymes studied. In sheep duodenal mucosa and rat liver and duodenal mucosa, males had higher aminopyrine N-demethylase than females. In rat liver and goat duodenal mucosa males had higher aniline 4-hydroxylase than females. Male rats had higher UDP-glucuronyltransferase in liver when compared to females.     

Characterization of distinct forms of cytochromes P-450, epoxide metabolizing enzymes and UDP-glucuronosyltransferases in rat skin

Study of drug metabolizing enzyme activity was undertaken in skin microsomal and cytosolic fractions of male and female rats. The presence of several isoforms was revealed from their activities towards selected substrates and from their cross immunoreactivity using antibodies raised against purified hepatic or renal cytochromes P-450, epoxide hydrolase and UDP-glucuronosyltransferases. Cytochrome P-450 content was precisely quantified by second derivative spectrophotometry, 23.1 and 16.5 pmol/mg protein in males and females, respectively. The monooxygenase activity associated to cytochromes P-450IIB1 and P-450IA1 was determined through O-dealkylation of ethoxy-; pentoxy- and benzoxyresorufin. The activity ranged between 4 and 2 nmol/min/mg protein for male and female rats, respectively. These results and Western blot analysis indicated that rat skin microsomes contain both monooxygenase systems associated with cytochromes P-450IIB1 and P-450IA1. By contrast lauric acid hydroxylation, supported by cytochrome P-450IVA1, was not detectable. Activities of epoxide metabolizing enzymes (microsomal and cytosolic epoxide hydrolases; glutathione S-transferase) were also characterized in skin. Microsomes catalysed the hydratation of benzo(a)pyrene-4,5-oxide and cis-stilbene oxide at the same extent, whatever the sex, although the specific activity was 10 times lower than in liver. The hydratation of trans-stilbene oxide by soluble epoxide hydrolase was four times lower than in the liver. Conjugation of cis-stilbene oxide with glutathione in skin and liver proceeded at essentially similar rates, as the specific activity of glutathione S-transferase in skin was only two times less than that measured in hepatic cytosol. Glucuronidation of 1-naphthol, bilirubin but not of testosterone could be followed in the microsomal fraction. Revelation by Western blot indicated that both the isoforms involved in conjugation of phenols and bilirubin were present in skin microsomes. By contrast, the isoform catalysing the conjugation of testosterone was apparently missing. When immunoblotting was carried out using specific antibodies raised against the renal isoforms, the same result was obtained. In addition, an intense staining corresponding to a 57 kD-protein was observed.     

Immunochemical characterization of multiple forms of cytochrome P-450 in rabbit nasal microsomes and evidence for tissue-specific expression of P-450s NMa and NMb

Two unique forms of cytochrome P-450 (P-450), designated NMa and NMb, were recently isolated in this laboratory from nasal microsomes of rabbits. In the present study, polyclonal antibodies to the purified nasal cytochromes were prepared. Immunochemical analysis with specific rabbit anti-NMa and sheep anti-NMb antibodies indicated that P-450 isozymes identical to or having a high structural homology with NMa are present in both olfactory and respiratory mucosa, as well as in liver, but NMb was detected only in the olfactory mucosa. Neither form was detected in other tissues examined, including brain, esophageal mucosa, heart, intestinal mucosa, kidney, and lung. The specific occurrence of NMb in the olfactory mucosa was further substantiated by the detection and specific inhibition by anti-NMb of the formation of unique NMb-dependent metabolites of testosterone in olfactory microsomes but not in microsomes from liver or respiratory mucosa. Similar experiments with antibodies to previously purified rabbit hepatic P-450 isozymes indicated that not all of the hepatic cytochromes are expressed in the nasal tissues. Thus, P-450 isozymes structurally homologous to hepatic forms 2, 3a, and 4, but not 3b and 6, were found in the olfactory mucosa. On the other hand, only form 2 was detected in the respiratory mucosa. Immunoquantitation experiments revealed that NMa and NMb are the major P-450 forms in olfactory microsomes, whereas NMa and P-450 form 2 (or its homolog) constitute the major portion of the respiratory nasal microsomal P-450. The level of NMa in the liver is relatively low, accounting for less than 3% of total microsomal P-450 in this tissue. In addition, evidence is provided that NMa is the major catalyst in the dealkylation of two nasal carcinogens, hexamethylphosphoramide and phenacetin, in both olfactory and respiratory nasal microsomes.     

Effects of phenobarbital and β-naphthoflavone on the activation of cyclophosphamide to mutagenic metabolites in vitro by liver and kidney from male and female rats

The activation of cyclophosphamide to metabolites that are mutagenic to Salmonella typhimurium TA 1535 by liver microsomes and kidney S9 fractions from male and female rats was studied. In addition, the effect of pretreatment with inducers of cytochromes P-450, phenobarbital and β-naphthoflavone on the activation of cyclophosphamide by these tissues was evaluated. The activation of cyclophosphamide to mutagenic metabolites with microsomes from male rat liver was three times that obtained with microsomes from female rat liver. Whereas pretreatment of either male or female rats with phenobarbital increased hepatic activation of cyclophosphamide to mutagenic metabolites about 10-fold, the increase in cytochrome P-450 content was only about 2-fold. β-Naphthoflavone pretreatment of either male or female rats decreased hepatic activation of cyclophosphamide by one-half despite an increase of 1.4-fold in hepatic cytochrome P-450 content. Kidney S9 fractions from male rats had one-third to one-half the ability of liver microsomes to activate cyclophosphamide to mutagenic metabolites; however, no enzymatic activation of cyclophosphamide to mutagenic metabolites by female rat kidney was noted. Neither phenobarbital nor β-naphthoflavone pretreatment altered the metabolic activation of cyclophosphamide to mutagenic metabolites by kidney fractions from either sex. These results demonstrate that enzymatic activation of cyclophosphamide to mutagenic metabolites differs markedly between the sexes, between different tissues (liver vs kidney), and in response to inducing agents.     

CYP1A1 and CYP1B1, two hydrocarbon-inducible cytochromes P450, are constitutively expressed in neonate and adult goat liver, lung and kidney.

The ontogeny of cytochrome P-450 isozymes (P450) in goat liver, lung and kidney was studied using anion exchange HPLC separation of solublized microsomal proteins and Western immunoblotting. Comparison of the overall HPLC profile of goat P450 isozymes between liver, lung and kidney showed that while the P450's of goat liver were equally separated into five peaks of isozyme(s), only two peaks constitute the majority of P450 isozyme(s) in lung and kidney, thus demonstrating the tissue specific differences in P450 isozyme distribution in goats. Immunoblotting analysis using polyclonal antibodies against rat CYP1B1, and mouse CYP1B1, polyaromatic hydrocarbon-regulated P450's, revealed that goat orthologs of CYP1A1 and CYP1B1 are expressed constitutively in goats. The CYP1A1 was expressed in goat liver and lung as early as 1st day of age, and the levels of its expression in adult lung and liver were, respectively, 1.3 and 5.5 pmol per mg microsomal proteins. CYP1B1 was expressed in goat livers in substantial levels as of 1 week of age and increased thereafter to reach approximately 4.5 pmol per mg microsomal proteins in adult livers, while low level was detectable only in adult but not neonate lung tissues. Furthermore, polyclonal antibodies against rat CYP1A2 detected very high levels of CYP1A2 in livers of adult and 6 week old goats. The Ah receptor which controls the expression of CYP1A1/1A2 and CYP1B1, was detected in cytosolic fractions from these tissues as a 104 kDa and a minor level of the 106 kDa form. These are potentially very important findings in light of the role of CYP1A1/1A2 and CYP1B1 in activation of polyaromatic hydrocarbons, heterocyclic amines and nitroaromatic hydrocarbons to genotoxic metabolites, and the health consequences of these metabolites on humans, as consumers of goat milk and meat. Using polyclonal antibodies against rat hepatic CYP2B1 and CYP3A1, the goat CYP2B and CYP3A forms were not detectable in livers of goats at any age, but lungs of adult and 6 week old goats expressed these two CYPs in levels equivalent to the livers of phenobarbital-induced rats. On the other hand, anti-rat CYP2C6 antibodies specifically detected two goat ortholog forms which were expressed in all three tissues and exhibited age-dependent changes. In conclusion, results from both immunoblot and HPLC analyses confirmed that, as in other species, the expression of P450 isozymes in goat is under both developmental- and tissue-specific regulatory factors.     

CYP1A1 and CYP1B1, Two Hydrocarbon-Inducible Cytochromes P450, are Constitutively Expressed in Neonate and Adult Goat Liver,Lung and Kidney *

Abstract: The ontogeny of cytochrome P-450 isozymes (P450) in goat liver, lung and kidney was studied using anion exchange HPLC separation of solublized microsomal proteins and Western immunoblotting. Comparison of the overall HPLC profile of goat P450 isozymes between liver, lung and kidney showed that while the P450's of goat liver were equally separated into five peaks of isozyme(s), only two peaks constitute the majority of P450 isozyme(s) in lung and kidney, thus demonstrating the tissue specific differences in P450 isozyme distribution in goats. Immunoblotting analysis using polyclonal antibodies against rat CYP1A1, and mouse CYP1B1, polyaromatic hydrocarbon-regulated P450's, revealed that goat orthologs of CYP1A1 and CYP1B1 are expressed constitutively in goats. The CYP1A1 was expressed in goat liver and lung as early as 1st day of age, and the levels of its expression in adult lung and liver were, respectively, 1.3 and 5.5 pmol per mg microsomal proteins. CYP1B1 was expressed in goat livers in substantial levels as of 1 week of age and increased thereafter to reach approximately 4.5 pmol per mg microsomal proteins in adult livers, while low level was detectable only in adult but not neonate lung tissues. Furthermore, polyclonal antibodies against rat CYP1A2 detected very high levels of CYP1A2 in livers of adult and 6 week old goats. The Ah receptor which controls the expression of CYP1A1/1A2 and CYP1B1, was detected in cytosolic fractions from these tissues as a 104 kDa and a minor level of the 106 kDa form. These are potentially very important findings in light of the role of CYP1A1/1A2 and CYP1B1 in activation of polyaromatic hydrocarbons, heterocyclic amines and nitroaromatic hydrocarbons to genotoxic metabolites, and the health consequences of these metabolites on humans, as consumers of goat milk and meat. Using polyclonal antibodies against rat hepatic CYP2B1 and CYP3A1, the goat CYP2B and CYP3A forms were not detectable in livers of goats at any age, but lungs of adult and 6 week old goats expressed these two CYPs in levels equivalent to the livers of phenobarbital-induced rats. On the other hand, anti-rat CYP2C6 antibodies specifically detected two goat ortholog forms which were expressed in all three tissues and exhibited age-dependent changes. In conclusion, results from both immunoblot and HPLC analyses confirmed that, as in other species, the expression of P450 isozymes in goat is under both developmental-and tissue-specific regulatory factors.     

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

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

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.     

Effect of metronidazole on the hepatic mixed function oxygenases (cytochromes b5 and P-450) in Swiss mice

Effect of metronidazole (MNZ) treatment (oral and ip) on activities of cytochrome b5 and P-450 was studied in male, virgin and pregnant female mice. Activities of both the cytochromes increased in virgin mice treated with 2 mg (ip or PO, per mouse) but not in male and pregnant females. 30 mg dose (per mouse) was toxic in pregnant mice but increased the cytochromes activities in males and virgin females. HPLC analysis of liver MNZ levels showed that virgin females had higher MNZ content than male and pregnant females when treated with ip injection of MNZ (250 mg/kg).     

Differential toxicity of aflatoxin B1 in male and female rats: relationship with hepatic drug-metabolizing enzymes

It is well known that distinct differences in the metabolism of xenobiotics exist between males and females of the same species. Male and female rats were treated with a single intraperitoneal dose of aflatoxin B₁ (AFB₁): 1 or 3 mg/kg for males, 3 or 6 mg/kg for females. Comparative changes in hepatic drug metabolizing and plasma enzymes had been studied. The obtained results show that, at the common dose of 3 mg/kg, AFB₁ induced an 18% mortality in males and none in females. In the plasma, total bilirubin concentration as well as the activity of transaminases and alkaline phosphatase (ALP), utilized as indicators of liver damage, were highly increased in both males and females due to the treatment with 3 or 6 mg AFB₁/kg. In the female, the plasma features rapidly declined. In contrast, in the male, the effect of AFB₁ was prolonged. Hepatic determinations revealed a pattern difference of drug metabolizing enzymes and cytochrome P-450 between males and females. The results also show that in the male, most of the drug metabolizing enzyme activities were decreased until the ninth day with the 3 mg/kg treatment. So, we observed a decrease in the activities of UDP-glucuronosyltransferase (UDPGT) with p-nitrophenol as substrate (PNP) and GSH S-transferase, 40 and 53% respectively; while the activity of epoxide hydrolase was increased up to 170%. In the meantime, the concentration of cytochrome P-450 decreased by 69%. By contrast, in the case of the female, these decreases were only 14, 43 and 23% for the UDPGT, GSH S-transferase and cytochrome P-450, respectively. Moreover, these decreases occurred only during the first three days after treatment. Thereafter, these enzyme activities significantly increased above the control values. This study suggests that the induction of detoxicating enzymes, more important in the female (72% increase in the activity of UDPGT, 480% in that of epoxide hydrolase and 42% for GSH S-transferase, may have a protective role against AFB₁ metabolites and could explain, partly, the lower sensitivity of the female to the toxic effects of AFB₁.     

Tolbutamide hydroxylation by human, rabbit and rat liver microsomes and by purified forms of cytochrome P-450

Tolbutamide hydroxylation has been investigated in human, rabbit and rat liver microsomes and by six purified forms of hepatic rabbit cytochromes P-450. These studies were carried out to investigate whether an appropriate animal model could be developed for the human cytochrome(s) P-450 metabolizing tolbutamide. Selective induction was used in rats and rabbits to indicate the isozymes primarily responsible for tolbutamide hydroxylation in these species. Microsomal tolbutamide hydroxylase activity was significantly induced only by phenobarbital pretreatment in the rat which induces P-450 forms b (P-450IIB1) and/or e (P-450IIB2). Only pretreatment of rabbits with rifampicin, which induces cytochrome P-450 form 3c (P-450IIIA6), significantly increased the microsomal hydroxylation of tolbutamide. However, the increase in tolbutamide hydroxylase activity in rifampicin-induced microsomes (congruent to 50%) appears low compared to known levels of induction of P-450IIIA6 following rifampicin pretreatment (5-10-fold). These data suggest that P-450IIIA6 is at least partially involved in tolbutamide hydroxylation in rabbit liver but that other form(s) may be relatively more important. Reconstitution experiments with six purified forms of rabbit cytochrome P-450 indicated that the highest activity occurred with P-450IIIA6 (form 3c). As isozymes from different gene families or subfamilies appeared to metabolize tolbutamide in the three species studied, catalytic similarities between the P-450s with respect to inhibition was further investigated in microsomes using sulfaphenazole, alpha-naphthoflavone and mephenytoin. These studies showed that the catalytic characteristics in relation to inhibition differ markedly between species. Hence, it appears that the animal model approach is not likely to be successful in the identification and characterization of the cytochrome P-450 form(s) metabolizing tolbutamide in humans.     

Metabolism of Chlorpromazine and p-Nitrobenzoic Acid in the Liver,Intestine and Kidney of the Human Foetus

The ability of the liver, intestine and kidney to metabolize chlor-promazine (CPR) and p-nitrobenzoic acid (NBA) was studied in the human foetus. Low levels of CPR metabolizing activity were present in all the tissues studied, but only the liver and intestine were capable of metabolizing NBA. We were not able to detect any cytochrome P-450 in the liver microsomal fraction. The enzymes metabolizing CPR and NBA are located in the liver microsomes, they require NADP and the 100,000 × g supernatant fraction or NADPH₂ for full activity, are inhibited by carbon monoxide, and have Michaelis constants of the same order of magnitude as found in the enzymes from experimental animals. The above mentioned characteristics of the foetal enzymes strongly suggest that they belong to the same class of NADPH₂-dependent mixed function oxidases which are detected in the livers of adult humans and animals and which are thought to be responsible for the greater part of oxidative and reductive drug metabolism.     

Effects of long-term choline deficiency on hepatic microsomal cytochrome P-450-mediated steroid and xenobiotic hydroxylases in the female rat

Total cytochrome P-450 levels decreased to about 80% of control in hepatic microsomes from female rats maintained for 30 weeks on a choline-deficient diet. Livers from these rats were fibrotic and had extensive fatty infiltration but, unlike livers of male rats on the same regimen, were not cirrhotic. Steroid hydroxylase activities were assessed in microsomes of female rats that received the choline-deficient diet and it was noted that the activity of the cytochrome P-450 UT-F-mediated steroid 7 alpha-hydroxylase was decreased to about 50% of the activity present in choline-supplemented control rat microsomes. Similar decreases were observed for microsomal androstenedione 6 beta-hydroxylase and aniline 4-hydroxylase activities. In female rat hepatic microsomes these two activities are probably mediated by the isozyme cytochrome P-450 ISF-G. In contrast to these findings, the activities of four other xenobiotic metabolising enzymes, as well as rates of microsomal steroid 16 alpha- and 16 beta-hydroxylation, were unchanged from control. Thus, in hepatic microsomes from choline-deficient female rats, it appears likely that levels of the non-sexually differentiated cytochromes P-450 UT-F and ISF-G are decreased. Unlike the situation in male rats, long term choline deficiency does not appear to influence levels of sexually-differentiated P-450 enzymes in the female rat.     

Is liver microsomal enzyme induction the cause of tolerance to barbiturates?]

In 12, 24 and 48 hours after a single injection of phenobarbital, barbital-sodium and pentabarbital-sodium in doses of 80 175 an 40 mg/kg respectively an increased synthesis of protein in the cell-free protein-synthetizing system and a rise in the level of cytochromes b5 and P-450 in the liver microsomes of female rats were noted. The maximal changes were registered following introduction of phenobarbital the inducing capacity of barbital-sodium and pentabarbital-sodium twice as low. With chronic introduction of the drugs the tolerance with respect to all of them develops at an equal rate, which excludes the dependence of this phenomenon upon the induction of microsomal metabolizing enzymes of the liver.     

Effects of tetraethyl lead on the activities of drug metabolizing enzymes in different tissues of the rat.

The present study describes the effects of tetraethyl lead on various drug metabolizing enzymes in different tissues of the rat. Tetraethyl lead was administered intraperitoneally to rats (250 mumol/kg) on two consecutive days. The animals were killed on day 3. Tetraethyl lead-treatment decreased the concentration of hepatic cytochrome P-450 (to 45 per cent of the control), the hepatic activity of aryl hydrocarbon hydroxylase (to 41 per cent of the control) and ethoxycoumarin deethylase (to 45 per cent of the control). Epoxide hydratase activity was enhanced in the liver (1.3-fold), kidney (3.3-fold), and small intestinal mucosa (4.7-fold). The activity of glutathione S-transferase decreased in the liver (to 69 per cent of the control) but increased in the kidney (1.5-fold) and small intestinal mucosa (1.7-fold). The glucuronidation of o-aminophenol was enhanced (2.2-fold) in the kidney of tetraethyl lead treated rats. It is concluded that exposure to tetraethyl lead brings about widespread changes in the ability of mammals to detoxify foreign compounds.     

Studies on sex related differences in elimination of theophylline in the rat after pretreatment with phenobarbital, chrysene, Aroclor 1254 or lindane.

Cytochrome P-450 in liver microsomes plays a central role as a drug metabolizing enzyme. The sex-dependent differences in the properties of cytochrome P-450 have been extensively studied using liver microsomes, and marked differences have been found in microsomal metabolism of xenobiotic. Effect of pretreatment with phenobarbital, chrysene, lindane and polychlorinated biphenyls (Aroclor 1254) on the theophylline elimination in male and female rats was studied. Levels of theophylline were measured spectrophotometrically.     

Aging and drug metabolism: alteration of liver drug metabolizing ability in male rats. Is it functional deterioration or feminization of the liver?]

The profiles of hepatic drug metabolism were obtained by using young and old male and female rats. The profile obtained from old male rats was completely different from that from young male rats, while it was almost identical to those of females of any ages. This was due to the selective decrease in male hepatic enzyme activities showing higher activities than females to the activity levels of females which did not alter much with aging. Castration of young adult male rats caused a decrease in enzyme activities but did not result in the feminization of the metabolic profile. Administration of testosterone to old male rats resulted in the recovery of the profile of young male rats, but the levels of activities were not as high as young male rats. Plasma testosterone levels were found to decrease in parallel with drug metabolizing activities of male rats during aging. These results suggest that sex hormones play important roles in the alteration of drug metabolizing activities in male rats with aging. The loss of male characteristics in profile of drug metabolism during aging was evaluated by use of antibody to the male specific cytochrome P-450, P-450 m1. Anti-P-450 ml strongly inhibited imipramine N-demethylase activity, which showed marked sex (male greater than female) and age (young greater than old) differences, while this did not inhibit imipramine 2-hydroxylase activity, which showed no sex or age differences. The portion of N-demethylase activity inhibited by this antibody decreased in old rats while the portion not inhibited did not decrease with age. These results indicate that the decrease of sex specific cytochrome P-450 is responsible for the age-associated decrease in at least one of the drug metabolizing enzyme activities in male rats. It is suggested that some processes of the control mechanism of the gene expression of male specific cytochrome P-450 may be altered with old age.     

Differences in the biochemical properties of aldrin epoxidase, a cytochrome P-450-dependent monooxygenase, in various tissues

Aldrin epoxidase, a cytochrome P-450-dependent monooxygenase, was studied in the lung and kidney of male rats. The sensitivity of the liver enzyme activity to different chemicals in vitro was influenced by the treatment of the animals with phenobarbital or methylcholanthrene. These results confirm that more than one form of cytochrome P-450 supports aldrin epoxidase in the liver. The lung and kidney aldrin epoxidase activity was not modified by the administration of chemical inducers to the rats. In vitro, the lung and kidney aldrin epoxidase activities were activated by tetrahydrofurane and progesterone, respectively. The results obtained from the lung and kidney indicate that one single species of cytochrome P-450, associated with aldrin epoxidase, exists in these organs, but it may be a different type, or regulated in a different manner in these tissues.     

Genotoxic and mutagenic activation of aflatoxin B1 by constitutive forms of cytochrome P-450 in rat liver microsomes

Cytochrome P-450-mediated activation of aflatoxin B1 (AFB1) to genotoxic and mutagenic products which subsequently cause induction of an umu gene expression in Salmonella typhimurium TA1535/pSK1002 has been studied in a rat liver microsomal or reconstituted monooxygenase system. Liver microsomes from male Sprague-Dawley rats had a 1.5-fold higher activity to catalyze AFB1 than did those from female rats. In addition, the activation was not increased in liver microsomes from rats pretreated with phenobarbital, 3-methylcholanthrene, a polychlorinated biphenyl mixture, or dexamethasone, suggesting that the constitutive forms of cytochrome P-450 have important roles for the activation of AFB1 in rat liver microsomes. Using 15 forms of cytochrome P-450 purified from liver microsomes of untreated and phenobarbital- and 3-methylcholanthrene-treated rats, three isozymes from untreated male rats and one isozyme from untreated female rats were found to have high reactivities in metabolizing AFB1 to genotoxic products. Cytochrome P-450 forms isolated from inducer-treated rats were relatively less active. The close correlation between induction of umu gene expression and mutagenicity with Ames/S. typhimurium TA98 system by activated metabolites of AFB1 in the reconstituted monooxygenase system suggested that the constitutive forms of cytochrome P-450 had major roles for genotoxic and mutagenic activation of AFB1 in rat liver microsomes.     

Induction of hepatic oxidative and conjugative drug metabolism in the hamster by N-substituted imidazoles

Three antimycotic N-substituted imidazoles, clotrimazole, tioconazole and miconazole, were able to induce hepatic microsomal cytochrome P-450 and monooxygenase reactions in both male and female hamsters to an extent similar to that seen with phenobarbital treatment. Imidazole treatment did not alter the cytochrome P-450 concentration, and ketoconazole treatment decreased it. Cytosolic sulfo- and glutathione transferases were not significantly altered by any imidazole. Induction of microsomal morphine glucuronosyltransferase activity by each compound generally paralleled the effect on cytochrome P-450 in females but induction was not evident in males. Clotrimazole treatment, in contrast to phenobarbital treatment, also caused a large induction of l-naphthol glucuronosyltransferase in females. The potential for antimycotic imidazoles to alter the hepatotoxicity of compounds will require consideration of the inductive changes in both Phase I and Phase II drug metabolizing enzymes in addition to their known inhibitory effects on Phase I oxidations. The inductive effects differ for each imidazole, and in the hamster model, depend upon the sex of the animal.