Search

Cytochrome P450 (CYP) enzymes constitute an essential xenobiotic metabolizing system that regulates the elimination of lipophilic compounds from the body. Convenient and affordable assays for CYP enzymes are important for assessing these metabolic pathways. In this study, 10 novel profluorescent coumarin derivatives with various substitutions at carbons 3, 6 and 7 were developed. Molecular modeling indicated that 3-phenylcoumarin offers an excellent scaffold for the development of selective substrate compounds for various human CYP forms, as they could be metabolized to fluorescent 7-hydroxycoumarin derivatives. Oxidation of profluorescent coumarin derivatives to fluorescent metabolites by 13 important human liver xenobiotic-metabolizing CYP forms was determined by enzyme kinetic assays. Four of the coumarin derivatives were converted to fluorescent metabolites by CYP1 family enzymes, with 6-methoxy-3-(4-trifluoromethylphenyl)coumarin being oxidized selectively by CYP1A2 in human liver microsomes. Another set of four compounds were metabolized by CYP2A6 and CYP1 enzymes. 7-Methoxy-3-(3-methoxyphenyl)coumarin was oxidized efficiently by CYP2C19 and CYP2D6 in a non-selective fashion. The advantages of the novel substrates were (1) an excellent signal-to-background ratio, (2) selectivity for CYP1 forms, and (3) convenient multiwell plate measurement, allowing for precise determination of potential inhibitors of important human hepatic forms CYP1A2, CYP2C19 and CYP2D6.

1. The effects of several CYP3A substrates (alpha-naphthoflavone (alphaNF), terfenadine, midazolam, erythromycin) on nifedipine oxidation and testosterone 6beta-hydroxylation activities were investigated in hepatic and intestinal microsomes from mouse and human. 2. alphaNF (10 microM) and terfenadine (100 microM) inhibited nifedipine oxidation activities (at substrate concentration of 100 microM) in mouse hepatic microsomes to approximately 50%, but not in mouse intestinal microsomes. alphaNF (30 microM) stimulated nifedipine oxidation activities in mouse and human intestinal microsomes and in human hepatic microsomes to approximately 1.3-1.8-fold. Inhibitory potencies (50% inhibition concentration, IC50) of midazolam and erythromycin for nifedipine oxidations were calculated to be approximately 90 microM in human intestinal microsomes. In contrast, testosterone (100 microM) stimulated the nifedipine oxidation activities approximately 1.5-fold in hepatic and intestinal microsomes from mouse and human. 3. alphaNF showed different effects on the kinetic parameters including the Hill coefficients of nifedipine oxidation and testosterone 6beta-hydroxylation catalysed by hepatic and intestinal microsomes from mouse and human. Cooperativity in nifedipine oxidation was increased by the addition of alphaNF to pooled human hepatic microsomes, but little effects of alphaNF could be observed in individual human intestinal microsomes. 4. These results suggest that CYP3A enzymes in liver and intestine might have different characteristics and that observations from hepatic microsomes should not be directly applicable to intestine metabolism in some cases. Studies of drug-drug interactions of CYP3A substrates are recommended to be performed using intestinal samples.

1. In the present study, nine cytochrome P450 enzyme activities in seven species were characterized to allow a practical means of comparing this important metabolic step between various test animals and man. 2. Enzyme activities and kinetic parameters were first determined towards marker substrates for human cytochrome P450 enzymes. Inhibition profiles were then determined with both antibodies directed against various cytochrome P450 enzymes and with chemical inhibitors. 3. Both the enzyme kinetic parameters/enzyme activities, and the inhibition profiles obtained for the animal species were compared with those obtained for human liver microsomes in order to postulate the animal species most similar to man with regard to each individual cytochrome P450 enzyme activity. 4. It was found that, as expected, none of the tested species was similar to man for all the measured P450 enzyme activities, but that in each species only some of the P450 enzyme activities could be considered as similar to man. 5. When it is known which human cytochrome P450 enzymes are involved in the metabolism of a compound, the comparative data presented here can be used for selecting the most suitable species for in vitro and in it no experiments.

The aims were to examine the kinetics of the oxidative metabolism of flunitrazepam in vitro when flunitrazepam was dissolved in dimethylformamide and acetonitrile, and to determine which cytochrome P450 isoform(s) are involved. The kinetics of the formations of 3'-hydroxyflunitrazepam and desmethyl-flunitrazepam were non-linear and best estimated using the Hill equation. Inhibition of their formation was studied using specific chemical inhibitors, expressed enzyme systems and specific antibodies. Ks, Vmax, Clmax and n (slope factor) for the formation of 3'-hydroxyflunitrazepam and desmethylflunitrazepam had ranges of 165-338 and 179 391 microM, 22-81 and 3-10 nmol x mg protein(-1) x h(-1), 6-17 and 0.9-1.9 microl x mg protein(-1) x h(-1), and 2.3-3.6 and 1.6-2.6 respectively when dimethylformamide was the organic solvent. When acetonitrile was the solvent, Ks, Vmax, Clmax and n (slope factor) for the formation of 3'-hydroxyflunitrazepam and desmethylflunitrazepam had ranges of 173-231 and 74-597 microM, 35-198 and 2.7-48 nmol.mg protein(-1) x h(-1), 1347 and 0.7-6.3 microl.mg protein(-1) x h(-1), and 1.5-3.6 and 1.1-2.7 respectively. CYP2C19, CYP3A4 and CYP1A2 mediated the formation of both 3'-hydroxyflunitrazepam and desmethylflunitrazepam. Investigators need carefully to consider the choice of organic solvent to avoid false CYP identification.

1. The roles of different human cytochrome P450s (CYP) in phenacetin O-deethylation were investigated using human liver microsomes and recombinant proteins. Phenacetin O-deethylase (POD) activities in human liver microsomes at substrate concentrations of 10 and 500 microM were inhibited by 0.1 and 1 microM alpha-naphthoflavone and activated by 10 and 100 microM alpha-naphthoflavone. The activation of POD activity in human liver microsomes by alphanaphthoflavone was inhibited by 100 microM aniline, anti-CYP2E1 antibody, 1 microM ketoconazole and anti-CYP3A4 antibody. 2. In recombinant CYP from human B-lymphoblast cells, POD activities at a phenacetin concentration of 500 microM were detected for CYP2E1 and CYP3A4, as well as CYP1A2, CYP1A1, CYP2C19, CYP2C9 and CYP2A6. In recombinant CYP from human B-lymphoblast cells or baculovirus-infected insect cells and in reconstituted systems, a requirement of cytochrome b5 (b5) for POD activities catalysed by CYP2E1 and CYP3A4 was observed. The activation of POD activity by alpha-naphthoflavone was observed for CYP3A4, but not for CYP2E1. Co-expression of b5 with CYP3A4 enhanced the activation of POD activity by alpha-naphthoflavone. 3. In the absence of alpha-naphthoflavone, the POD activity in pooled human liver microsomes at 500 microM phenacetin was significantly inhibited (p<0.0001) by 10 microM fluvoxamine, but not by 1 microM ketoconazole. In the presence of alpha-naphthoflavone, the activity was significantly inhibited (p<0.0001) by 1 microM ketoconazole, but not by 10 microM fluvoxamine. 4. Inter-individual differences in the effects of alpha-naphthoflavone on POD activity in human liver microsomes were observed, and the involvement of CYP3A4 as well as CYP1A2 in POD activity in human liver was identified even at a low substrate concentration.

1. To identify the cytochrome P450 (CYP) isoenzymes responsible for the major metabolic pathways of S-2-[4-(3-methyl-2-thienyl)phenyl] propionic acid (S-MTPPA) in man, the metabolism of S-MTPPA was examined using human liver microsomes and microsomes containing cDNA-expressed CYP isozymes (CYP1A2, 2A6, 2B6, 2C9-Arg, 2C9-Cys, 2C19, 2D6-Val, 2E1 and 3A4). 2. S-MTPPA was mainly oxidized to the 5-hydroxylated metabolite of the thiophene ring (MA6) with human liver microsomes in the presence of NADPH. The formation of MA6 was inhibited by SKF 525-A, suggesting that CYP plays role in the formation of MA6. 3. Eadie-Hofstee plots for the 5-hydroxylation of S-MTPPA in the range 5-100 microM were linear for all samples studied, suggesting that the formation of MA6 by human liver microsomes follows simple Michaelis-Menten kinetics. Apparent Vmax = 1.42+/-0.64 nmol/min/mg protein; Km = 12+/-5 microM. 4. Among the CYP inhibitors examined (alpha-naphthoflavone, sulphaphenazole, omeprazole, quinidine and troleandomycin), sulphaphenazole (a CYP2C9 inhibitor) showed the most potent inhibitory effect on the 5-hydroxylation of S-MTPPA by human liver microsomes. 5. When incubated with microsomes containing cDNA-expressed CYP isozymes, S-MTPPA was substantially oxidized to MA6 only by CYP2C9. 6. These results suggest that formation of the major metabolite of S-MTPPA, MA6, in human liver microsomes is catalysed predominantly by a single CYP isoenzyme, namely CYP2C9.

1. To aid in the prediction of drug interactions with alprazolam, the human CYP involved in the 1'- and 4-hydroxylation of alprazolam were characterized using human liver microsomes, expressed enzymes and selective chemical inhibitors. 2. The formation of 4-hydroxyalprazolam and 1'-hydroxyalprazolam at an alprazolam concentration of 62.5 microM were reduced by the prototypic CYP3A inhibitor, troleandomycin (50 microM), by 97 and 9900 respectively. Only microsomes from B-lymphoblastoid cells expressing CYP3A4 were capable of catalysing the 1'- and 4-hydroxylation of alprazolam. 3. The formation rates of 1'-hydroxyalprazolam and 4-hydroxyalprazolam at an alprazolam concentration of 1 mM were significantly correlated (n = 19, r = 0.95, p<0.01) indicating that the same enzyme(s) mediated these biotransformations. A significant (p<0.01) correlation was observed between alprazolam 4- and 1'-hydroxylase activity and CYP3A-mediated midazolam 4-hydroxylase, midazolam 1'-hydroxylase, dextromethorphan N-demethylase and erythromycin N-demethylase activities. 4. In conclusion, in adult human liver the CYP3A subfamily members are the principal enzymes involved in the 1'- and 4-hydroxylation of alprazolam. Thus, clinically significant drug drug interactions between alprazolam and other CYP3A substrates are to be expected.

1. The effect of some xenobiotics on levels of selected cytochrome P450 (CYP) isoenzymes determined by Western immunoblotting and associated enzyme activities has been studied in 72-h cultured rat and human precision-cut liver slices. 2. In cultured rat liver slices, 0.5 mM sodium phenobarbitone (PB), 25 microM beta-naphthoflavone (BNF), and 20 micrograms/ml Aroclor 1254 (ARO) induced mixed-function oxidase enzyme activities. Western immunoblotting of liver slice microsomes was performed with antibodies to rat CYP1A2, 2B1/2 and 3A. Compared with 72-h control (dimethyl sulphoxide only treated) rat liver slice microsomes, PB induced CYP2B1/2 and 3A, BNF induced CYP1A2, and ARO induced CYP1A2, 2B1/2, and 3A. 3. The peroxisome proliferators methylclofenapate (MCP), ciprofibrate (CIP) and Wy-14,643 (WY) induced palmitoyl-CoA oxidation in 72-h cultured rat liver slices. Compared with 72-h control rat liver slice microsomes, MCP, CIP, and WY all induced levels of CYP4A. 4. In cultured human liver slices, 20 micrograms/ml ARO, but not 0.5 mM MCP, induced 7-ethoxyresorufin O-deethylase activity. Neither ARO nor MCP had any effect on homogenate palmitoyl-CoA oxidation and microsomal lauric acid 11- and 12-hydroxylase activities. Compared with 72-h control human liver slice microsomes, ARO induced CYP1A2, and MCP appeared to induce CYP4A. Further studies would be required to confirm that CYP4A isoenzymes could be induced by xenobiotics in human liver slices. 5. These results demonstrate that cultured liver slices may be used in evaluating the effect of xenobiotics on both rat and human CYP isoenzymes.

1. Molecular and electronic structural parameters have been determined, by molecular orbital (MO) calculations, for a homologous series of 8 alkoxyresorufins (methoxy- to octoxy-). 2. Quantitative structure-activity relationships (QSARs) between these structural parameters and the rates of metabolism of the alkoxyresorufins in hepatic microsomes from the 3-methylcholanthrene (MC)-, and phenobarbital (PB)-pretreated mouse, and the beta-naphthoflavone (beta NF)-pretreated rat have been established. 3. The most significant single relationship is between beta NF-induction of cytochrome P4501 (CYP1A) and the total nucleophilic superdelocalizability (sigma SN) for the eight compounds in the series. 4. For double regressions, the electronic charge on the alkoxy oxygen, Q(O), or alpha-carbon Q(C), is important when combined with the hydrophobic substituent constant (pi). 5. These findings indicate that the rates of metabolism of these alkoxyresorufins are dependent upon their ability to cross cellular membranes, to fit the relevant CYP1A binding site, and on their ability to accept electrons from a donor nucleophilic species. 6. A different set of parameters correlated with CYP2B activity, namely, parameters of overall shape, which indicates that the way in which the alkoxyresorufins fit the CYP2B site, determines their differences in specificity. 7. Computer graphic interactive docking studies of the alkoxyresorufins with their affinity-specific cytochromes P450, namely, methoxy- with CYP1A2; ethoxy- with CYP1A1; pentoxy- with CYP2B1; and benzyloxy- with CYP3A, have also been undertaken to show the specific interactions of the alkoxyresorufins with the binding sites of the individual P450s.

1. The effects of beta-naphthoflavone, dexamethasone, phenobarbitone and isosafrole on the metabolism of theophylline by rat liver microsomes have been studied. Only beta-naphthoflavone, a known P4501A inducer, increased the rate of 1-methylxanthine formation (3-fold), whereas all the inducers studied increased the rate of 1,3-dimethyluric acid production (2.5-3-fold). 2. To study the effects of a range of fluoroquinolones on theophylline metabolism, beta-naphthoflavone-induced microsomes were used, as the ratio for metabolite production rates was similar to that of untreated microsomes (4:1,3-dimethyluric acid: 1-methylxanthine at 2 mM theophylline). High concentrations of fluoroquinolones (0.5-1.5 mM) were required to affect microsomal theophylline metabolism. 1-Methylxanthine was more sensitive to fluoroquinolone inhibition by enoxacin, ciprofloxacin, norfloxacin and pipemidic acid than 1,3-dimethyluric acid; CP67015, had a significant effect on 1,3-dimethyluric acid production only; binfloxacin had no effect on either pathway. 3. Ethoxycoumarin, a rapidly metabolized substrate, was also investigated as a surrogate for theophylline in in vitro experiments. Fluoroquinolone inhibition of ethoxycoumarin O-de-ethylation in beta-naphthoflavone-induced microsomes was quantitatively greater but qualitatively similar to theophylline metabolism (IC50s 440-870 microM at 2 microM 7-ethoxycoumarin). 4. These data are comparable with previous rat experiments in vivo, indicating that enoxacin, ciprofloxacin and norfloxacin have similar intrinsic activity in the inhibition of theophylline metabolism.

1. The in vitro metabolism of 3,3',4,4'-tetrachloro-[14C]-biphenyl ([14C]-TCB) by hepatic microsomes from the Wistar rat was investigated with liver microsomes from the male, pregnant female and foetus. 2. Three hydroxylated metabolites (4-OH-3,3',4,5'-tetrachlorobiphenyl, 5-OH-3,3',4,4'-tetrachlorobiphenyl, and 6-OH-3,3',4,4'-tetrachlorobiphenyl) were identified by hplc and gc-ms after incubations of liver microsomes from the beta-naphthoflavone-pretreated male rat and TCB-treated pregnant rat. No metabolites of [14C]-TCB were found after incubation with foetal liver microsomes from dams pretreated with [14C]-TCB. The results indicate that the in vivo accumulation of 4-OH-tetraCB in the foetal compartment is probably due to transplacental transport rather than the formation of this metabolite in the foetus. 3. Pretreatment of the male rat with beta-naphthoflavone substantially induced the formation of hydroxylated metabolites, but pretreatment with phenobarbital and dexamethasone was without effect. Based on in vitro incubations of liver microsomes from the beta-naphthoflavone pretreated male rat, an apparent Km and Vmax of 4.5 microM and 240 pmol/mg protein/min respectively was determined for the metabolism of [14C]-TCB. The formation of phenolic metabolites of [14C]-TCB was most likely dependent on P4501A induction.

1. D-Limonene, a monoterpenoid constituent of citrus fruit oil, blocks tumour induction by chemical carcinogens in laboratory animals, apparently by preventing bioactivation of procarcinogens and by enhancing conjugation of proximal carcinogenic metabolites. 2. Inhibitory effects of D-limonene were measured in vitro using cytochrome P450 isoform-specific substrates. D-Limonene inhibited p-nitrophenol hydroxylase (pNP) activity in vitro in liver microsomes from acetone-, phenobarbital (PB)- and beta-naphthoflavone (BNF)-treated mouse, and 7-ethoxyresorufin O-deethylase (EROD) activity in microsomes from PB- and BNF-treated mouse. p-Nitrophenol and ethoxyresorufin are substrates for cytochromes P2E1 and P1A1, respectively. No inhibition of benzphetamine (BNZP) or aminopyrine (AP) demethylases by D-limonene was observed. 3. EROD, BNZP and AP activities in liver microsomes were increased 18 h after i.p. administration of D-limonene to acetone-induced mouse, while pNP activity was unchanged. The immunodetectable protein level of cytochrome P2B1 in non-acetone treated mouse was increased 18 h after D-limonene, with no differences in P2E1 or P1A1. 4. Acute D-limonene did not protect against paracetamol (acetaminophen)-induced depletion of liver reduced glutathione (GSH). A prolonged paracetamol challenge (0.6% diet for 10 days) elevated liver cytosolic GSH-S-transferase activity (GST) two-fold and decreased liver GSH to 46% of control values. Dietary D-limonene (1.0% diet for 10 days) maintained liver GSH concentrations at 92% of control values in the paracetamol-challenged mouse without altering GST activity. D-Limonene also increased liver GSH concentration (23%) in mouse fed 1.0% D-limonene alone.

1. The effects of phenobarbital (PB), beta-naphthoflavone (beta-NF), omeprazole (Omep) and rifampicin (Rif) on drug-metabolizing activities in dog hepatocytes, cultured with William's medium E, were examined. 2. The drug metabolizing activities of the hepatocytes decreased during culture; 7-ethoxycoumarin O-deethylase (ECOD) activity was nearly 70% of initial value at 72 h, but 7-methoxycoumarin O-demethylase (MCOD), 7-propoxycoumarin O-depropylase (PCOD), progesterone 6 beta-hydroxylase (6 beta-OH-P), progesterone 16 alpha-hydroxylase (16 alpha-OH-P), progesterone 21-hydroxylase (21-OH-P), 7-ethoxyresorufin O-deethylase (EROD) activities and total cytochrome P450 content were approx. 50%. 3. When the hepatocytes were cultured with PB, the enzyme activities increased time- and dose-dependently. MCOD, ECOD and PCOD activities increased 5-8 fold with 2 mM PB in 96 h. Similar results were obtained for 6 beta-OH-P, 16 alpha-OH-P and 21-OH-P activities, and total cytochrome P450. The effect of PB was abolished when 2.5 microM cycloheximide or 0.1 microM actinomycin D was included in the culture. 4. Treatment of hepatocytes with 40 microM beta-NF for 72 h resulted in 25-fold elevation of EROD activity. beta-NF enhanced PCOD activity approx. six-fold, while ECOD increased only slightly, and 7-MCOD negligibly. 5. Omep (100 microM) increased EROD activity nearly 10-fold, and 25 microM Rif increased 6 beta-OH-P activity approx. 8-fold, but ECOD only slightly. 6. Western blot analysis of microsomes from cultured dog hepatocytes with anti-rat CYP 2B1 antibodies indicated that PB increased an immunochemically-reactive protein. The protein showed the same mobility as the major dog P450 isozyme (cytochrome P450 PBD-2 or CYP 2B11) purified from liver microsomes of PB-treated male beagle dog. In a similar manner, induction of cytochrome P450 PBD-1 (CYP 3A12) by PB was confirmed.

1. The effect of beta-naphthoflavone (BNF), given i.p. (n = 9) and orally (n = 9), on the metabolism of antipyrine and metronidazole was investigated in rats. 2. The clearances of antipyrine and metronidazole were determined on a single saliva sample. The rates of formation of antipyrine and metronidazole metabolites were determined from a 20 h urine sample and saliva clearance. 3. Administration of beta-naphthoflavone i.p. was significantly more effective than oral dosage on the induction of antipyrine and metronidazole metabolism (p < 0.05). 4. The capacity of extrahepatic tissues to metabolize antipyrine and metronidazole was quantitatively assessed in rats with and without pretreatment with beta-naphthoflavone immediately after sham operation or 70% partial hepatectomy (n = 40). 5. Antipyrine and metronidazole clearances correlated with liver weight in induced and non-induced rats. Linear regression of antipyrine and metronidazole clearances did show a non-significant Y-intercept (p > 0.05), indicating a negligible extrahepatic metabolism in both induced and in non-induced rats. 6. From a quantitative point of view this study indicates that induction of extrahepatic cytochrome P450 metabolism of antipyrine and metronidazole is negligible.

1. The effects of oxygen concentration were studied on the metabolic pathways of anisole homologues (anisole, phenetole and isopropoxybenzene) catalysed by liver microsomes from phenobarbital-treated rats. 2. With increase of oxygen concentration, the rate of anisole o-hydroxylation reached a plateau at about 35 microM O2, while the rates of O-demethylation and aromatic p-hydroxylation were still increasing at 223 microM O2 (air). 3. The rates of all three metabolic reactions of phenetole reached plateau levels at about 80 microM O2. 4. The rates of all three metabolic reactions of iso-propoxybenzene were still increasing as 223 microM O2 (air). 5. The ratio of aromatic p-hydroxylation or O-dealkylation to aromatic o-hydroxylation decreased in anisole metabolism, and showed no uniform change in phenetole and isopropoxybenzene metabolism with decreasing oxygen concentration. 6. The ratio of aromatic p-hydroxylation to O-dealkylation was essentially constant over the range of oxygen concentration studied in anisole and phenetole metabolism, while in iso-propoxybenzene metabolism the ratio was different between higher and lower oxygen concentrations than 60 microM. 7. This series of compounds with increasing chain length did not show homologous changes in rates of product formation or O2 dependent of product formation.

omega-Hydroxylation of the ethyl moiety of phenacetin by rabbit-liver microsomal preparations was slow, but was increased 10-fold by pretreatment of the animals with phenobarbitone (PB), and was decreased 2.8-fold by treatment with 3-methylcholanthrene (3-MC) or beta-naphthoflavone (beta-NF). N-[4-(2-hydroxyethoxy)phenyl]acetamide (beta-HAP), the omega-hydroxylation product, which was detected in trace amounts only in the urine of rabbits injected with phenacetin, was converted into [4-(acetylamino)phenoxy]acetic acid (4-APA) by the microsomal and cytosolic fraction of liver homogenate and NADP+ or NAD+. Rabbits excreted 56% of a dose of beta-HAP as 4-APA in the 48 h urine. Phenacetin, injected i.p. into rabbits previously treated with PB, was excreted in the urine as 4-APA (12.2% of dose). beta-HAP formed endogenously or added as substrate in vitro was recovered as the O-acetyl derivative, when ethyl acetate was used for extraction of metabolites from microsomal incubation mixtures. (omega-1)-Hydroxylation of the ethyl moiety of phenacetin, which gave 4-acetamido-phenol, occurred rapidly with rabbit-liver microsomal preparations, and was not increased significantly after pretreatment of animals with either PB or 3-MC. omega-Hydroxylation of the acetic moiety of phenacetin by rabbit-liver preparations to give N-(4-ethoxyphenyl)glycolamide (4-GAP) was slow, but was increased three-fold after pretreatment of animals with 3-MC or beta-NF, whereas PB had no effect. 4-GAP was detected in trace amounts only in the urine of rabbits injected i.p. with phenacetin. N-Hydroxylation of phenacetin by rabbit-liver microsomal preparations was slow, but increased three-fold after treatment of animals with 3-MC, and was unchanged by PB. N-Hydroxylation of phenacetin by hepatic microsomes from 3-MC-treated rabbits was 26 times slower than that of 2-acetylaminofluorene; no N-hydroxy derivatives of N-(4-chlorophenyl)acetamide and propanil were detected in vitro.

The effect of four inhibitors of cytochrome P-450-mediated drug oxidations (SKF 525A, cimetidine, metyrapone and alpha-naphthoflavone) on the urinary metabolite pattern and 14CO2 exhalation rate (CER)-time profile following [N-methyl-14C]antipyrine administration has been investigated. The CER-time profiles indicated that inhibition of antipyrine metabolism was in the rank order SKF 525A greater than cimetidine greater than metyrapone greater than ANF. The urinary metabolite patterns showed selectively in action towards particular pathways, 3-hydroxylation being primarily decreased by SKF 525A and cimetidine, and N-demethylation by ANF. The results provide further evidence for involvement of multiple forms of cytochrome P-450 in antipyrine metabolism.

Treatment of rats with 3-methylcholanthrene increased Vmax of the high-affinity component of 7-ethoxycoumarin O-deethylase activity 60-fold. There was also an increase in Vmax of the low-affinity component. Treatment with phenobarbitone increased Vmax of the high-affinity component six-fold whilst not affecting the Km of this component. Modest changes were also observed in the kinetics of the low-affinity component. Following treatment with 3-methylcholanthrene, the sensitivity of both the high- and low-affinity components of activity to inhibition by alpha-naphthoflavone was considerably increased, with the IC50 decreasing from greater than 250 microM to less than 10 microM in both instances. Following treatment with phenobarbitone, the sensitivity of the low-affinity component to inhibition by metyrapone was considerably increased, with the IC50 decreasing from greater than 1000 microM to 96 microM. There was also a modest, but significant, increase in sensitivity of the high-affinity component to metyrapone. These results indicate that both components of 7-ethoxycoumarin O-deethylase activity are catalysed by more than one form of cytochrome P-450.

The metabolism of the hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB) is catalysed by selective forms of cytochrome P-450. DAB metabolism has been studied using microsomes from regenerating rat liver prepared 1, 2, 3, 7 and 10 d after partial hepatectomy. Greatly decreased N-demethylation of DAB was seen during liver regeneration, while virtually no effect on ring-hydroxylation was observed. Glutathione stimulated N-demethylation and ring-hydroxylation of DAB, while metabolism of the corresponding secondary amine N-methyl-4-aminoazobenzene (MAB) was not affected. During regeneration, response to the thiol was depressed in the early stages but later returned to normal. beta-Naphthoflavone (BNF) specifically induced N-demethylation of DAB. Induced activity was not depressed during liver regeneration. Phenobarbital (PB) induced total metabolism, which was depressed during regeneration. This indicates greater stability of BNF-induced cytochrome P-450 compared to control and PB-induced cytochrome P-450. The results indicate that during liver regeneration the metabolism of DAB associated with activation (N-demethylation) is depressed, whereas that associated with detoxication (ring-hydroxylation) is only slightly affected. This confirms the involvement of different forms of cytochrome P-450 in DAB metabolism.

1. By several criteria, macrolide antibiotics constitute a new class of nitrogenous cytochrome P-450 metabolic-intermediate complex-forming compounds. 2. Macrolide antibiotic metabolic-intermediate complexes are only formed in livers induced with phenobarbital or with the macrolide antibiotics themselves. The extent of metabolic-intermediate complex formation in microsomes from phenobarbital-induced rats is lower than that seen for members of the amphetamine and SKF 525-A classes of compounds. 3. Cytochrome P-450 induced by macrolide antibiotics, of which troleandomycin is the most potent, is extensively sequestered as a metabolic intermediate complex in vivo. 4. Cytochrome P-450 induced by troleandomycin differs, using several criteria, from those induced by phenobarbital, beta-naphthoflavone or SKF 525-A, and those present in uninduced rats.

1. Metabolism of 14C-labelled benzo[a]pyrene (-)trans-7,8-dihydrodiol to protein- and DNA-binding products in a reconstituted enzyme system proceeds 5 to 10 times faster with rabbit cytochrome P-450 LM4 than with LM2. 2. Either cytochrome converts the substrate to ethyl acetate- and water-soluble metabolites, identified by h.p.l.c. Water-soluble metabolites comprise 78% of the total products with cytochrome P-450 LM2, but only 50% of those formed by LM4. The relative proportion of the two types of metabolites is differentially affected by certain modifiers such as 7,8-benzoflavone. 3. Half of the radioactivity in the aqueous phase of reaction mixtures containing cytochrome P-450 LM4 represents (-)trans-7,8-diol metabolites in complex primarily with NADPH and phosphate. The remaining water-soluble products are bound covalently to proteins in the reconstituted system. 4. Polyacrylamide gel electrophoresis, autoradiography, and measurement of the radioactivity in individual bands indicate that a larger fraction of metabolites is bound to cytochrome P-450 LM4 than to NADPH-cytochrome P-450 reductase, and only marginal binding to cytochrome P-450 LM2 is seen. Metabolite binding to added DNA is likewise substantially greater in magnitude when cytochrome P-450 LM4, as opposed to LM2, catalyses (-)trans-7,8-diol oxygenation. Thus, the degree of metabolite binding to monoxygenase proteins and to DNA correlates well with the catalytic activity of cytochrome P-450 LM4 and LM2 towards (-)trans-7,8-diol. 5. DNA causes a dramatic enhancement in the activity of cytochrome P-450 LM4 with (-)trans-7,8-diol, indicating that the cytochrome and/or the reductase may be functionally impaired by metabolites of this substrate. Such an effect may alter the balance between detoxication and activation of the carcinogenic benzo[a]pyrene.

1. The activities of 7-methoxycoumarin (7-MCOD), 7-ethoxycoumarin (7-ECOD) and 7-propoxycoumarin (7-PCOD) O-dealkylases decreased by 75-90% during culture of rat hepatocytes for 72 h. 2. The addition of dexamethasone (D) produced a stabilization or modest enhancement of 7-ECOD and 7-PCOD activities depending on the medium used; D was without effect on 7-MCOD activity. 3. The addition of nicotinamide (N) produced some stabilization of 7-ECOD and 7-PCOD activities but not of 7-MCOD activity. 4. The addition of D + N was associated with large increases in 7-ECOD and 7-PCOD activities, again with little effect on 7-MCOD activity. 5. Ethoxyresorufin O-dealkylase activity correlated with 7-ECOD/7-PCOD activities, whereas pentoxyresorufin O-dealkylase activity correlated with 7-MCOD activity. 6. It is concluded that D exerts a selective effect on certain forms of cytochrome P-450, but that more than one mechanism is probably implicated in this effect. 7. The magnitudes of induction of 7-ECOD activity by phenobarbitone and beta-naphthoflavone were blunted when cells were treated in culture when compared to treatment in vivo; the presence of D in the culture medium did not modify this phenomenon.

1. Single sample clearance estimates, Cl, were calculated for each of five drugs employed as probes of hepatic drug-metabolizing activity in rats. Probe drugs were theophylline, phenytoin, valproic acid, antipyrine, and S-warfarin. Cl values were calculated for each probe in animals pretreated with phenobarbital, isosafrole, beta-naphthoflavone, or clofibrate. Control animals were pretreated with vehicle only. 2. A clearance index (c.i., probe Cl after pretreatment divided by probe Cl control) was calculated for each probe and each pretreatment regimen, and data were consolidated to give different probe-based handprints of the pretreatment effects. 3. S-Warfarin was the least specific probe as its c.i. was greater than 1.0 subsequent to each pretreatment. Theophylline appeared to be the most selective probe since its c.i. deviated significantly from unity (3.56) only after beta-naphthoflavone pretreatment. Phenytoin exhibited c.i. values less than unity after each pretreatment indicating that it may not, when used as a single sample probe of hepatic drug-metabolizing activity, effectively discriminate between inductive or inhibitory effects of xenobiotics. 4. Multi-probe-based handprints of hepatic drug-metabolizing activity structured from simple single sample estimates of probe clearance have potential in the rapid screening of xenobiotic-induced alterations of drug-metabolizing enzyme activity.

1. 1-(2-Chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide (RP 52028), an antagonist of peripheral type benzodiazepine binding sites, a potential anticonvulsant, has been shown to have an inducing effect on drug-metabolizing enzymes. 2. RP 52028 was administered orally at 20-800 mg/kg for 1 week, and enzymic activities were determined using a panel of substrates. Western blot analyses were performed using several specific polyclonal and monoclonal antibodies directed against cytochrome P-450 isozymes. 3. RP 52028 appears to be an inducer of cytochrome P-450 II B1 (P-450b) and related enzymic activities; i.e. benzphetamine, ethylmorphine and aminopyrine demethylation.

Pretreatment of rats with phenobarbitone increased hepatic microsomal 7-methoxy-and 7-ethoxy-coumarin O-dealkylase activities. Pretreatment with beta-naphthoflavone increased only the 7-ethoxycoumarin O-dealkylase activity. The addition of metyrapone in vitro inhibited the O-dealkylations to different extents. Similar results were obtained with diphenyloxazole and ethanol. These results are taken to indicate that different forms of cytochrome P-450 are involved in the O-dealkylation of these two substrates. The pattern of metabolism (Phase I and Phase II) of each alkoxycoumarin in rat isolated hepatocytes was very similar. The sulphate conjugate was the major metabolite produced, the amount of which approached a plateau as the rate of O-dealkylation increased. It is concluded that the type of cytochrome P-450 involved in the initial Phase I metabolism does not influence the subsequent pattern of conjugation.

The metabolism of benzo[a]pyrene (BP) in hepatic microsomes isolated from rats exposed to chlordecone or mirex was compared to that of untreated rats and rats treated with 3-methylcholanthrene (3-MC) or phenobarbital (PB). Treatment with chlordecone resulted in a two- to three-fold increase in cytochrome P-450 content but the BP-hydroxylase activity per mg microsomal protein was unaffected. Addition of alpha-naphthoflavone (alpha-NF) or chlordecone caused changes in BP-hydroxylase activity indicating that chlordecone-induced cytochromes P-450 were similar to control. H.p.l.c. analyses of BP metabolites confirmed this similarity. Treatment with mirex caused a two-fold induction of cytochrome P-450, and BP-hydroxylase activity expressed per mg microsomal protein was increased 1.3-fold. Addition of chlordecone or alpha-NF caused changes in BP-hydroxylase activity, indicating differences between control and mirex-induced cytochromes P-450. H.p.l.c. analyses of BP metabolites confirmed this difference. Treatment with chlordecone or mirex increased microsomal epoxide hydrolase activity three-fold. Chlordecone accumulated in hepatic nuclei.

1. Addition of 7,8-benzoflavone (7,8-BF) inhibited both hepatic and renal constitutive activity of aryl hydrocarbon (benzo[a]pyrene) hydroxylase in vitro for a number of avian species. Inhibition of hepatic benzo[a]pyrene hydroxylase was 70-94% in wild pigeon; 46-88% in homing pigeon; 85-93% in house crow; 18-53% in common kite; 56-86% in cattle egret; and for the renal enzyme was 43-84, 52-88, 18-73 and 13-48% respectively. 2. 3-Methylcholanthrene treatment of homing pigeons resulted in the induction of liver and kidney benzo[a]pyrene hydroxylase activity. Induced enzyme activity was also significantly inhibited by 7,8-BF.

2,5-Diphenyloxazole (PPO) is metabolized to one major fluorescent product by human liver and rat liver microsomes. PPO metabolism by human-liver microsomes involves more than one cytochrome P-450 isozyme, termed low-affinity and high-affinity components. At a substrate concentration of 0.1 microM, 95% of activity is due to the high-affinity component whereas at 100 microM 69% of activity is due to the low-affinity component. Inhibition studies with metyrapone and alpha-naphthoflavone at 0.1 microM and 100 microM suggest that the high-affinity component may reflect a 3-methylcholanthrene-inducible form of cytochrome. Therefore studies at low substrate concentrations may be a useful tool for cytochrome P-450 studies in man. Rat liver microsomes show linear kinetics indicating the involvement of one major form of cytochrome P-450.

The ability of metyrapone to induce hepatic cytochrome P-450 and xenobiotic metabolism in rats and rat hepatocyte culture has been studied. Metyrapone is a phenobarbitone-type inducer in rats. When rat hepatocytes were cultured with beta-naphthoflavone, a polycyclic hydrocarbon-type induction pattern was observed. In contrast, the observed induction pattern of both phenobarbitone and metyrapone in cell culture differed from that obtained with these compounds in vivo. The relationship between the nature of the induction of hepatic xenobiotic metabolism by metyrapone in vivo and its effects in liver-cell culture are discussed.

The rate of production of 4-hydroxybiphenyl from 4-methoxybiphenyl in hepatocytes isolated from untreated rats was essentially identical to that from biphenyl in hepatocytes isolated from rats pretreated with beta-naphthoflavone at 40 mg/kg. Similar results were obtained using liver microsomes isolated from untreated or treated rats. The selective inhibition of these reactions by metyrapone, alpha-naphthoflavone and ethanol demonstrated that different forms of cytochrome P-450 are responsible for O-demethylation of 4-methoxybiphenyl in livers of untreated rats and 4-hydroxylation of biphenyl in livers of pretreated rats. The pattern of conjugation of 4-hydroxybiphenyl generated from biphenyl in hepatocytes isolated from pretreated rats was different from that for 4-hydroxybiphenyl generated from 4-methoxybiphenyl in hepatocytes isolated from untreated rats, there being a 60% decrease in sulphation measured after 60 min incubation with biphenyl. Glucuronidation of 4-hydroxybiphenyl was not affected. The sulphation of 4-hydroxybiphenyl added directly was significantly decreased in hepatocytes isolated from pretreated rats. The initial rate of glucuronidation of 4-hydroxybiphenyl added directly was not altered by the pretreatment, but there was a significant decrease in overall glucuronidation measured over a 60 min incubation. Similar results were obtained using liver slices. beta-Naphthoflavone added directly to liver slices significantly decreased the extent of sulphation of 4-hydroxybiphenyl but did not influence glucuronidation. There was evidence of a late decrease in biphenyl 4-hydroxylase activity in hepatocytes isolated from pretreated rats. It is concluded that the differences observed in the cellular metabolism of biphenyl and 4-methoxybiphenyl can be ascribed to competition between beta-naphthoflavone and/or its metabolites retained within the cells following pretreatment, and biphenyl and/or its metabolites for the pathways common to their metabolism. It is also concluded that the type of cytochrome P-450 involved in the generation of 4-hydroxybiphenyl does not, per se, influence the subsequent pattern of conjugation.

The characteristics of induction of rat-hepatic microsomal mono-oxygenase activity by promethazine and isosafrole have been investigated and compared with the classic inducers phenobarbitone and beta-naphthoflavone. Both promethazine and isosafrole pretreatments result in increased cytochromes P-450, and enhanced NADPH-cytochrome c reductase, aminopyrine N-demethylase, dichloronitroanisole O-demethylase, ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activity. Isosafrole but not promethazine increased the liver to body weight ratio. It is concluded that promethazine and isosafrole pretreatment produces an induction of the rat-hepatic microsomal mono-oxygenase system which shows both phenobarbitone- and polycyclic aromatic hydrocarbon-type characteristics.

A series of 2-arylalkyl- and 2-(4'-alkyl)phenoxymethylbenzimidazoles was synthesized and evaluated as inhibitors of mixed-function oxidase activity in phenobarbitone- and beta-naphthoflavone-induced rat liver microsomes. Higher homologues of the 2-arylalkyl series were more potent inhibitors than lower homologues against all mono-oxygenase activities except aniline p-hydroxylation. Smaller 2-substituents were associated with relatively low-affinity reverse type I spectral binding behaviour, whereas larger substituents were associated with type I binding of high affinity.

A series of eight 4-n-alkoxymethylenedioxybenzene (AMDB) derivatives were evaluated for their inhibitory effects on several mono-oxygenase reactions and their capacity to form metabolite complexes with cytochrome P-450 in vitro in hepatic microsomes from phenobarbital (PB)-and Beta-naphthoflavone (Beta NF)-induced rats. Ethoxyresorufin O-deethylase in Beta NF-induced microsomes and aminopyrine N-demethylase in PB-induced microsomes were most susceptible to inhibition by the test compounds. In contrast, aldrin epoxidation and arylhydrocarbon hydroxylase in PB-and Beta NF-induced microsomes, respectively, were not inhibited by derivatives of AMDB. All AMDB derivatives elicited spectral complexes with cytochrome P-450, the characteristics of which were influenced by the microsomes employed and by the length of the AMDB alkoxy side-chain. Derivatives containing short-chain alkoxy substituents (C1 to C3) formed unstable metabolite complexes and generated substantial quantities of carbon monoxide (CO), those with intermediate length alkoxy groups (C4 to C6) generated little CO and rapidly formed intense spectral complexes (large delta A max), and those with the largest alkoxy groups (C7 and C8) formed no CO and elicited complexes of high stability. Quantitative structure-activity analyses showed that the biological data could be described by parabolic equations in II, the hydrophobic constant of the alkoxy substituent, and suggested the importance to AMDB interactions of a lipophilic-binding region at the active centre of the cytochrome P-450. The alkoxy chain length for optimal mono-oxygenase inhibition and complex formation with cytochrome P-450 appeared to be about five or six carbon atoms. The data suggest that the capacity of AMDB compounds to form stable inhibitory complexes with cytochrome P-450 may not always be associated with their ability to inhibit mono-oxygenase activity.

1. Inhibitory activity of dihydrosafrole towards benzo[a]pyrene (BP) hydroxylase activity in hepatic microsomes from beta-naphthoflavone (BNF)-induced rats, and in reconstituted systems containing cytochrome P-450c, increased dramatically on preincubation of the inhibitor with NADPH; no inhibition occurred without preincubation. The level of BP hydroxylase inhibition was associated with the progressive formation of the 456 nm dihydrosafrole metabolite-cytochrome P-450c spectral complex during preincubation. 2. Inhibition of BP hydroxylase by dihydrosafrole in control microsomes, and inhibition of ethoxyresorufin O-deethylase (EROD) in microsomes (control or BNF-induced) and in reconstituted systems with cytochrome P-450c, did not require preincubation and apparently was not dependent on prior formation of the dihydrosafrole metabolite-cytochrome P-450 complex. 3. Kinetic studies established that, following preincubation with NADPH, dihydrosafrole was a noncompetitive inhibitor of both BP hydroxylase and EROD activities. In the absence of preincubation, dihydrosafrole was an effective competitive inhibitor of EROD in BNF-induced microsomes and in reconstituted systems with cytochrome P-450c. 4. Both ethoxyresorufin and benzo[a]pyrene inhibited the development of the type I optical difference spectrum of dihydrosafrole in reconstituted systems containing cytochrome P-450c. Inhibition by ethoxyresorufin was competitive while that caused by benzo[a]pyrene was noncompetitive in nature. 5. The type II ligand phenylimidazole was an effective noncompetitive inhibitor of EROD activity but failed to exert any inhibitory effect on cytochrome P-450c-mediated BP hydroxylase activity. Phenylimidazole inhibited formation of the dihydrosafrole type I optical difference spectrum non-competitively. 6. The results indicate that ethoxyresorufin and benzo[a]pyrene may occupy different binding sites on cytochrome P-450c and that dihydrosafrole binds primarily to the site utilized by ethoxyresorufin.

Felodipine, a 1,4-dihydropyridine diester, is metabolized to its corresponding pyridine analogue by rat-liver microsomes. Kinetic studies showed similar Km, Vmax and t1/2 for the formation of the pyridine metabolite and the disappearance of felodipine, indicating that oxidation of felodipine to the corresponding pyridine analogue is the major pathway of metabolism. Response to inhibitors such as CO, SKF 525-A and metyrapone indicates participation of cytochrome P-450 in the aromatization of felodipine. Phenobarbital pretreatment markedly increased the metabolism of felodipine and its pyridine analogue. Felodipine pretreatment had no effect on the cytochrome P-450 concn. in rat-liver microsomes, nor on the rate of its own metabolism, but a slight increase was observed in the rate of metabolism of four standard substrates.

1. Pretreatment of male Syrian golden hamsters with 7,8-benzoflavone (7,8-BF, 0.4% in the diet) for 4 and 20 weeks resulted in a significant increase in the amount of cytochrome P-450 and cytochrome b5 in hepatic microsomes. The activities of microsomal 7-ethoxycoumarin-O-deethylase (ECOD) and 7-ethoxyresorufin-O-deethylase (EROD) increased by a factor of 2 and 5, respectively, whereas aryl hydrocarbon hydroxylase (AHH) activity was only marginally enhanced. 2. Because the relative increase in cytochrome b5 exceeded that of cytochrome P-450, the ratio of P-450 to b5 decreased from 2.2 in controls to 1.6 in 7,8-BF-treated animals. 3. Hepatic microsomes from untreated and 7,8-BF-pretreated hamsters metabolize E-diethylstilboestrol (E-DES) to its stereoisomer Z-DES and to several oxidative metabolites, of which 3-hydroxy-DES and Z,Z-dienestrol were unambiguously identified by g.l.c.-mass spectrometry. 4. Pretreatment with 7,8-BF led to a marked decrease in the formation of all oxidative DES metabolites but not in the isomerization to Z-DES. The possible implication of these data for the mechanism of liver tumour formation by the combined treatment of hamsters with 7,8-BF and DES is discussed.