N-demethylase Activity Research Articles

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 The animals received a single dose of TMO (4 mg kg −1) and plasma samples were withdrawn before this administration and again at 0.08, 0.25, 0.5, 1 and 2 h later.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.

P3B65 - Increase in renal metabolizing enzyme activities induced by heapatic damage

1. The drug-metabolizing activities (aminopyrine N-demethylase and p-nitroanisole O-demethylase activities) have been measured at monthly intervals throughout the year in liver microsomes of male and female Japanese bullfrogs, Rana catesbeiana.2. The aminopyrine n-demethylase activity based on cytochrome P-450 of both sexes was significantly higher in May–July (spring-summer) than in August–October (summer-autumn).3. On the contrary, the p-nitroanisole O-demethylase activity based on cytochrome P-450 of males was significantly higher in July–October (summer-autumn) than in May–June (spring). However, there was no seasonal changes in the O- demethylase activity in females.4. There were significant differences between males and females in the n-demethylase activity in August–October and in the O -demethylase activity in May–June (or July–October).

Effect of the degree of hydrogenation of dietary fish oil on the trans fatty acid content and enzymatic activity of rat hepatic microsomes.

The degree of fat hydrogenation and the trans fatty acid content of the diet affect the fatty acid composition of membranes, and the amount and the activity of some membrane enzymes. We describe the effects of four isocaloric diets containing either sunflower oil (SO, 0% trans), fish oil (FO, 0.5% trans), partially hydrogenated fish oil (PHFO, 30% trans), or highly hydrogenated fish oil (HHFO, 3.6% trans) as fat sources on the lipid composition and the trans fatty acid content of rat hepatic microsomes. We also describe the effect of these diets on the cytochrome P-450 content and on the aminopyrine N-demethylase, aniline hydroxylase, and UDP-glucuronyl transferase microsomal activities. Cytochrome P-450 content was dependent on the degree of unsaturation of the diet, being higher for the FO-containing diet and lower for the HHFO diet. Aminopyrine N-demethylase activity also correlated with the degree of unsaturation of the diet as did the cytochrome P-450 content did (FO > SO > PHFO > HHFO). Aniline hydroxylase activity appeared to be independent of the degree of unsaturation of the dietary fat, but correlated with the trans fatty acid content of the diet, which was also reflected in the trans content of the microsomal membranes. UDP-glucuronyl transferase activity was higher for the FO-containing diet than for the SO diet, showing intermediate values after the PHFO and HHFO diets.

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.

Temporal variation in hepatotoxicity and metabolism of acetaminophen in mice

Temporal variation in metabolism and hepatotoxicity of acetaminophen (APAP) was examined using male ICR mice. Animals were injected with a single dose of APAP (400 mg/kg, i.p.) at 08:00, 14:00 or 20:00 h. APAP at this dose was markedly hepatotoxic to mice when administered at 20:00 h as determined by increases in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and by decreases in hepatic glucose-6-phosphatase (G-6-Pase) activity. However, mice appeared to be entirely insensitive to an identical dose of APAP given either at 08:00 or 14:00 h. Hepatic glutathione (GSH) level was significantly higher at 08:00, but no difference in GSH levels between 14:00 and 20:00 h was observed in normal mice. APAP and its metabolites in blood were monitored using HPLC for 3 h following the treatment. There were no significant differences in the plasma concentrations of APAP, APAP-glucuronide, APAP-sulfate, or APAP-mercapturate among the mice treated with this drug at 08:00, 14:00 or 20:00 h. However, the APAP-cysteine and APAP-GSH levels measured at 1 h following the APAP treatment were significantly lower in mice treated with this analgesic either at 14:00 or 20:00 h. In vitro hepatic microsomal p-nitrophenol hydroxylase activities were not different between 08:00, 14:00 and 20:00 h. But ethoxyresorufin O-deethylase and aminopyrine N-demethylase activities measured at 14:00 h were significantly lower than those of 08:00 or 20:00 h. Thus, the greater hepatotoxicity of APAP administered at 20:00 h appears to be related to the marked decrease in hepatic GSH at this time period, whereas the simultaneous reduction in APAP activation may be responsible for the lack of hepatotoxicity in mice treated with this analgesic at 14:00 h. These results suggest that the temporal variation in hepatotoxicity and metabolism of APAP is determined by interactions of multiple factors including the hepatic GSH level and drug metabolizing activities.

Influence of Some Novel N-Substituted Azoles and Pyridines on Rat Hepatic CYP3A Activity

A series of N-substituted heteroaromatic compounds structurally related to clotrimazole was synthesized, and the effects of these compounds on ethosuximide clearance in rats were determined as a measure of their abilities to induce cytochrome P4503A (CYP3A) activity. Ethosuximide clearance and in vitro erythromycin N-demethylase activity were shown to correlate. In this series, imidazole or other related heteroaromatic “head groups” were linked to triphenylmethane or other phenylmethane derivatives. Within the series, it was found that 1-triphenylmethane-substituted imidazoles elicited the greatest increase in CYP3A activity, and that among the triphenylmethyl-substituted imidazoles, the highest activities were achieved by the substitution of F- or Cl- in either the meta or para position of one of the phenyl rings. Diphenylmethyl-substituted pyridine was effectively devoid of activity. Compounds eliciting the largest increase in CYP3A activity (viz. 1-[(3-fluorophenyl)diphenylmethyl]imidazole, 1-[(4-fluorophenyl)diphenylmethyl]imidazole, and 1-[tri-(4-fluorophenyl)methyl]imidazole) produced little or no increase in ethoxyresorufin O-dealkylase (EROD) activity (i.e. CYP1A), whereas benzylimidazole, which elicited only a small increase in CYP3A activity, produced an almost 9-fold increase in CYP1A activity. For a series of eleven compounds exhibiting a wide range of influence on CYP3A activity, a positive correlation was found between ethosuximide clearance and hepatic CYP3A mRNA levels.

Susceptibility of C57BL/6 mice to tumorigenicity induced by dimethylnitrosamine and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine in the neonatal bioassay

Male C57BL/6 neonates were treated on days 8 and 15 with 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP, 6.5 or 26.2 mg/kg) or dimethylnitrosamine (DMN, 2.6 or 10.5 mg/kg). No tumors were seen in PhIP-treated animals at 15 months of age. Liver and lung tumor incidences in DMN-treated animals were 67–79 and 0–7%, respectively. In comparison with data from other strains, our results indicate that (1) neonatally-treated C57BL/6 mice are resistant to the induction of liver and lung tumors by PhIP and lung tumors by DMN and (2) the susceptibility of this strain to induced liver tumors correlates with the activity of hepatic DMN N-demethylase and PhIP N-hydroxylase in the (untreated) neonates.

Modulation of Snake Hepatic Cytochrome P450 by 3-Methylcholanthrene and Phenobarbital

Induction mode of the hepatic drug-metabolizing enzymes was studied in Corn snake ( Elaphe guttata emoryi). Treatment of snakes with 3-methylcholanthrene or phenobarbital produced no effects on liver weight and total content of cytochromes P450 and b 5. Treatment with 3-methylcholanthrene significantly induced the activities of arylhydrocarbon hydroxylase, 7-ethoxyresorufin O-deethylase and 7-pentoxyresorufin O-dealkylase, whereas those of ethoxycoumarin O-deethylase, benzphetamine N-demethylase, erythromycin N-demethylase and testosterone hydroxylases were not affected. 3-Methylcholanthrene-induced activities of 7-ethoxyresorufin O-deethylase and 7-pentoxyresorufin O-dealkylase were inhibited by 20 μM α-naphthoflavone by 98% and 73%, respectively. Phenobarbital-treatment caused a significant induction of the activities of erythromycin N-demethylase and testosterone 6 β-hydroxylase, but did not affect those of the other phase I enzymes and the other testosterone hydroxylases. The activities of UDP-glucuronyltransferase and glutathione S-transferase were not affected by either 3-methylcholanthrene or phenobarbital administration. Immunoblotting showed that 3-methylcholanthrene-treatment induced a protein band related to hamster CYP1A2, and decreased the intensity of the two bands detected with anti-rat CYP2B1. Phenobarbital-treatment did not affect the intensity of CYP2B-related proteins. The results suggest that snake liver has multiple forms of cytochrome P450, notably those inducible by 3-methylcholanthrene.

Ethylmorphine N-demethylase activity as a marker for cytochrome P450 CYP3A activity in rat hepatic microsomes

The purpose of this study was to evaluate the selectivity and sensitivity of ethylmorphine N-demethylase (EMD) as an indicator of chemically-induced cytochrome P450 CYP3A activity in liver microsomes of rats following treatment with selective enzyme inducers. Male and female Sprague–Dawley (CD®) rats were dosed with either pregnenolone-16 α-carbonitrile (PCN; 50 mg/kg per day for 5 days), phenobarbital (PB; 100 mg/kg per day for 4 days), beta-naphthoflavone ( βNF; 100 mg/kg per day for 3 days), clofibrate (CF; 300 mg/kg per day for 14 days), isoniazid (ISO; 100 mg/kg per day for 3 days), or dexamethasone (DEX; 50 mg/kg per day for 4 days). Microsomes were isolated, frozen and subsequently assayed for protein, cytochrome P450 content and EMD activity. In males, significant elevations ( P<0.01) in EMD activity were observed in microsomes from PB-, DEX- and PCN-dosed animals compared with untreated controls. Microsomes from ISO- and βNF-dosed males showed a reduction ( P<0.05) in EMD activity when compared with control microsomes, and CF was without effect. In females, EMD activities were significantly increased in microsomes from PCN, DEX and PB-dosed but not βNF, ISO, or CF-dosed animals. As expected on the basis of sex-related differences in gene expression, EMD activities in untreated animals were considerably higher in males than females, attributable to constitutive CYP3A and CYP2C11 activities. The selectivity of EMD for induced CYP3A was confirmed on the basis of inhibition studies with selected steroid substrates of CYP3A, polyclonal anti-CYP3A1 antibodies and triacetyloleandomycin (TAO), a selective inhibitor of CYP3A. In conclusion, for both sexes, the greatest elevations (≈3–13-fold) in EMD activity were observed in microsomes from rats dosed with DEX, a potent archetypal inducer with lesser but significant increases noted for PCN and PB, indicating that EMD is a reliable indicator of induced rat hepatic cytochrome P450 CYP3A activity.

Effect of interleukin 1β-induced fever on hepatic drug metabolism in rat

1. A fever-induced modelinrat was created by repeated injection of interleukin 1β(IL-1β) in the cerebroventricle and the influence of fever on hepatic drug metabolism was investigated. Fever apparently decreased the content of cytochrome P450 (CYP) and the activities of NADPH-ferrihaemoprotein reductase (ƒp2), aminopyrine N-demethylase, aniline hydroxylase, FAD-monooxygenase, p-nitrophenol UDP-glucuronosyl-transferase and glutathione S-transferase. Immunoblot analysis of the CYP isozymes indicated that CYP2C11 and CYP3A were extensively decreased in the IL-1β-induced fevered rat. 2. Repeated administration (5 days) of mefenamic acid in the fevered rat could not restore the activities of ƒp2, aminopyrine N-demethylase and aniline hydroxylase to control plevels, although their hyperthermic state had been improved. The CYP content in the mefenamic acid-treated fevered rat was also lower than that in the control. 3. These findings suggest that fever impairs the hepatic drug-metabolizing capacity (both oxidation and some conjugations) and that the fever-induced impairments are partially retained, even if the hyperthermia has been offset by the administration of antipyretics.

Evaluation of the Effects of Risedronate on Hepatic Microsomal Drug Metabolizing Enzyme Activities Following Administration to Rats for 14 Days: Lack of an Induction Response

ABSTRACTRisedronate ([1-hydroxy-2-(3-pyridinyl)-ethylidene[bis]phosphonic acid] monosodium salt) was evaluated for induction of hepatic microsomal drug metabolizing enzymes in male and female Sprague Dawley rats (N-4/sex/dose group). Main study animals received water (vehicle control), risedronate (0.1, 0.8, 4, or 16 mg/kg/day) or phenobarbital (80 mg/kg/day, positive control) by daily oral gavage for 14 consecutive days. Recovery study animals received water, risedronate (16 mg/kg/day) or phenobarbital (80 mg/kg/day) by daily oral gavage for 14 consecutive days and then were maintained drug-free for 14 days to evaluate the reversibility of any observed effects. At the conclusion of each study the animals were sacrificed, the liver removed, weighed and the microsomal subcellular fraction prepared. the hepatic microsomal fraction was then evaluated for protein content, cytochrome P450, and the activities of aniline hydroxylase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase and p-nitrophenol UDP-glucuronosyltransferase. Risedronate was well tolerated during the dosing phase of the study as evidenced by clinical observations, body weight gain and food consumption which were not significantly different from the vehicle controls. Risedronate did not significantly increase (P>0.05) liver weight, liver/body weight ratio, protein content, P450, aniline hydroxylase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase or p-nitrophenol UDP-glucuronosyltransferase in rats of either sex when compared to vehicle controls. As expected, the hepatic microsomal enzyme inducer phenobarbital significantly increased (P>0.05) liver weight, liver/body weight ratio, protein content (males only), P450, aniline hydroxylase (males only), aminopyrine N-demethylase (males only), ethoxycoumarin O-deethylase and p-nitrophenol UDP-glucuronosyltransferase in rats relative to vehicle controls. Following the 14 day drug-free recovery period the induction parameters increased by phenobarbital reversed to vehicle control levels. the results obtained in this well controlled study indicate that risedronate is not an inducer of hepatic microsomal drug metabolizing enzymes in the rat.

Characterization of the cytochrome P450 isoenzymes involved in the in vitro N-dealkylation of haloperidol.

The present study was carried out to identify the cytochrome P450 isoenzyme(s) involved in the N-dealkylation of haloperidol (HAL). In vitro studies were performed using human liver microsomes and c-DNA-expressed human P450 isoforms. N-dealkylation of HAL was assessed by measuring the formation of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP). There was a tenfold variation in the extent of CPHP formation amongst the nine human liver microsomal preparations. The CPHP formation rates as a function of substrate concentration, measured in three livers, followed monophasic enzyme kinetics. Km and Vmax values ranged respectively from 50 to 78 microM and from 180 to 412 pmol mg-1 min-1 CPHP formation rates in the nine liver preparations were significantly correlated with dextromethorphan N-demethylase activity (a marker of CYP3A4 activity), but not with the activity of dextromethorphan O-demethylase (CYP2D6), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). Ketoconazole, an inhibitor of CYP3A4, inhibited competitively CPHP formation (Ki=0.1 microM), whereas sulphaphenazole (CYP2C9), furafylline (CYP1A2) and quinidine (CYP2D6) gave only little inhibition (IC50 > 100 microM). CPHP formation was, moreover, enhanced by apha-naphtoflavone, an effect common to CYP3A4 mediated reactions. Anti-CYP3A4 antibodies strongly inhibited CPHP formation, whereas no inhibition was observed in the presence of CYP2D6 antibodies. Among the recombinant human CYP isoforms tested, CYP3A4 exhibited the highest activity with respect to CPHP formation rate, with no detectable effect of other CYP isoforms (CYP1A2, CYP2D6 and CYP2C9). HAL inhibited dextromethorphan O-demethylase (CYP2D6) with IC50 values between 2.7 and 8.5 microM, but not (IC50 > 100 microM) dextromethorphan N-demethylase (CYP3A4), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). These results strongly suggest that the N-dealkylation of HAL in human liver microsomal preparations is mediated by CYP3A4.

In vitro analysis of the activity of the major human hepatic CYP enzyme (CYP3A4) using [ N-methyl- 14C]-erythromycin

Although the N-demethylation of erythromycin has found widespread use in a noninvasive assay with which to phenotype hepatic CYP3A function, currently, the routine in vitro analysis of erythromycin N-demethylase activity relies on the quantitation of liberated formaldehyde by relatively labor-intensive and insensitive colorimetric or fluorimetric detection. This report describes the development of a rapid, sensitive, and reproducible radioassay for human CYP3A4 using solid-phase extraction (SPE). The kinetics of erythromycin N-demethylation were best described by a one-site Michaelis-Menten model with autoinhibition and the apparent kinetic parameters for pooled human liver microsomes (HLM; Km = 88 μM, Vmax = 345 pmol/min/mg) and expressed CYP3A4 (Km = 33 μM, Vmax = 130 pmol/min/mg) were in good agreement. Erythromycin N-demethylase activity was found to vary 14-fold in HLM and correlated with the rate of testosterone 6β-hydroxylation (r 2 = 0.92, p < 0.001; N = 9). Ketoconazole was a potent inhibitor of the N-demethylation of erythromycin, and the estimated IC 50 value (104 ± 23 nM) agreed well with that obtained using testosterone as a probe for CYP3A (71 ± 4 nM). The addition of this radioassay to those established for human CYP1A2, 2C9, 2D6, and 2E1 and its subsequent automation should enable the routine use of this methodology in the analysis of CYP-dependent reactions.

Protein Engineering Using Molecular Assembly: Functional Conversion of Cytochrome c via Noncovalent Interactions

The structure−function relationship of cytochrome c (Cyt-c) interacted with the lipid bilayer membranes was studied by various spectroscopic methods, the reaction−products analysis, and its kinetics. Ultrafiltration binding assay, UV−visible, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies showed that Cyt-c was tightly bound to the lipid bilayer membranes bearing a phosphate head group. The anisotropic and nonnatural complexation with the phosphate−lipid membranes caused a spin-state change of the heme in the active center of Cyt-c. Depending on the membrane fluidity, two classes of the structurally altered Cyt-c were prepared and they showed the greatly enhanced N-demethylase activity. Products analysis by HPLC demonstrated that the lipid membrane bound Cyt-c performs a clean enzymatic reaction similar to native hemoenzymes. Kinetics studies established that there are two different activation manners via the phosphate lipid bilayer membranes: namely, simple enhancement of the affinity for H2O2, or the increased catalytic efficiency (kcat) in addition to the enhanced affinity for H2O2. The membrane fluidity again significantly affected the N-demethylation kinetics. A potential of the lipid membrane assembly to functionalize native proteins and enzymes with noncovalent but specific interactions is also discussed.

Suppression of microsomal cytochrome P450-dependent monooxygenases and mitochondrial oxidative phosphorylation by fullerenol, a polyhydroxylated fullerene C 60

The acute toxicity of fullerenol-1 was determined using mice pretreated intraperitoneally (i.p.) with polyhydroxylated C 60 derivatives. The LD 50 value of fullerenol-1 was estimated to be 1.2 g/kg. Pretreatments with 0.5 and 1.0 g/kg fullerenol-1 decreased cytochromes P450 and b5 contents, and NADPH-cytochrome P450 reductase, benzo[ a]pyrene hydroxylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase activities in liver microsomes. Pretreatments with 0.01 and 0.1 g/kg fullerenol-1 had no effect on these monooxygenases. Additions of fullerenol-1 to mouse liver microsomes suppressed monooxygenases activities toward benzo[ a]pyrene, 7-ethoxycoumarin, aniline, and srythromycin with IC 50 values of 42, 94, 102 and 349 μM, respectively. Fullereno1-1 exhibited noncompetitive and mixed-type of inhibition in benzo[ a]pyrene hydroxylation and 7-ethoxycoumarin O-deethylation, respectively. Additions of fullereno1-1 to rat liver mitochondria resulted in a dose-dependent inhibition of ADP-induced uncoupling and markedly inhibited mitochondrial Mg 2+-ATPase activity with an IC 50 value of 7.1 μM. These results demonstrate that fullerenol-1 can suppress the levels of the microsomal enzymes in vivo and decrease the activities of P450-dependent monooxygenase and mitochondrial oxidative phosphorylation in vitro.

Effects of horminone on liver mixed function mono-oxygenases and glutathione enzyme activities of Wistar Rat

The present study reports on the effects of horminone on serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, on hepatic cytochrome P450 (P450) and cytochrome b 5 (cyt b 5) contents and on the activities of NADPH-cytochrome P450 reductase (NR), mixed function mono-oxygenases (MFO), glutathione- S-transferase (GST) and glutathione reductase (GR) of Wistar male rat. Horminone is a diterpenoid quinone (7,12-dihydroxyabiet-8,12-diene-11,14-dione) present in several species of the Labiatae family and used as medicinal plants in folk medicine. In this study, horminone was administered by the intraperitoneal route (i.p.) at a concentration of 1 or 10 mg/kg to each group of six mice, using water as a vehicle. On the one hand, results showed that horminone increased serum ALT and AST levels and cyt b 5 content and induced the activities of ethylmorphine N-demethylase (EMD). On the other hand, horminone decreased P450 content and inhibited the activities of 7-ethoxyresorufin O-deethylase (ERD), 7-ethoxycoumarin O-deethylase (ECD), aniline 4-hydroxylase (AH) and NR. Based on these results, the possibility of toxic effects occurring after administration of plant extracts containing horminone must be considered.

Diverse Function of Aromatase and the N-terminal Sequence Deleted Form

The diverse function of human placental aromatase including estradiol 6α-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6α-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli . Aromatase cytochrome P450 was reconstituted and incubated with [6α,7α- 3 H 2 ,4- 14 C]estradiol, 7-ethoxycoumarin, and [N-methyl- 3 H 3 ]cocaine. 6α-Hydroxy[7α- 3 H,4- 14 C]estradiol was isolated as the metabolite of estradiol and the 3 H-water release method based on the 6α- 3 H label was established. The initial rate kinetics of the 6α-hydroxylation gave K m of 4.3 μM, V max of 4.02 nmol min −1 mg −1 , and turnover rate of 0.27 min −1 . Testosterone competed dose-dependently with the 6α-hydroxylation and showed the K i of 0.15 μM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed K m of 200 μM, V max of 12.5 nmol min −1 mg −1 and turnover rate of 1.06 min −1 . The N-demethylation of cocaine was analysed by the 3 H-release method, giving K m of 670 μM, V max of 4.76 nmol min −1 mg −1 , and turnover rate of 0.49 min −1 . All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 ± 83 pmol min −1 mg −1 ( n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min −1 .

Diverse function of aromatase and the N-terminal sequence deleted form

The diverse function of human placental aromatase including estradiol 6α-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6α-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6α,7α-3H2,4-14C]estradiol, 7-ethoxycoumarin, and [N-methyl-3H3]cocaine. 6α-Hydroxy[7α-3H,4-14C]estradiol was isolated as the metabolite of estradiol and the 3H-water release method based on the 6α-3H label was established. The initial rate kinetics of the 6α-hydroxylation gave Km of 4.3 μM, Vmax of 4.02 nmol min−1mg−1, and turnover rate of 0.27 min−1. Testosterone competed dose-dependently with the 6α-hydroxylation and showed the Ki of 0.15 μM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed Km of 200 μM, Vmax of 12.5 nmol min−1mg−1 and turnover rate of 1.06 min−1. The N-demethylation of cocaine was analysed by the 3H-release method, giving Km of 670 μM, Vmax of 4.76 nmol min−1mg−1, and turnover rate of 0.49 min−1. All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 ± 83 pmol min−1mg−1 (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min−1.

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.

Localization of Multiple Forms of Inducible Cytochromes P450 in Rat Liver Mitochondria: Immunological Characteristics and Patterns of Xenobiotic Substrate Metabolism

Hepatic mitochondria contain inducible cytochromes P450 that cross-react with antibodies to P4501A1/2 and 2B1/2. In the present study, we present evidence for the occurrence of additional P450 forms in rat liver mitochondria that cross-react with antibodies to microsomal P4503A1/2 and 2E1. Protease protection and also immunoelectron microscopy studies were carried out to support the mitochondrial location of the immunoreactive P450s. The solubility of immunoreactive proteins in 0.1 mNa 2CO 3suggests that the mitochondrial P450 forms tested are not membrane-integral proteins. The mitochondrial-associated P450 forms are capable of metabolizing resorufin derivatives, erythromycin, and p-nitrophenol in an adrenodoxin- and adrenodoxin reductase-supported system. Treatment of rats with phenobarbital (PB) resulted in the induction of mitochondrial pentoxyresorufin Odeethylase (PROD), benzoxyresorufin O-deethylase (BROD), and erythromycin N-demethylase (ERND) activities by 17-, 23-, and 2-fold, respectively. These activities were inhibited by 33 to 64% by antibodies to P4502B1/2 and P4503A1/2. The induction of the above monooxygenase activities correlated with the levels of mitochondrial proteins cross-reacting with antibodies to P4502B1/2 and P4503A1/2 in PB-treated livers. Similarly, administration of β-naphthoflavone (BNF) resulted in a marked elevation of O-deethylation of ethoxy-, benzoxy-, and methoxyresorufins and a 2-fold increase in ERND activity. Immunoblot and immunoinhibition experiments using P4501A1/2, P4502B1/2, P4503A1/2, and P4502E1 antibodies revealed the presence of P450 forms closely related to the microsomal inducible forms. Results of immunoinhibition studies, using antibodies to adrenodoxin and reconstitution of enzyme activity with purified P450 forms, suggested a role for the mitochondrial P450 in the metabolism of xenobiotic substrates. The purified mitochondrial P450s also exhibited overlapping substrate specificities for resorufin derivatives and erythromycin.This research was supported in part by NIH Grant GM34883.