Sex difference in the daily rhythm of hepatic P450 monooxygenase activities in rats is regulated by growth hormone release

Daily fluctuations have been observed in the hepatic cytochrome P450 monooxygenase activities of rodents. It was confirmed in our previous study that the P450 activities assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) showed obvious daily fluctuations in male rats with high values during the dark period. However, there is still very little information about the daily fluctuation of P450 activities in female rats. In the first experiment of the present study, the daily fluctuation of ACD activities in female rats was examined. The results indicated the absence of any apparent daily fluctuation in the hepatic ACD activities of the females, so the study confirmed a sex difference in the daily rhythm of the ACD activities. In the second experiment using hypophysectomized (Hpx) rats, it was investigated whether this sex difference in daily rhythm was attributable to growth hormone (GH) release, an activity which is known to affect the sex difference in the expression of P450 isoforms. It was found that the Hpx rats, given subcutaneous injections of GH twice during the light period to mimic a male pattern of GH secretion, showed obvious light-dark fluctuation in ACD activities with high values in the dark period, and similar results were obtained in sham-operated males. Conversely, the Hpx rats given continuous infusion of GH to mimic a female pattern of GH secretion did not show the light-dark fluctuation in ACD activities, and similar results were obtained in sham-operated females. In conclusion, there is a sex difference in the daily rhythm of the hepatic P450 activities in rats and this difference is apparently influenced by the difference in the patterns of GH secretions.     

Daily fluctuation of hepatic P450 monooxygenase activities in male rats is controlled by the suprachiasmatic nucleus but remains unaffected by adrenal hormones

Hepatic P450 monooxygenase activities, which strongly influence the efficacy and/or toxicity of drugs, are known to fluctuate daily. We also know that the P450 activities assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) activities fluctuate daily, with apparently high values during the dark period in male rats. However, there is little knowledge about the factors that regulate daily fluctuation of P450 monooxygenase activities. In the present study using rats, we induced lesions in the suprachiasmatic nucleus (SCN) of the brain, the known site of the body's internal clock, and examined the effects on the daily fluctuation of the ACD activities to clarify the relationship between the SCN and the daily fluctuation of P450 monooxygenase activities. In addition, adrenalectomy was performed to re-evaluate the influence of adrenal hormones on the P450 activities. Our results indicated that daily fluctuations of the hepatic ACD activities were completely eliminated in the SCN-lesioned rats. However, the ACD activities in the adrenalectomized rats showed apparent daily fluctuations with high values during the dark period and low values during the light period. Therefore, this study demonstrated that the daily fluctuation of the hepatic P450 monooxygenase activities in male rats is controlled by the SCN but remains unaffected by the adrenal hormones. Received: 11 May 1999 / Accepted: 19 July 1999     

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.     

Unaltered drug metabolizing enzyme systems in type II diabetes mellitus before and during glyburide therapy

Diabetes mellitus is associated with alterations in hepatic drug metabolizing enzyme activities in experimental animals. To determine whether Type II diabetes or glyburide affect hepatic drug metabolism, the authors used 13C-labeled aminopyrine and caffeine breath tests as in vivo probes of the hepatic cytochrome P450 and P(1)450 enzyme activities respectively in six subjects with Type II diabetes (4 women, 2 men). These subjects had been treated previously with diet, had an age range of 41-63 years, had a body mass index range of 24.1-43.3 kg/m2 and had a mean fasting plasma glucose of 14.6 +/- 1.2 mM and a mean hemoglobin A1c of 12.2 +/- 0.7%. These subjects did not drink alcohol or take drugs known to affect hepatic drug metabolism. The caffeine and aminopyrine breath tests (CBT, ABT) were performed sequentially while fasting before the start of glyburide treatment (5 mg daily) and at weekly intervals for 4 weeks. ABT and CBT values are expressed as cumulative percentage of dose exhaled through 2 hours. Before beginning glyburide, the mean ABT and CBT results were 10.2 +/- 0.7% and 4.2 +/- 0.7% respectively, well within the normal ranges for these tests (ABT 6.5-15%; CBT 2.5-10%). The ABT and CBT values remained unaltered during 4 weeks of glyburide therapy. However, FBS decreased to 10.2 +/- 1.1 mM and HbA1C to 10.1 +/- 0.8% by the end of drug treatment. Type II diabetes that is poorly controlled by diet alone is not associated with alterations of the hepatic drug metabolizing enzymes as judged by the caffeine and aminopyrine breath tests. Furthermore, glyburide does not induce or inhibit the drug metabolizing enzyme systems in the liver, thereby precluding drug-drug interactions of this type.     

Liver Enzyme Activities after Multiple Administration of Nifedipine in Mice

Abstract: The effect of multiple nifedipine administration on hexobarbital sleeping time, liver monooxygenase and synthetase activities, lipid peroxidation and microsomal membrane fluidity were studied in male albino mice. The drug was administered orally at a dose of 25 mg/kg daily for 14 and 21 days. Nifedipine caused enzyme induction, demonstrated by shortened hexobarbital sleeping time, enhanced ethylmorphine N-demethylase, aniline 4-hydroxylase, ethoxycoumarine O-deethylase, UDP-glucuronyl transferase, glutathione S-transferase and NADPH-cytochrome c reductase activities and increased content of cytochrome P450 and cytochrome b₅. This effect persisted until the 7th day after the last dose of nifedipine. There were no changes in lipid peroxidation and fluidity of the microsomal membranes after 14-day nifedipine administration. The increased cytochrome P450 content and drug metabolizing enzyme activities could be not associated with changes in these liver microsomal membrane properties.     

Hepatic drug metabolizing enzymes induced by clofibrate in rasH2 mice

Hepatic drug metabolizing enzyme activities were determined, after treatment with clofibrate, in transgenic mice carrying human c-Ha-ras (rasH2 mice). Changes in the drug metabolizing enzyme activities in these mice by gene integration were also evaluated. Male and female rasH2 mice (Tg) and the litter mates not carrying the gene (non-Tg) received orally 500 mg/kg of clofibrate or the vehicle for 12 consecutive days. Liver homogenate and microsomes were prepared and the contents and activities of cytochrome P450 (CYP), cytochrome b5 content and enzyme activities related to peroxisome proliferation were determined. Relative liver weights, CYP4A and activities of catalase and carnitine palmitoyl transferase increased to the same extent in Tg and non-Tg mice treated with clofibrate. In Tg and non-Tg groups that received vehicle, contents and activities of CYP and cytchrome b5 contents were comparable. It was concluded that gene integration did not alter drug metabolizing enzymes and responses to clofibrate.     

Comparison of effects of acetaminophen on liver microsomal drug metabolism and lipid peroxidation in rats and mice.

Studies were conducted to determine the in vivo effect of acetaminophen (AAP) on the lipid peroxidation, drug metabolizing enzyme activity and microsomal electron transfer system of rat and mouse liver. AAP was found to inhibit ethylmorphine N-demethylase activity in the presence of NADPH and this inhibition of the enzyme was due to decrease in cytochrome P-450 content, but not due to change in lipid peroxidation in liver microsomes. Kinetical data showed that AAP administration had no effect on Km values of ethylmorphine N-demethylase, however, a decrease in the Vmax values was seen in rats and mice. There was no significant effect of AAP on both NADPH-cytochrome c reductase and the content of cytochrome b5 3 hours after this administration to rats and mice. On the other hand, AAP induced a significant decrease in NADH-ferricyanide reductase in mice, but not in rats. The greatest decrease in cytochrome P-450 observed among the components of the liver microsomal electron transfer system of rats and mice.     

In vitro and in vivo studies of the effect of vitamin E on microsomal cytochrome P450 in rat liver

Administration of a diet supplemented with 0.06% vitamin E acetate to male rats over a 6-week period doubled hepatic microsomal stores of alpha-tocopherol over those in control (vitamin E adequate) rat liver. Total cytochrome P450 content and NADPH-cytochrome P450 reductase activity were significantly elevated in hepatic microsomes from vitamin E-supplemented rats to 111% and 123% of respective control values. Androstenedione 16 alpha-hydroxylase activity was increased in these fractions (2.57 +/- 0.31 nmol product/min/mg protein vs 1.81 +/- 0.38 in controls) whereas activities of the 6 beta-, 7 alpha- and 16 beta-hydroxylase pathways were unchanged. Immunoquantitation of the microsomal 16 alpha-hydroxylase, P450 IIC11, indicated a corresponding increase in the hepatic content of the enzyme. In view of the established antioxidant role of tocopherols, the effects of dietary vitamin E manipulation on the concentration of protein sulphydryl groups and the susceptibility of microsomes to ferric sulphate-ADP-NADPH-mediated lipid peroxidation were also assessed. Dietary supplementation did not influence microsomal protein sulphydryl content (68 +/- 10 nmol glutathione equivalents/mg protein) but decreased the extent of lipid peroxidation produced by the ferric sulphate-ADP-NADPH system in vitro. Further in vitro experiments demonstrated that vitamin E acetate (2 microM) protected protein sulphydryl groups and lipids against peroxidation in control microsomes and partially reduced the associated losses of P450-mediated steroid hydroxylase activities. Western immunoquantitation of P450 IIC11 revealed that exogenous vitamin E acetate protected completely against peroxidation-induced apoprotein loss. These studies establish that the in vitro protective effects of vitamin E acetate against sulphydryl and lipid peroxidation extend to protection of the P450 apoprotein but that enzyme activity is only partially protected. This finding suggests that peroxidation-dependent loss of P450 in vitro is mediated by haem degradation from the P450 holoenzyme and is not directly related to lipid/sulphydryl oxidation. In contrast, the in vivo effects of dietary vitamin E on drug metabolizing enzymes are regulatory in nature and are unrelated to effects on lipid peroxidation.     

Zinc mediates normalization of hepatic drug metabolizing enzymes in chlorpyrifos-induced toxicity

The present study investigated the protective effects of zinc in attenuating the altered activities of drug metabolizing enzymes in the livers of rats intoxicated with chlorpyrifos. Male Sprague-Dawley rats received oral chlorpyrifos treatment (at a dose level of 13.5 mg/kg body weight in corn oil every alternate day), zinc supplementation alone (at a dose level of 227 mg/l in drinking water), or combined chlorpyrifos plus zinc treatments for a total duration of 8 weeks. The effects of different treatments were studied on the specific activities of various drug metabolizing enzymes including cytochrome P(450), cytochrome b(5), NADPH cytochrome-c-reductase, NADH cytochrome-c-reductase, aminopyrene-N-demethylase (APD) and aromatic hydrocarbon hydroxylase (AHH). Additionally, serum zinc levels were also determined in each of the treatment groups at the end of the study. Chlorpyrifos treatment resulted in a significant decrease in the serum zinc concentrations. Analogous to these changes, we observed significant depression in the activities of majority of the drug metabolizing enzymes investigated in the present study, except for AHH, where the decrease in enzyme activity was not statistically significant. However, zinc treatment to chlorpyrifos treated animals effectively restored the depressed serum zinc levels to within normal limits. Similarly, co-administration of zinc to chlorpyrifos intoxicated animals normalized the enzymatic activities of cytochrome P(450), NADPH cytochrome-c-reductase and NADH cytochrome-c-reductase within normal range. Collectively, these findings suggest that zinc plays an important role in regulating the hepatic activities of drug metabolizing enzymes in chlorpyrifos intoxicated animals, although it remains to be determined whether such protective effects of zinc are regulated directly, or through some indirect mechanism.     

Effects of flutropium bromide, a new antiasthma drug, with repeated administration on bronchomotor response and hepatic drug metabolizing enzymes

Effects of flutropium bromide, a new antiasthma drug possessing the quarternary ammonium structure of atropine derivatives, were investigated on the bronchodilatory activity with repeated inhalations in guinea pigs and on the hepatic drug metabolizing enzyme activities with repeated i.v. administration in rats. Single inhalation of flutropium bromide (0.03%) inhibited the ACh-induced bronchoconstriction without changing the fall in blood pressure induced by ACh. Flutropium bromide (0.03%) was inhaled for 5 min a day by placing the animal in an inhalation box, successively during periods of 14 and 28 days. The bronchodilatory effect of flutropium bromide after 14- or 28-day repeated inhalations was almost the same potency as that after single inhalation. Twenty-eight-day repeated inhalations did not change the body weight curve in guinea pigs; and in addition, 14-day repeated i.v. administration did not change the hepatic drug metabolizing enzyme activities in rats. From the above results, it is indicated that flutropium bromide causes neither reduction in response nor cumulative effect after repeated inhalations, and that the agent maintains the bronchodilatory effect without change in the hepatic drug metabolizing enzyme activities.     

The effect of galactosamine on rat liver cytochrome P-450 activities

The effect of galactosamine on hepatic drug metabolizing activities was examined in rats. In the microsomal fraction, the contents of cytochrome P-450 (P-450) and cytochrome b5 (b5) and the activity of NADPH-cytochrome c reductase (reductase) were examined for 7 days after galactosamine administration. In addition, substrate metabolizing activities in damaged microsomes were examined using four substrates: aminopyrine, aniline, benzo(a)pyrene (B(a)P) and 7-ethoxycoumarine (7-EC). The contents of P-450 and b5 and the activity of reductase showed a minimal value after 3 days of galactosamine administration and then gradually increased, reaching to the control level after 7 days. All four substrate metabolizing activities showed a similar response as the content of P-450, but the decrement among the four activities was not uniform. The activities of B(a)P hydroxylation and 7-EC deethylation were more impared than those of aminopyrine demethylation and aniline hydroxylation. This nonuniformity was clear on the activity based on P-450. This result suggested that galactosamine disturbed the population of multiple P-450 subtypes, and each P-450 subtype was damaged to the various extent by galactosamine administration.     

苯巴比妥诱导下大鼠肝微粒体药酶活性与膜流动性变化的相关性

本文用ANS和DPH为荧光探剂,研究苯巴比妥(PB)诱导下大鼠肝微粒体膜脂区流动性与膜药酶活性变化的相关性。结果表明,经PB诱导后在增加肝微粒体蛋白质含量,P-450含量及NADPH-细胞色素C还原酶等酶活性的同时,肝微粒体膜流动性明显增大,且膜深层流动性的增大与膜氨基比林N-脱甲基酶、细胞色素C还原酶活性增加有明显的直线正相关。膜胆固醇/碑脂比值明显降低。此结果提示,肝微粒体膜流动性的适当增大与PB增加单胺氧化酶系统活性之间可能存在着某种联系。     

Induction and recovery of hepatic drug metabolizing enzymes in rats treated with Ginkgo biloba extract.

Herb-drug interactions, especially cytochrome P450 (CYP)-mediated interactions, cause an enhancement or attenuation in efficacy of co-administered drugs. In a previous study, we reported that repeated oral ingestion of Ginkgo biloba extract (GBE) markedly induced hepatic drug metabolizing enzymes in rats. In this study, we focused on the recovery of GBE-induced hepatic drug metabolizing enzymes after the discontinuation of GBE in rats. Feeding of a 0.5% GBE diet to rats for 1 week markedly increased liver weight, content of total CYP, activities of 6 CYP subtypes and glutathione S-transferase (GST). The content and activities of CYP enzymes were recovered to almost basal levels within 1 week after the discontinuation of GBE, while the activity of GST gradually decreased and recovered to the control level after 3 weeks. These results indicated that GBE-induced hepatic drug metabolizing enzymes in rats, especially CYPs, were rapidly recovered by discontinuation of GBE in rats even after excess treatment, and suggested that interactions of GBE with drugs could be avoided by discontinuation of GBE.     

Metabolic fate of the new anti-ulcer drug enprostil in animals. 4th communication: effect on hepatic drug metabolizing enzyme system in rats

Effects of multiple oral administration of enprostil ((+/-)-11 alpha,15 alpha-dihydroxy-9-oxo-16-phenoxy-17,18,19,20-tetranorprosta- 4,5,13(t)-trienoic acid methyl ester, TA 84135) (20 micrograms/kg/d) and its solvent propylene carbonate (PC, 100 microliters/kg/d) on body weight gain, liver weight, hepatic drug metabolizing enzyme system and hexobarbital sleeping time were investigated in male rats during a 14-day period. Cytochrome P-450 content (as compared to the untreated control) and cytochrome b5 content (as compared to PC treated group) were slightly, but significantly, reduced in the group given a single oral dose of enprostil. However, these slight reductions were not augmented significantly by repeated administrations of enprostil. Slight but significant increase in microsomal protein content was observed in the group given 14 oral doses of enprostil and PC. Enprostil did not affect the other indicators used to evaluate the status of the hepatic drug metabolizing enzyme system. Additionally, single or multiple oral doses of enprostil or PC showed no effect on the hexobarbital-induced sleeping time. It therefore may be safely concluded that multiple oral administration, both of enprostil and of PC, has very little effect on drug metabolizing enzyme inducing or inhibiting activity in rats.     

The effect of prolonged sodium phenobarbitone treatment on hepatic xenobiotic metabolism and the urinary excretion of metabolites of the d-glucuronic acid pathway in the rat

Male Sprague-Dawley rats were fed a 0.1 % (w/w) sodium phenobarbitone (PB) diet for periods of 4, 12 and 24 weeks. Phenobarbitone treatment resulted in a marked increase in liver size which was accompanied by the induction of several parameters of hepatic xenobiotic metabolism including mixed function oxidase enzyme activities, UDP-glucuronyltransferase, cytochrome P450 and the microsomal protein content. In addition, treatment with the barbiturate led to the stimulation of the urinary excretion of d-glucaric acid, l-gulonic acid, free d-glucuronic acid, l-ascorbic acid and xylitol. The levels of stimulation of both the hepatic and urinary parameters remained relatively constant throughout the period of treatment. On cessation of PB treatment the parameters of hepatic xenobiotic metabolism reverted to control levels at a faster rate than the excretion of the d-glucuronic acid metabolites. The results demonstrate a correlation between the urinary excretion of metabolites of the d-glucuronic acid pathway and hepatic xenobiotic metabolizing enzyme activities during the prolonged administration of PB.     

The effect of heat stress on the induced hepatic drug metabolizing system in rats.

The activity of the hepatic oxidative drug metabolizing system has been investigated in an experimentally-induced heat stress animal model pretreated with phenobarbitone. Female rats, unacclimatized and untrained were pretreated for 3 days with phenobarbitone as the inducing agent for the drug metabolizing systems. On the fourth day, they were restrained and exposed to an ambient temperature of 40 degrees C. One hour after acute exposures to such conditions, the activities of hepatic cytochrome P450, cytochrome b5 and NADPH cytochrome c reductase were significantly decreased in the induced animal model. Further, cytochrome P450 isozymes observed by SDS-gel electrophoresis were significantly decreased. In addition, the hypnotic effect of pentobarbitone was significantly increased. It is concluded that the activity of the hepatic oxidative drug metabolizing enzymes was decreased in induced drug metabolism systems exposed to heat stress conditions.     

Action of styrene and its metabolites styrene oxide and styrene glycol on activities of xenobiotic biotransformation enzymes in rat liver in vivo

The effects of the administration of styrene and its metabolites, styrene oxide and styrene glycol, on drug metabolizing enzymes of rat liver were studied. Styrene (three or six daily doses of 500 g/kg) doubled the activities of microsomal p-nitroanisole O-demethylase, epoxide hydrase (styrene oxide as substrate), and UDP glucuronosyltransferase (p-nitrophenol) when measured in microsomes pretreated in vitro with digitonin or trypsin. On the other hand, glycine conjugation, the cytochrome P-450 content, and activities of NADPH cytochrome c reductase and benzpyrene hydroxylase, were less affected by styrene. Styrene oxide was more toxic to rats than styrene or styrene glycol and it also caused (one dose of 375 mg/kg) a significant decrease in the activities of benzpyrene hydroxylase and p-nitroanisole O-demethylase and in the cytochrome P-450 content. Epoxide hydratase NADPH cytochrome c reductase, and UDP glucuronosyltransferase were more resistant towards styrene oxide. Styrene glycol did not significantly alter the activities of drug metabolizing enzymes. The current data show that styrene causes an increase in the activities of some enzymes involved in its own metabolism.     

Induction of hepatic cytochrome P-450c-dependent monooxygenase activities by dantrolene in rat

The effect of dantrolene sodium, a skeletal muscle relaxant, on drug metabolizing enzymes has been investigated after treatment of rats with a dose of 200 mg/kg for five days. We observed an induction of cytochrome P-450c and epoxide hydrolase in immunoassays and activities. An enhancement of the UDP-glucuronosyltransferase (GT1) activity was observed. We also reported a decrease of both liver cytochrome P-450 content and microsomal cytochrome P-450b dependent N-demethylation activities. On the other hand, the binding of dantrolene on microsomal cytochrome P-450 produced a type I difference spectrum, these data were obtained with liver microsomal cytochrome P-450c induced by 3-methylcholanthrene.     

Further studies on the persistence of neonatal androgen imprinting on sex-specific cytochrome P-450, testosterone and drug oxidations

Neonatal castration completely suppressed the expression of P-450-male and expressed P-450-female; and testosterone treatment in a neonatal period partially reversed the effect of castration, i.e., neonatal imprinting (Kamataki et al., 1984; Waxman et al., 1985). In the present communication, we investigate the reversibility and persistency of neonatal imprinting on the expression of P-450-male and P-450-female. To our surprise, testosterone treatment at adulthood (8 weeks old) caused full expression of P-450-male and restored the activities of 2 alpha- and 16 alpha-testosterone hydroxylases in neonatally castrated rats. The levels of ethylmorphine N-demethylation, propoxycoumarin O-depropylation and benzo(a)pyrene hydroxylation were increased to the levels of adult male rats by adult testosterone-treatment. Moreover, treatment with testosterone of neonatally castrated rats at the age of 19 weeks did not cause a complete recovery of P-450-male content and drug-metabolizing activities. Testosterone administration into neonatal female rats did not significantly alter the contents of sex-dependent cytochrome P-450 and drug and steroid metabolizing activities in adulthood. Additional testosterone treatment in adulthood only slightly affected these parameters. All these results indicate that neonatal androgen imprinting on sex-dependent cytochrome P-450 and drug and steroid metabolizing activities in rat liver microsomes is not a permanent programming process and is modified by the presence and absence of sex-steroid hormones.     

Differential selectivity in carbamazepine-induced inactivation of cytochrome P450 enzymes in rat and human liver

Oxidative metabolism of carbamazepine results in covalent binding of its reactive metabolite to liver microsomal proteins, which has been proposed as an important event in pathogenesis of the hypersensitivity reactions to this drug. Although the proposed reactive metabolites are produced by cytochrome P450 enzymes (P450 or CYP), the impact of the formation of unstable metabolites on the enzyme itself has not been elucidated. The present study examines the alteration of P450 enzyme activities during the metabolism of carbamazepine. Liver microsomes from rats and humans were preincubated with carbamazepine in the presence of NADPH, and subsequently assayed for monooxygenase activities representing several P450s. No evidence was obtained for inactivation of CYP2C11, CYP3A, CYP1A1/2 or CYP2B1/2 in rat liver microsomes during the carbamazepine metabolism, whereas the CYP2D enzyme was inactivated in a manner related to the preincubation time. Interestingly, under the same protocol human liver microsomes did not exhibit inactivation of CYP2D6, as well as there being no CYP2C8, CYP2C9 or CYP3A4 inactivation, whereas CYP1A2 was inactivated. Reduced glutathione could not protect against the observed inactivation of the P450s. These results suggest that CYP2D enzyme(s) in rats and CYP1A2 in humans biotransform carbamazepine into reactive metabolites, resulting in inactivation of the enzyme themselves, and raise the possibility that the P450 isoforms participate in toxicity induced by the drug in both animal species.