Benzphetamine N-demethylase Activity Research Articles

녹차가 다이옥신계 TCDD(2,3,7,8-tetrachlorodibenzo-p-dioxin)에 노출된 흰쥐의 지질대사 및 간 독성물질대사에 미치는 영향

This study investigated the effects of green tea on the disorders of lipid metabolism, oxidative system and hepatic functions induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), using adult male rats (SD) for 3 weeks. These 36 animals were divided into four groups. TCDD (50 ㎍/㎏ BW) was intraperitoneally injected at the beginning of experiment. Green tea powder was added 1% or 3% levels in basal diets respectively. Relative weights of thymus were decreased about one-third of control group, but those of liver, brain and testis were significantly increased in rats treated TCDD. Neutrophill% and lymphocyte% by TCDD treatment was improved by green tea diets. In liver functional enzyme, elevation of glutamic oxaloacetic transaminase (GOT) and alkaline phosphatase (ALP) activities due to TCDD treatment was lowered by green tea diets. The concentrations of serum and liver lipids were significantly increased by TCDD treatment, however, those of serum and liver triglyceride tended to decrease by green tea diets. Fecal lipid excretion was increased in rats fed green tea diets. Especially, fecal total cholesterol level was significantly elevated by 3% green tea diets. The activities of superoxide dismutase (SOD) were increased in rats fed 3% green tea diets. Increment of benzphetamine N-demethylase (BPND) activity by TCDD treatment was declined by 1% green tea diets. These results indicate that green tea can exert improving effects on liver lipid accumulation and unfavorable hepatic functions, and elevate antioxidation.

Differential effects of diabetes on CYP2E1 and CYP2B4 proteins and associated drug metabolizing enzyme activities in rabbit liver

The effects of diabetes on cytochrome P450 (CYP)-dependent drug metabolizing enzymes are yet to be clarified. The most widely used animals in these studies have been rats, and information on the effects of diabetes on rabbit liver drug metabolizing enzymes have been unavailable until now. In this study, for the first time, a significant induction of liver CYP2E1 is demonstrated via immunoblot analysis in alloxan-induced rabbits. The CYP2E1 content of diabetic microsomes was highly correlated with the activities of liver aniline 4-hydroxylase (r=0.82, p<0.05), and p-nitrophenol hydroxylase (r=0.86, p<0.01), and diabetes increased the activities of the enzymes associated with CYP2E1. The activities of aniline 4-hydroxylase and p-nitrophenol hydroxylase were significantly increased by 1.7 and 1.8-fold, respectively compared to those of control rabbits. In marked contrast, diabetes had no effect on the protein levels of CYP2B4 as determined by immunoblotting and on benzphetamine N-demethylase activity, which is known to be specifically metabolized by CYP2B4 in rabbit liver. The present study demonstrates that diabetes increases the activities of CYP2E1 and associated enzymes but does not change the activity levels of CYP2B4 and associated enzymes in diabetic rabbits. These findings are in contrast to those of mice, hamsters and rats, and that suggest the presence of species-dependent responses of CYP-dependent drug metabolizing enzymes to diabetes.

Effect of 4-(4-chlorobenzyl)pyridine on rat hepatic microsomal cytochrome P450 and drug-metabolizing enzymes in vivo and in vitro.

The effect of 4-(4-chlorobenzyl)pyridine (4-CBP) on rat hepatic microsomal cytochrome P450 (P450) and its molecular species (CYP2B1, 2E1, 3A2, 2C11, and 2C12), and on drug-metabolizing enzyme activities were examined in vivo and in vitro. Treatment of rats with 4-CBP resulted in the induction of P450 and drug-metabolizing enzymes in a dose-dependent manner, but it was markedly inhibitory at higher dose levels. Immunoblot analyses revealed that 4-CBP induces both CYP2B1 and 2E1; however, both were decreased by increasing the dose of 4-CBP. The in vitro inhibitory experiment revealed that 4-CBP strongly inhibited benzphetamine N-demethylase activity, but not dimethylnitrosamine N-demethylase activity. The present findings provide information on the induction and inhibition effect of chlorinated benzylpyridine on hepatic microsomal P450s and drug-metabolizing enzymes in vivo and in vitro.

Bimodal Expression of CYP2D16 in the Guinea Pig Adrenal Cortex

Prior studies revealed an apparent bimodal distribution of adrenal xenobiotic-metabolizing activities in outbred guinea pigs. High activities were characteristic of most animals but a minority had very low rates of metabolism. The present studies were done to determine the basis for this distribution. Among the enzymatic activities abundant in guinea pig adrenal microsomes is bufuralol 1′-hydroxylation, a marker for CYP2D isozymes. Western blot analyses revealed that adrenal expression of CYP2D16, the guinea pig CYP2D analogue, was highly correlated with xenobiotic-metabolizing activities. Of 12 animals studied, 4 had low levels of adrenal CYP2D16 expression and correspondingly low bufuralol 1′-hydroxylase and benzphetamine N-demethylase activities. In contrast, adrenal expression of steroidogenic P450 isozymes (CYP17 and CYP21) and the corresponding steroid hydroxylase activities were similar in all animals. There was also no evidence of polymorphism in hepatic xenobiotic metabolism. The results suggest that the bimodal expression of adrenal CYP2D16 is at least partly responsible for the variability in adrenal xenobiotic metabolism, and that this pattern of expression is not applicable to other adrenal P450 isozymes or to hepatic CYP2D16. Further studies are needed to determine if genetic polymorphism is responsible for the mode of adrenal CYP2D16 expression.

Effect of hyperoxia on rat pulmonary and hepatic cytochrome P450 monooxygenases.

Exposure of adult male rats to hyperoxia (O(2) > 95%) resulted in a tendency for all of the components of the pulmonary cytochrome P450 (P450) system to increase at 48 h after the exposure. However, the most pronounced effect of hyperoxia was observed on pulmonary ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activities which were induced 4- and 25-fold respectively after 48 h. In the liver, P450 and NADH b(5) reductase were increased after 48 h, while other components of the monooxygenase system remained unchanged. In the hepatic microsomes, contrary to the lungs, aminopyrine N-demethylase activity was decreased after 24 h of hyperoxic exposure (P < 0.05) and returned to the control level by 48 h. Similar changes were observed in benzphetamine N-demethylase activity. Aniline hydroxylase activity was decreased after 8 h of hyperoxic exposure (P < 0.01) and remained decreased at 24 h (P < 0. 01) and 48 h (P < 0.05). The level of induction of ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activities, however, was almost similar in the liver to that observed in the lungs.

Identification, Functional Characterization, and Regulation of a New Cytochrome P450 Subfamily, the CYP2Ns

The screening of liver and heart cDNA libraries from the teleost Fundulus heteroclitus with degenerate oligonucleotide probes to conserved alpha-helical regions in mammalian P450s resulted in the identification of two cDNAs that together represent a novel P450 subfamily, the CYP2Ns. Northern analysis demonstrated that CYP2N1 transcripts are most abundant in liver and intestine, whereas CYP2N2 mRNAs are most abundant in heart and brain. CYP2N1 and CYP2N2 proteins were co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 insect cells, and their ability to metabolize arachidonic acid and xenobiotic substrates was examined. Both CYP2N1 and CYP2N2 metabolize arachidonic acid to epoxyeicosatrienoic acids. Epoxidation is highly regio- and enantioselective with preferential formation of (8R,9S)-epoxyeicosatrienoic acid (optical purities are 91 and 90% for CYP2N1 and CYP2N2, respectively) and (11R, 12S)-epoxyeicosatrienoic acid (optical purities are 92 and 70% for CYP2N1 and CYP2N2, respectively). CYP2N1 and CYP2N2 also catalyze the formation of a variety of hydroxyeicosatetraenoic acids. Both P450s have benzphetamine N-demethylase activities but show minimal alkoxyresorufin O-dealkylase activities. To investigate factors affecting CYP2N expression in vivo, CYP2N transcripts were examined following starvation and/or treatment with 12-O-tetradecanoyl phorbol-13-acetate. Intestinal CYP2N1 mRNAs decrease in starved and/or phorbol ester-treated fish, whereas intestinal CYP2N2 transcripts decrease only following phorbol ester treatment. Interestingly, cardiac CYP2N2 expression decreases following phorbol ester treatment but increases following starvation. These results demonstrate that members of this novel P450 subfamily encode early vertebrate forms of arachidonic acid catalysts that are widely expressed and are regulated by environmental factors. Given the wealth of information on the functional role of P450-derived arachidonate metabolites in mammals, we postulate that CYP2N1 and CYP2N2 products have similar biological functions in early vertebrates. The identity of the mammalian orthologue(s) of the CYP2Ns remains unknown.

The effects of antirhino- and enteroviral vinylacetylene benzimidazoles on cytochrome P450 function and hepatic porphyrin levels in mice

In an ongoing effort to identify an orally bioavailable compound for the treatment of rhino- and enteroviral infections, a series of vinylacetylene benzimidazoles was recently examined. Previous studies demonstrated the potential for these compounds to possess both good in vitro antiviral activity as well as acceptable oral plasma concentrations in mice. Optimization of these properties led to four compounds as candidates for further evaluation. In view of the recognized potential for certain acetylenic drugs both to inhibit cytochrome P450 enzymes by mechanism-based inactivation and to possibly perturb heme metabolism, information regarding drug effects on cytochromes P450 and hepatic porphyrin levels was sought. In an initial single-dose pharmacokinetic study, the four selected compounds were given orally to mice, and both plasma concentrations and porphyrin levels were determined. Two of the compounds, 4 and 5, caused a pronounced increase in liver porphyrin levels whereas compounds 6 and 7 exhibited almost no effect on porphyrin levels. Analysis of plasma concentrations showed that only 4 and 5 gave significant exposure and that 6 and 7 produced negligible levels of drug in the plasma even at the highest dose tested (500 mg/kg). A multiple dose study was then initiated in which compounds 4 and 5 were given for 1 week in daily oral doses to mice. Upon completion of dosing, liver was analyzed for cytochrome P450-dependent 7-ethoxyresorufin O-deethylase (EROD) and benzphetamine N-demethylase (BND) activities, total cytochrome P450 content, and porphyrin levels. Both vinylacetylenes showed dose–dependent inhibitory and induction effects on EROD and BND activities. In addition, these compounds caused a marked increase in hepatic porphyrin levels. Therefore, while all four selected compounds displayed potent antiviral activity and two of the compounds exhibited acceptable pharmacokinetic properties, the hepatic effects of these latter two compounds suggest the potential for drug induced porphyria with multidose therapeutic use.

Trimethadione metabolism and microsomal monooxygenases in untreated and phenobarbital-treated rhesus monkeys

The contribution of induced cytochrome P450 (P450) isozymes (CMLa; CYP2B, CMLb; CYP2A and CMLc; CYP3A) and related enzymes to trimethadione (TMO) metabolism in phenobarbital-treated rhesus monkey were investigated. The animals received a single dose of TMO (4 mg kg −1) and plasma samples were withdrawn before this administration and again at 0.08, 0.25, 0.5, 1 and 2 h later. Phenobarbital-treatment (20 mg kg −1 day −1 for 3 days; i.p.) significantly increased the plasma dimethadione (DMO)/TMO ratios at 0.08, 0.5, 1 and 2 h one’s appropriate controls. Phenobarbital treatment also increased the P450 content (1.7-fold) and activity of aniline p-hydroxylase (1.3-fold), p-nitroanisole O-demethylase (1.8-fold) and benzphetamine N-demethylase (2.3-fold). The content of CMLa, CMLb and CMLc were increased about 12.8, 2.3 and 2.7-fold by phenobarbital pretreatment, respectively. The activity of TMO N-demethylation was inhibited by anti-P450 CMLa and anti-P450 CMLb. However, the anti-P450 CMLc antibody had no effect on this activity in liver microsomes. The results of both in vivo and in vitro studies of the effects of phenobarbital treatment on TMO metabolism indicate that these effects may be attributed to the induction of CMLa. These findings suggest that plasma DMO/TMO ratio in a single blood sampling after TMO administration is very useful for determination the degree of hepatic induction in clinical study.

Time-dependent effects of Klebsiella pneumoniae endotoxin on hepatic drug-metabolizing enzyme activity in rats.

The time-dependent effects of Klebsiella pneumoniae endotoxin on hepatic cytochrome P450-dependent drug-metabolizing capacity (cytochrome P450 and b5 content, activity of aminopyrine N-demethylase, p-nitroanisole O-demethylase, aniline hydroxylase and benzphetamine N-demethylase) and on the pharmacokinetics of antipyrine have been determined in rats. Measurement of enzyme activity and antipyrine (after intravenous injection of 20 mg kg(-1)) were performed 2, 24 and 96 h after a single intraperitoneal injection of endotoxin (1 mg kg(-1)) and after repeated doses (once daily for 4 days). The contribution of tumour necrosis factor alpha (TNFalpha) to the endotoxin-induced changes was also examined in rats pretreated with granulocyte colony-stimulating factor (G-CSF). The systemic clearance of antipyrine and the activity of hepatic cytochrome P450-dependent drug-metabolizing enzymes were dramatically reduced 24 h after a single injection of endotoxin, but had returned to control levels by 96h. The magnitudes of these decreases in these measurements after repeated doses of endotoxin were similar to those seen 24h after the single dose. The systemic clearance of antipyrine correlated significantly with cytochrome P450 content and aminopyrine N-demethylase activity. In histopathological experiments, moderate hypertrophy of Kupffer cells was observed, with no evidence of severe liver-tissue damage. G-CSF pretreatment suppressed the increased plasma concentrations of TNFalpha produced 2 h after single endotoxin injection, but did not eliminate the endotoxin-induced decrease in the systemic clearance of antipyrine, suggesting that TNFalpha is not the sole component responsible for the reduction of cytochrome P450-mediated drug-metabolizing enzyme activity. These results provide evidence that a single intraperitoneal injection of 1.0 mgkg(-1)K. pneumoniae endotoxin in rats reduces hepatic P450 and b5 levels, and reduces the activity of various cytochrome P450-mediated drug-metabolizing enzymes without causing severe liver-tissue damage. This suggests that the effect of endotoxin on hepatic cytochrome P450-mediated drug-metabolizing isozymes is non-selective.

Suppression of male-specific cytochrome P450 isoforms by bisphenol A in rat liver.

We examined the effect of bisphenol A (BPA) on microsomal cytochrome P450 (P450) enzymes in rats. Rats were treated intraperitoneally with BPA daily for 4 days, at doses of 10, 20, and 40 mg/kg. Among the P450-dependent monooxygenase activities, testosterone 2alpha-hydroxylase (T2AH) and testosterone 6beta-hydroxylase (T6BH) activities, which are associated with CYP2C11 and CYP3A2 respectively, were remarkably decreased by 40 mg/kg BPA. The levels of the control activities were 13 and 50%, respectively. Furthermore, immunoblotting showed that BPA (20 or 40 mg/kg) significantly reduced CYP2C 11/6 and CYP3A2/1 protein levels in rat liver microsomes. In addition, estradiol 2-hydroxylase (ED2H) and benzphetamine N-demethylase (BZND) activities were significantly decreased by BPA at 20 and 40 mg/kg (by 19-73%). The Km values for T2AH and T6BH in 20 and 40 mg/kg BPA-treated rats were significantly high compared with that in control rats. The Vmax for T2AH was dose-dependently decreased by BPA treatment, whereas that of T6BH was only decreased by BPA at 40 mg/kg. On the other hand, lauric acid omega-hydroxylase (LAOH) activity was significantly increased by BPA at 20 and 40 mg/kg (1.5- and 1.7-fold, respectively). Immunoblot analysis showed that 20 and 40 mg/kg BPA induced CYP4A1/2 protein expression. However, the activities 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), chlorzoxazone 6-hydroxylase (CZ6H), erythromycin N-demethylase (EMND), and testosterone 7alpha-hydroxylase (T7AH) were not affected by BPA at any dose. These results suggest that BPA affects male-specific P450 isoforms in rat liver, and that these changes closely relate to the toxicity of BPA.

Effects of cytochrome P450 inducers on tamoxifen genotoxicity in female mice in vivo

We recently reported that administration of the antiestrogen tamoxifen (TAM) gives rise to two groups of DNA adducts in female mouse liver in vivo, as measured by 32P-postlabeling, and provided evidence that 4-hydroxytamoxifen and α-hydroxytamoxifen are proximate carcinogenic metabolites leading to group I and group II adducts, respectively (Randerath et ed., Carcinogenesis 15: 2087-2094, 1994). Because cytochrome P450 (CYP) enzymes play an important role in TAM metabolism, in this investigation we tested the hypothesis that induction of liver CYP enzymes may affect TAM metabolism profoundly, resulting in increased or decreased TAM-DNA adduct formation in vivo. To this end, we treated female ICR mice with TAM either alone or in combination with one of several classic CYP inducers, i.e. phenobarbital (PB), ß-naphthoflavone (BNF), and pregnenolone-16α-carbonitrile (PCN), and determined the levels of 32P-postlabeled TAM-DNA adducts and the activities of several CYP-dependent enzymes. Each of the inducers greatly diminished levels of group II, but did not affect group I adducts. TAM elicited induction of benzphetamine N-demethylase activity in liver, while activities of other enzymes were not affected. TAM, when given in combination with BNF, elicited a synergistic induction of ethoxyresorufin O-deethylase (EROD) (CYP1A1) and methoxyresorufin O-demethylase (MROD) (CYP1A2) activities. Likewise, PCN given along with TAM caused synergistic induction of EROD and ethylmorphine N-demethylase activities. There was no synergism between PB and TAM, however. Overall, the results further support the existence of two pathways of TAM metabolism to DNA-reactive electrophiles and strongly suggest that the classic CYP inducers tested enhance detoxication of TAM to non-genotoxic metabolites.

Distinct responses of mouse hepatic CYP enzymes to corn oil and peroxisome proliferators

We studied the response of male DBA/2N mouse liver monooxygenases to acute (one-day) and subacute (7-day) exposure to clofibrate, gemfibrozil, and com oil. The day following a single treatment with clofibrate (200 mg/kg), coumarin 7-hydroxylase (COH) activity decreased significantly (by 70%) with a concomitant decrease in the CYP2A4/5 protein and mRNA levels. The 7-day treatment schedule also decreased COH activity but only by 30%, though the levels of CYP2A4/5 protein and mRNA were still low. Treatment 1 and 7-day with clofibrate decreased 7-pentoxyresorufin O-dealkylase (PROD) activity by 40%. No changes were seen in testosterone 15α-hydroxylase (TISαOH) activity after 1 day of treatment with clofibrate but, after 7 days, it was decreased by 50%. Clofibrate treatment had no significant effects on testosterone 7α-hydroxylase (T7αOH), 7-ethoxyresorufin O-deethylase (EROD), or benzphetamine N-demethylase (BZDM) activities. Gemfibrozil (200 mg/kg) did not alter COH activity or CYP2A4/5 protein content after a single treatment, but a slight decrease was seen in the mRNA level. Treatment for 7 days significantly increased (2.5-fold) the activity and mRNA content but the amount of protein remained unchanged. Gemfibrozil enhanced (2–2.7-fold) PROD and EROD (2–2.5-fold) activities by both treatments, whereas T15αOH, T7αOH, or BZDM activities were not significantly affected. Treatment with corn oil for 7 days significantly decreased (65%) COH activity and CYP2A4/5 protein and mRNA levels. PROD (55%) and T15αOH (65%) activities were significantly decreased even after a single dose although injection for 7 days had no effect. Neither of the corn oil schedules had any marked effect on T7αOH, EROD, or BZDM activities. These results demonstrate: 1. a decrease in the expression of CYP2A4/5 gene by clofibrate and corn oil; 2. substantial differences within the CYP2A subfamily in their responses to corn oil, clofibrate, and gemfibrozil; and 3. distinct responses of other xenobiotic metabolizing CYP subfamily enzymes to clofibrate and gemfibrozil.

Effect of Chronic Exposure to Ozone and Nitric Acid on Cytochrome P-450 Monooxygenase System of Rat Lung and Liver

AbstractMale F344/N rats were exposed to 0.15 ppm ozone (O3) and 50 μg/m3 nitric acid (HNO3) vapor alone and in combination for 4 h/day, 3 days/wk for a total of 40 wk to investigate the effects of these environmental pollutants on cytochrome P-450 monooxygenases in pulmonary and hepatic microsomes. Exposure to HNO3 vapor alone caused a significant increase of 7-ethoxyresorufin O-deethylase (EROD) activity in the hepatic microsomes (p < .01). Immunoinhibition assays with monoclonal anti-cytochrome P-450 (CYP) 1A1/1A2 (1-7-1) showed that the monoclonal antibody-inhibitableEROD activity in the hepatic microsomes was increased by 59% (p < .01) and 40% (p < .05), respectively, in the HNO3 vapor and O3 plus HNO3 vapor groups. Benzphetamine N-demethylase activity was not significantly affected by exposure to either O3 or HNO3 vapor in the hepatic microsomes. However, in the lung, O3 exposure caused a 72% increase in benzphetamine N-demethylase activity (p < .01), whereas this activity was decreased by 15% by HN...

Species differences in the renal toxicity of the antiarthritic drug, gold sodium thiomalate.

Two gold compounds, gold sodium thiomalate (AuTM) and auranofin, are presently in clinical use in therapy of rheumatoid arthritis. In these studies, AuTM administered to Sprague-Dawley rats and three strains of mice, Swiss-Webster, C3H/HeJ, and DBA/2J, were studied with regard to its effect on liver and renal monooxygenases, metallothionein contents, and serum levels of alanine aminotransferase and urea nitrogen. These effects of AuTM were compared to those of cadmium, since the latter metal has exhibited tissue and species differences in the induction of metallothionein. Benzo(a)pyrene hydroxylase and benzphetamine N-demethylase activities were not altered by AuTM in livers of rats and the three strains of mice. Benzo(a)pyrene hydroxylase activity was significantly decreased in rat kidney, whereas this enzyme activity was not affected in the kidneys of mice. In rats, AuTM caused a sevenfold induction in liver metallothionein, while in mice, liver metallothionein was induced twofold in Swiss-Webster mice and about fivefold in the inbred strains. AuTM caused minimal changes in renal metallothionein contents in the three strains of mice studied. Serum alanine amino-transferase, an indicator of hepatotoxicity, was not altered by AuTM in rats and mice studied. Blood urea nitrogen, an indicator of kidney dysfunction, was increased threefold in rats, but not in AuTM-treated mice. These data demonstrate that AuTM, a nephrotoxic agent in rats and humans, showed no nephrotoxic effects in the mouse strains studied here.

Ketone potentiation of haloalkane-induced hepato- and nephrotoxicity. II. Implication of monooxygenases.

Previous results in Sprague-Dawley rats indicate that acetone (A), methyl ethyl ketone (MEK), and methyl isobutyl ketone (MiBK) pretreatment (3 d, po) at dosages of 6.8 and 13.6 mmol/kg potentiate CCl4 hepatotoxicity and CHCl3 nephrotoxicity, respectively. The potentiation potency profile observed was MiBK > A > MEK for liver and A > MEK > or = MiBK for kidney toxicity (Raymond & Plaa, 1995). In the present study, hepatic and renal microsomes from A-, MEK-, and MiBK-pretreated rats (6.8 or 13.6 mmol/kg) were examined for cytochrome P-450 content, substrate-specific monooxygenase activity (aminopyrine and benzphetamine N-demethylase, aniline hydroxylase) and in vitro covalent binding of 14CHCl3 and 14CCl4. Of the three ketones, only MiBK significantly increased P-450 content of liver and renal cortical microsomes. Similarly, 14CCl4 covalent binding under aerobic and anaerobic conditions was significantly increased by MiBK pretreatment only. 14CHCl3 covalent binding by renal cortical microsomes was significantly increased only under aerobic conditions by MiBK pretreatment. MiBK (13.6 mmol/kg) increased (threefold) aminopyrine N-demethylation in both liver and kidney, but only benzphetamine N-demethylation (two-fold, at 6.8 and 13.6 mmol/kg) in liver; A and MEK had no effect on either monooxygenase. All ketones at dosages of 6.8 and 13.6 mmol/kg increased aniline hydroxylation in liver (two-fold) and kidney (fivefold). Comparable profiles for P-450 induction, haloalkane covalent binding, and aminopyrine or benzphetamine N-demethylase activity were observed in liver and kidney microsomes. This profile was consistent with the ketone potentiation potency ranking profile observed in vivo for liver but not kidney injury. These findings affirm the importance of ketone-enhanced bioactivation for potentiation of CCl4 hepatotoxicity but suggest an alternative mechanism for CHCl3 nephrotoxicity.

Role of Acidic Residues in the Interaction of NADPH-Cytochrome P450 Oxidoreductase with Cytochrome P450 and Cytochrome c

Site-directed mutagenesis of the acidic clusters 207Asp-Asp-Asp209 and 213Glu-Glu-Asp215 of NADPH-cytochrome P450 oxidoreductase demonstrates that both cytochrome c and cytochrome P450 interact with this region; however, the sites and mechanisms of interaction of the two substrates are clearly distinct. Substitutions in the first acidic cluster did not affect cytochrome c or ferricyanide reductase activity, but substitution of asparagine for aspartate at position 208 reduced cytochrome P450-dependent benzphetamine N-demethylase activity by 63% with no effect on KP450m or KNADPHm. Substitutions in the second acidic cluster affected cytochrome c reduction but not benzphetamine N-demethylase or ferricyanide reductase activity. The E213Q enzyme exhibited a 59% reduction in cytochrome c reductase activity and a 47% reduction in KCyt cm under standard conditions (x0.27 M potassium phosphate, pH 7.7), as well as a decreased KCyt cm at every ionic strength and a shift of the salt dependence of cytochrome c reductase activity toward lower ionic strengths. The E214Q substitution did not affect cytochrome c reductase activity under standard conditions, but shifted the salt dependence of cytochrome c reductase activity toward higher ionic strengths. Measurements of the effect of ionic strength on steady-state kinetic properties indicated that increasing ionic strength destabilized the reductase-cytochrome c3+ ground state and reductase-cytochrome c transition state complexes for the wild-type, E213Q, and E214Q enzymes, suggesting the presence of electrostatic interactions involving Glu213 and Glu214 as well as additional residues outside this region. The ionic strength dependence of kcat/KCyt cm for the wild-type and E214Q enzymes is consistent with the presence of charge-pairing interactions in the transition state and removal of a weak ionic interaction in the reductase-cytochrome c transition-state complex by the E214Q substitution. The ionic strength dependence of the E213Q enzyme, however, is not consistent with a simple electrostatic model. Effects of ionic strength on kinetic properties of E213Q suggest that substitution of glutamine stabilizes the reductase-cytochrome c3+ ground-state complex, leading to a net increase in activation energy and decrease in kcat. Glu213 is also involved in a repulsive interaction with cytochrome c3+. Cytochrome c2+ Ki for the wild-type enzyme was 82.4 microM at 118 mM ionic strength and 10.8 microM at 749 mM ionic strength; similar values were observed for the E214Q enzyme. Cytochrome c Ki for the E213Q enzyme was 17.6 microM at 118 mM and 15.7 microM at 749 mM ionic strength, consistent with removal of an electrostatic repulsion between the reductase and cytochrome c2+.

Different influences of two fractions of lung cytochrome b5 on reconstituted lung benzphetamine N-demethylase system

Chromatography of lung microsomal cytochrome b5 obtained from DEAE-cellulose columns, yielded two distinct cytochrome b5 fractions. These cytochrome b5 fractions were further purified by Sephadex G-100 gel filtration chromatography. The specific cytochrome b5 content of fraction 1 and fraction 2 was found to be 16.5 and 16.4 nmol/mg protein respectively. Both fractions were free of cytochrome P-450, NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase activities. The effects of lung cytochrome b5 (fraction 1 and fraction 2) and liver cytochrome b5 on benzphetamine N-demethylase activity were examined. Four different reconstitution systems were used. Lung cytochrome b5 fraction 2 and liver cytochrome b5 stimulated N-demethylase activity in all four systems when b5: P-450 molar ratio was low, i.e. 0.25 or 0.5. Both cytochrome b5 samples inhibited N-demethylase activity when b5: P-450 ratio exceeded 1:1 molar ratio. In contrast lung cytochrome b5 fraction 1 stimulated N-demethylase activity in all four systems. Maximal enhancement of the activity was obtained when b5: P-450 ratio was 0.5. The greatest increase in N-demethylation activity was observed in the reconstitution system with the lowest concentration of cytochrome P-450 reductase, conditions which most closely resemble intact microsomes.

Inhibition of adrenal steroid metabolism by administration of 1-aminobenzotriazole to guinea pigs.

Prior in vitro investigations demonstrated that the P450 suicide substrate, 1-aminobenzotriazole (ABT), was a potent inhibitor of xenobiotic metabolism but had no effect on steroidogenic enzymes in the guinea pig adrenal cortex. Studies were done to determine if ABT administration of guinea pigs in vivo also selectively inhibited adrenal xenobiotic metabolism. At single doses of 25 or 50 mg/kg, ABT effected rapid decreases in spectrally detectable adrenal P450 concentrations. The higher dose caused approx. 75% decreases in microsomal and mitochondrial P450 levels within 2 h. The decreases in P450 were sustained for 24 h but concentrations returned to control levels within 72 h. Accompanying the ABT-induced decreases in adrenal P450 content were proportionately similar decreases in P450-mediated xenobiotic and steroid metabolism. Microsomal benzo(a)pyrene hydroxylase, benzphetamine N-demethylase, 17 alpha-hydroxylase and 21-hydroxylase activities were decreased to 20-25% of control values by the higher dose of ABT. Mitochondrial 11 beta-hydroxylase and cholesterol sidechain cleavage activities were similarly diminished by ABT treatment. Adrenal 3 beta-hydroxysteroid dehydrogenase activity, by contrast, was not affected by ABT, indicating specificity for P450-catalyzed reactions. The results demonstrate that ABT in vivo is a non-selective inhibitor of adrenal steroid- and xenobiotic-metabolizing P450 isozymes. The absence of ABT effects on steroid metabolism in vitro suggests that an extra-adrenal metabolite may mediate the in vivo inhibition of steroidogenesis.

Escherichia coli Expression of Site-Directed Mutants of Cytochrome P450 2B1 from Six Substrate Recognition Sites: Substrate Specificity and Inhibitor Selectivity Studies

Cytochrome P450 2B1 wild-type and eight site-directed mutations at positions 114, 206, 236, 302, 363, 367, and 478 have been expressed in an Escherichia coli system. Solubilized membrane preparations yielded 100-180 nmol of P450/L of culture. The metabolism of a number of substrates including androstenedione, progesterone, (benzyloxy)resorufin, pentoxyresorufin, and benzphetamine was analyzed. The E. coli-expressed enzymes displayed the same androstenedione metabolite profiles previously observed with a COS cell expression system. Several of the mutants exhibited an increased rate of progesterone hydroxylation, possibly as the result of an enlarged substrate binding pocket and increased D-ring alpha-face binding. (Benzyloxy)resorufin and pentoxyresorufin O-dealkylation by the P450 2B1 mutants exhibited activities ranging from 10% to 99% and 3% to 71% of wild-type, respectively. Interestingly, the Val-363-->Leu mutant showed markedly suppressed pentoxyresorufin but unaltered (benzyloxy)resorufin dealkylase activity. Benzphetamine N-demethylase activities ranged from 28% to 110% of wild-type. Mechanism-based inactivation of the P450 2B1 mutants showed that susceptibility to inactivation by chloramphenicol and D-erythro- and L-threo-chloramphenicol was abolished in the Val-367-->Ala mutant. The Val-363-->Leu mutant was refractory to L-threo-chloramphenicol. Studies of chloramphenicol covalent binding and metabolism by the Val-367-->Ala mutant showed that its resistance to inactivation is largely attributable to an inability to bioactivate the inhibitor. The expression of P450 2B1 wild-type and mutants in E. coli provides an excellent opportunity to study structure/function relationships by site-directed mutagenesis.

Inactivation of chick embryo hepatic cytochrome P450 1A, 2H and 3A following in ovo administration of 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine and 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone

Rat hepatic cytochrome P450 (P450) isozymes 1A1, 2C6, 2C11, 3A1 and 3A2 are targets for mechanism-based inactivation by the porphyrinogenic compound 3,5-diethoxycarbonyl-1,4-dihydro-2, 6-dimethyl-4-ethylpyridine (4-ethyl DDC). It is of interest to determine whether similar P450 isozymes are targets of porphyrinogenic drugs in the chick embryo liver. The chick embryo expresses P450 2H1/2 isozymes, which are similar to the rat P450 2B1/2 isozymes, a polycyclic aromatic hydrocarbon-inducible P450 1A isozyme, and a pregnenolone 16α-carbonitrile-inducible P450 3A isozyme. We have found previously that chick embryo hepatic P450 1A and 3A isozymes are targeted for in vitro mechanism-based inactivation by 4-ethyl DDC and by the sydnone 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS). Marked differences have been observed between the in vitro and in vivo effects of porphyrinogenic drugs on P450 isozymes. Thus, the first objective of this study was to determine whether chick embryo hepatic P450 1A and 3A isozymes are subject to in ovo inactivation by these porphyrinogenic compounds. Our second objective was to determine whether the chick embryo hepatic P450 2H isozyme(s) was subject to in ovo and in vitro inactivation by 4-ethyl DDC and TTMS. Using hepatic microsomes prepared from β-naphthoflavone-, dexamethasone-, phenobarbital-, and glutethimide-induced 19-day-old chick embryos, we found that total P450 content was decreased significantly in microsomes prepared from all treatment groups following in ovo administration of 4-ethyl DDC and TTMS. Moreover, in ovo administration of both 4-ethyl DDC and TTMS caused a significant decrease of 7-ethoxyresorufin O-deethylase, erythromycin N-demethylase, and benzphetamine N-demethylase activities, which are selective catalytic markers for chick embryo hepatic P450 1A, 3A and 2H isozymes, respectively. In addition, in vitro administration of 4-ethyl DDC and TTMS caused mechanism-based inactivation of benzphetamine N-demethylase activity in microsomes from phenobarbital- and glutethimide-treated chick embryos, showing that the chick embryo hepatic P450 2H isozyme is a target for mechanism-based inactivation. Therefore, it was concluded that the chick embryo hepatic P450 1A, 2H and 3A isozymes serve as targets for both in ovo and in vitro mechanism-based inactivation by 4-ethyl DDC and TTMS.