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Oxidation and uptake of ascorbate show similar time courses in rat liver microsomal vesicles: a rapid burst phase is followed by a slower process. Inhibitors of ascorbate oxidation (proadifen, econazole or quercetin) also effectively decreased the uptake of ascorbate. The results show that dehydroascorbate is the transport form of ascorbate at the membrane of the endoplasmic reticulum.
Recent reports have suggested the involvement of arachidonic acid (AA) and its metabolites in the control of body pattern, head and tentacle regeneration and bud formation in Hydra spp. Here we describe for the first time the biosynthesis of hydroxyeicosatetraenoic acids (HETEs) in vitro by hydroid cytosolic extracts. Incubation of both unlabelled and tritiated AA with homogenates of Hydra vulgaris led to the conversion of up to 11% of the exogenous fatty acid into mainly two metabolites. These were characterized as 11-hydroperoxyeicosatetraenoic acid (11-HPETE) and 11-HETE by means of a combination of chromatographic, chemical, 1H-n.m.r. and electron-impact m.s. techniques. Trace amounts of 9-HETE and 12-HETE were also found. Analysis of 11-HETE by chiral-phase h.p.l.c. revealed that this metabolite was composed mainly of the R enantiomer. The production of 11-HPETE and 11-HETE was found to be: (1) associated with the cytosolic fraction of Hydra homogenates; (2) dependent on AA concentration, incubation time and protein amount in the homogenates; (3) unaffected by co-incubation with the 5- and 12-lipoxygenase inhibitors, 5,8,11-eicosatriynoic acid and nordihydroguaiaretic acid, the cyclo-oxygenase inhibitor, indomethacin, or the cytochrome P-450 inhibitors, proadifen and methoxalen. These results strongly suggest the presence of a very active (R)-11-lipoxygenase in H. vulgaris. The activity of both R and S enantiomers of synthetic 9-, 11- and 12-HETE and of 'endogenous' 11-HETE was studied on tentacle regeneration and bud formation in decapitated Hydra. Although almost all compounds tested inhibited budding, only endogenous 11-HETE and synthetic (R)-11-HETE significantly enhanced the average number of tentacles, thus suggesting that this eicosanoid might be one of the cellular regulators of regeneration in H. vulgaris.
1. The effects of the inhibition of the metabolism of arachidonic acid (AA) on the constrictor responses to noradrenaline (NA) were studied in the rat perfused mesenteric bed. The inhibitor of all the pathways of AA metabolism, 10 microM eicosatetraynoic acid (ETYA), reduced the constrictor responses to all the concentrations of NA assayed. 2. The constrictor responses to NA were also reduced by the cyclooxygenase (COX) inhibitor, indomethacin (10 microM), as well as by the lipoxygenase inhibitor, nordihidroguaiaretic acid (1 microM; NDGA), whereas they were unmodified by the cytochrome P450 monooxigenase inhibitors, clotrimazole (10 microM), metyrapone (10 microM) and proadifen (10 microM). 3. The reduction in NA contractility induced by indomethacin was reverted with a decreasing order of potency by the thromboxane A2 analogue, U-46619 > prostaglandin (PG) E2 > PGF2alpha. The exposure of the mesenteric bed to NA increased the production of PGF2alpha, whereas it did not modify the production of the remaining AA metabolites. 4. The increase in the NA-induced contractions caused by endothelium removal, as well as by the inhibition of nitric oxide synthase (NOs) with NG-nitro-L-arginine methyl ester (400 microM; L-NAME), was suppressed by indomethacin but not by NDGA. These observations suggest that the lipoxygenase-derived metabolites are formed in the endothelium, whereas the COX-derived metabolites are formed in the vascular smooth muscle. 5. The TP receptor antagonist, SQ29548, did not modify the NA-induced contractions, either in the presence or in the absence of the endothelium. 6. Contractions elicited by KCI (60-100 mM) were unmodified by the AA metabolism inhibitors, ETYA, NDGA and indomethacin. 7. In summary, these results show that metabolites of AA, through both the COX and the lipoxygenase pathways, are involved in the NA-induced contractions in the rat mesenteric bed. The lipoxygenase metabolites are likely to be formed in the vascular endothelium, whereas the COX metabolite, which could be PGF2alpha, is apparently formed within the vascular smooth muscle.
This study examined the role of calcium channels for the uptake of cadmium (Cd) into Madin-Darby canine kidney (MDCK) cells. Maitotoxin, an activator of different types of calcium channels, increased accumulation of 109Cd and 45Ca in MDCK cells. We found that maitotoxin increased accumulation by stimulating 109Cd influx because it did not affect efflux. An inhibitor of store-operated Ca channels, SKF96365, partially blocked 45Ca influx but did not affect 109Cd influx. Ni and Mn, and loperamide and proadifen (SKF 525a), inhibited 45Ca and 109Cd influx in cells stimulated with maitotoxin, but La and nifedipine did not. Overnight treatment with phorbol 12, 13-ibutyrate (PDBu) to activate protein kinase C resulted in a decrease in the concentration of maitotoxin needed to stimulate 45Ca and 109Cd influx. The effect of PDBu was blocked by treating cells with the protein kinase C inhibitor GF109203X. Additionally, the effect of PDBu was lost in cells treated with an inhibitor of RNA synthesis actinomycin D. These results suggest that a Ca permeable cation channel different from voltage-dependent and store-operated Ca channels mediates the uptake of Cd in MDCK cells. The expression of this channel is regulated by protein kinase C.
Proliferation of mouse fibrosarcoma cells G:5:113 was studied in vitro after affecting particular pathways of arachidonic acid metabolism by selected inhibitors. After 48 hours of cultivation with nonspecific lipoxygenase inhibitors, nordihydroguaiaretic acid (NDGA) and esculetin; a specific 12-lipoxygenase inhibitor, baicalein; and inhibitor of five-lipoxygenase activating protein, MK-886, markedly suppressed the number of cells and induced significant changes in cell cycle distribution in a dose-dependent manner. While proadifen, an inhibitor of cytochrome P-450-monooxygenase, applied in low concentrations, increased the cell number, at higher concentrations, it inhibited cell proliferation and significantly changed the cell cycle. Cyclooxygenase inhibitors, ibuprofen, flurbiprofen, and diclofenac suppressed cell numbers only moderately without any changes in the cell cycle. The occurrence of apoptosis was not significant for any of the selected drugs in comparison with untreated control cells. Moreover, not even one of the drugs caused the specific cleavage of poly (ADP-ribose) polymerase to the 89-kDa fragment, however, a decrease in total amount of this protein was observed after treatment with NDGA and esculetin. We conclude that the proliferation ability of fibrosarcoma cells G:5:113 in vitro depends on intact functions of 5-lipoxygenase, 12-lipoxygenase, and cytochrome P-450-monooxygenases, and that the effects of inhibitors do not include regulation of apoptosis.
Absorption, metabolism and DNA binding of 2-nitrofluorene (NF) was studied in isolated, perfused and ventilated rat lungs and in lung microsomal incubations. Comparisons were made between control animals and animals treated with beta-naphthoflavone (BNF), a 2,3,7,8-tetrachlorodibenzo(rho)dioxin (TCDD) receptor ligand and inducer of cytochrome P450IA1. Clearance of NF increased significantly in the isolated, perfused and ventilated lungs after BNF dosage, from 0.55 +/- 0.06 ml/min to 2.37 +/- 0.62 ml/min (P less than 0.05, n = 5-6). As a consequence of this, the mean residence time (MRT) for NF decreased when NF was dosed directly to the perfusion buffer, from 213 +/- 23 min (n = 6) to 48 +/- 9 min (n = 6), and after intratracheal dosage from 289 +/- 101 min (n = 5) to 135 +/- 72 min (n = 5). Irreversible binding of NF metabolites to DNA increased 2-fold after treatment with BNF when NF was dosed to the lung perfusion buffer. Treatment with BNF increased the rate of lung microsomal NF metabolism significantly, from 54 +/- 5 to 106 +/- 11 pmol/min/mg protein (P less than 0.05, n = 6-12). Formation of the monohydroxylated metabolite X-OHNF was inhibited in vitro by addition of alpha-naphthoflavone (50 microM), by 89 and 98% with lung microsomal fractions from control and BNF-treated rats respectively. In contrast, proadifen (50 microM) preferentially inhibited formation of 9-OHNF, by 42 and 33% in incubations with lung microsomal fractions from control and BNF-treated animals. Anti-P450IIB1-IgG inhibited formation of 9-OHNF by 96 and 45% with lung microsomes from control and BNF-treated rats respectively. Formation of X-OHNF was unaffected by addition of anti-P-450IIB1-IgG in both cases. These results show that both constitutive and inducible microsomal rat lung enzymes metabolize NF. A constitutive enzyme, most likely cytochrome P450IIB1, catalyzes metabolic attack on NF with high preference for the 9-position. A BNF-inducible microsomal enzyme, most likely cytochrome P450IA1, catalyzes hydroxylation of NF both in the 9-position and in other positions. Increased metabolic clearance, metabolism and DNA binding of NF after BNF treatment suggest that the level and specificity of cytochrome P450 isozymes may be important determinants for toxicity and availability of NF in the rat lung.
Northern and Western blot analyses, and analyses of microsomal metabolism of the carcinogen 2-nitrofluorene (NF) were conducted with the aim of studying age dependent cytochrome P-450b levels in the rat lung. The level of P-450b homologous mRNA and corresponding protein is very low in lungs from fetal and newborn rats. The levels then increase between 3 and 4 weeks of age, and reach adult levels at 6-8 weeks. No sex differences were detected with regard to lung P-450b mRNA levels or catalytical activities. Lung microsomal metabolism of NF increased in parallel with the accumulation of P-450b homologous mRNA and microsomal cytochrome P-450b protein concentration. Formation of the major metabolite, and potent mutagen, 9-hydroxy-2-nitrofluorene (9-OHNF) was significantly inhibited by addition of polyclonal anti-P-450b-IgG, and by addition of the inhibitor proadifen to incubations with lung microsomal protein. It is postulated that the observed, profound age-related differences in level and activity of lung cytochrome P-450b are likely to affect both availability and the ratio of metabolic detoxification and activation of chemical carcinogens deposited in the lung.
Medullipin I causes a delayed onset depressor response when injected intravenously into rats. The glyceryl compounds selachyl alcohol (SA) and monoolein (MO) cause similar vasodepression. The neutral lipid 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG) was suggested by Blank et al to be medullipin I (Med I, formerly ANRL). Biologic comparisons were made between Med I and various glyceryl compounds, including SA, MO, HAG, alkyl glyceryl ethers of phosphatidyl choline (termed APRL by us), diacylated SA, and the n-butyl boronic acid derivative of SA and MO. The n-butyl boronic acid derivative of Med I also was evaluated. The delay in onset of the depressor response to Med I was reduced by the injection of Med I into the portal vein; that of SA and MO was not. Med I, SA, and MO were activated by the liver, while APRL and HAG were not. Tween 20 inhibited Med I, SA, and MO, but not APRL and HAG. Proadifen (SKF 525A) inhibited Med I, but not SA and MO. The n-butyl boronic acid derivatives of SA, MO, and Med I were inactive. Med I, like SA and MO, appeared to have two hydroxyl groups in close proximity. It was concluded that Med I is neither HAG, APRL, SA, nor MO.
BACKGROUND: Inhalation anesthetics may interfere with the synthesis or action of endothelium-derived vasoactive factors. We investigated the effects of desflurane, enflurane, halothane, isoflurane, and sevoflurane on the release of nitric oxide and endothelium-derived hyperpolarizing factor (EDHF) in the isolated endothelium-intact carotid artery of the rabbit. METHODS: Isolated segments of the carotid artery were suspended in Krebs-Henseleit solution (37 degrees C) and preconstricted with phenylephrine (1 microM). Relaxations caused by acetylcholine (ACh) (0.03-10 microM) or sodium nitroprusside (0.01-10 microM) were compared in the presence or absence of the nitric oxide synthase inhibitor NG-nitro-L-arginine (0.1 mM) in segments exposed to desflurane (8%), enflurane (2-4%), halothane (2-3.5%), isoflurane (2-4%), or sevoflurane (2%) as well as in NG-nitro-L- arginine-treated segments exposed to enflurane (2%) in combination with the KCa(+)-channel blocker tetrabutylammonium (0.3 mM) or the cytochrome P450 inhibitor clotrimazole (3 microM). RESULTS: Desflurane, enflurane, and sevoflurane selectively inhibited the ACh-induced release of EDHF. Halothane and isoflurane also weakly affected the nitric oxide-mediated relaxant response to ACh. The inhibitory effect of these two anesthetics on EDHF release was concentration-dependent. Relaxations induced by sodium nitroprusside were not inhibited by any of the anesthetics tested. Three structurally unrelated cytochrome P450 inhibitors clotrimazole (0.1 mM), metyrapone (1 mM), and SKF525a (proadifen, 0.1 mM) abolished the EDHF-mediated relaxation elicited by ACh. The pharmacologic profile of the inhibitory effect of enflurane on the release of EDHF closely resembled that of clotrimazole but not that of tetrabutylammonium. Moreover, all anesthetics inhibited the cytochrome P450-catalyzed O-dealkylation of 7-ethoxycoumarin by rabbit liver microsomes in a concentration-dependent manner. CONCLUSIONS: Inhalation anesthetics significantly attenuate the EDHF-mediated relaxant response to ACh in the rabbit carotid artery. This effect appears to be attributable to inhibition of the cytochrome P450-dependent synthesis of EDHF by the endothelium.
The heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is a potent mutagen and is a mammary carcinogen in rodents. In man, hepatic activation is carried out by cytochrome P450 (CYP) 1A2 and the ultimate DNA-reactive species is thought to be a nitrenium ion formed via an acetoxy ester of an exocyclic amino group. Because most human breast tumours are ductal in origin, we investigated the ability of cell types present in the mammary gland (breast epithelial cells and neutrophils present in milk) to activate IQ to DNA-binding species using 32P-postlabelling. Phorbol myristate acetate-stimulated neutrophils produced a similar pattern of IQ-DNA adducts to that produced by human mammary epithelial cells. Adduct formation in stimulated neutrophils was inhibited 80% by the myeloperoxidase inhibitor sodium azide (1 mM) but was not affected by proadifen (100 microM), indomethacin (100 microM), or eicosatetraynoic acid (100 microM), inhibitors of cytochrome P450, prostaglandin synthetase, and lipoxygenase, respectively. Similar experiments in human mammary epithelial cells showed no azide inhibition of IQ-DNA adduct formation. Analysis of gene expression by reverse transcription-polymerase chain reaction showed that CYP1A1 and CYP1B1, but not CYP1A2, were expressed at detectable levels in untreated mammary epithelial cells, whereas in neutrophils cytochrome P450 expression was confined to low levels of CYP1A1. In cultured epithelial cells, IQ-DNA adduct formation and CYP1A1, but not CYP1B1 expression were induced threefold by benz[a]anthracene treatment; IQ-DNA adduct formation was inhibited by alpha-naphthoflavone. Our results indicate possible mechanisms for the metabolic activation of dietary carcinogens in the human breast.
ET-743 is a marine anti-cancer drug and is currently in phase I trials in which the effect of combination therapies will be investigated. Its dose-limiting toxicity in patients is hepatotoxicity. In-vitro studies have shown that ET-743 is mainly metabolized by cytochrome P450 (CYP) 3A4, but also by 2C9, 2C19, 2D6 and 2E1, and the phase II enzymes uridine diphosphoglucuronosyl transferase and glutathione-S-transferase. Based on this metabolic profile, there is a risk of drug-drug interactions possibly influencing the hepatotoxicity of ET-743. Therefore, the effect of CYP and phase II activity on the cytotoxicity of ET-743 was investigated in vitro in a human cell line model system. The effect of different CYP and phase II inhibitors and CYP inducers on ET-743 cytotoxicity was studied after 48 and 120 h of treatment in Hep G2 cells using different assays. Furthermore, the toxicity of ET-743 metabolites was investigated. Potent cytotoxic activity of ET-743 after 120 h treatment was observed, which could be increased in combination with the CYP inhibitors metyrapone (3A4), phenanthrene (substrate for 2E1, 3A4), piperonyl butoxide (3A), proadifen (2C9, 2E1, 3A4), ritonavir (3A4), and warfarin (2C9, 2C19). No effect on the cytotoxicity of ET-743 was observed in combination with phase II enzyme inhibition and CYP induction. CYP metabolites of ET-743 were less toxic compared with ET-743. These findings indicate that combination therapy of ET-743 with CYP inhibitors, e.g. other anti-cancer drugs, could lead to changes in the hepatotoxicity of ET-743 and are therefore of clinical importance.
By techniques involving differential centrifugation and specific precipitation with CaCl2, it was shown that dimethylamine and trimethylamine mono-oxygenase activities co-sediment with NADPH-cytochrome c reductase activity in sphaeroplast lysates of Candida utilis grown on trimethylamine as sole nitrogen source. Since the active fraction also contained low levels of cytochromes P-450 and P-420, it was concluded that the two amine mono-oxygenases are located in the smooth endoplasmic reticulum and thus end up in the microsomal fraction on cell fractionation. Ten to twenty-fold enrichment of mono-oxygenase specific activity could be achieved by separation of activity from soluble protein by centrifugation or gel filtration. Cell-free extracts prepared in the absence of FAD showed only very low mono-oxygenase activity for either substrate. Some activity could be restored by addition of flavin nucleotides: there was a fivefold stimulation by FAD and a fourfold stimulation by FMN. All trimethylamine mono-oxygenase activity was lost when a partially purified preparation containing both activities was incubated for more than 24 h at 0 degrees C, suggesting that separate enzymes are responsible for the oxidation of secondary and tertiary amines. The enzyme preparation oxidized a wide range of secondary alkylamines up to dibutylamine and tertiary alkylamines up to tributylamine. Primary amines, choline, di- and triethanolamine, spermine, spermidine and substituted anilines were not oxidized. NADH had a lower apparent Km value and higher Vmax value than NADPH. Secondary and tertiary alkylamines containing more than one kind of alkyl group gave more than one kind of aldehyde on oxidation. Stoicheiometry determinations showed a consumption of 1 mol NAD(P)H and 1 mol O2 per mol aldehyde formed. Carbon monoxide, cyanide, proadifen hydrochloride (SKF 525-A), mercurials and mercaptoethanol all inhibited both activities.
1. Calcium responses induced by brief stimulation with acetylcholine (ACh) were assessed from the fluorescence changes in fura-2 loaded submucosal arterioles of the guinea-pig small intestine. 2. Initially, 1-1.5 h after loading with fura-2 (fresh tissues), ACh increased [Ca2+]i in a concentration-dependent manner. This response diminished with time, and finally disappeared in 2-3 h (old tissues). 3. Ba2+ elevated [Ca2+]i to a similar extent in both fresh and old tissues. ACh further increased the Ba2+-elevated [Ca2+]i in fresh tissues, but reduced it in old tissues. Responses were not affected by either indomethacin or nitroarginine. 4. In fresh mesenteric arteries, mechanical removal of endothelial cells abolished the ACh-induced increase in [Ca2+]i, with no alteration of [Ca2+]i at rest and during elevation with Ba2+. 5. In the presence of indomethacin and nitroarginine, high-K+ solution elevated [Ca2+]i in both fresh and old tissues. Subsequent addition of ACh further increased [Ca2+]i in fresh tissues without changing it in old tissues. 6. Proadifen, an inhibitor of the enzyme cytochrome P450 mono-oxygenase, inhibited the ACh-induced changes in [Ca2+]i in both fresh and Ba2+-stimulated old tissues. It also inhibited the ACh-induced hyperpolarization. 7. In fresh tissues, the ACh-induced Ca2+ response was not changed by apamin, charybdotoxin (CTX), 4-aminopyridine (4-AP) or glibenclamide. In old tissues in which [Ca2+]i had previously been elevated with Ba2+, the ACh-induced Ca2+ response was inhibited by CTX but not by apamin, 4-AP or glibenclamide. 8. It is concluded that in submucosal arterioles, ACh elevates endothelial [Ca2+]i and reduces muscular [Ca2+]i, probably through the hyperpolarization of endothelial or smooth muscle membrane by activating CTX-sensitive K+ channels.
12-Hydroxyeicosatetraenoic acid (12-HETE) production from arachidonic acid by cerebral microvessels isolated from perfused adult murine brain was reduced by the lipoxygenase inhibitors baicalein, esculetin, gossypol, nordihydroguaiaretic acid, and quercetin. Except for quercetin and gossypol, the IC50 did not exceed 10 microM. Each inhibitor, except baicalein, also decreased microvessel prostaglandin production when present in concentrations above their IC50 value for 12-HETE. In contrast, inhibitors of the cytochrome P450 monooxygenase system, clotrimazole, metyrapone, and proadifen (SKF-525A), had little effect on microvessel 12-HETE production. Chiral phase HPLC analysis revealed that only the (S) enantiomer of 12-HETE was formed. The major microvessel metabolite of eicosapentaenoic acid co-eluted with 12-hydroxyeicosapentaenoic acid (12-HEPE) on reverse-phase HPLC and the (S) enantiomer of 12-HEPE on chiral phase HPLC. Furthermore, like 12-HETE, 12-HEPE production was blocked by lipoxygenase inhibitors. These studies demonstrate that brain microvessels produce only the (S) enantiomeric 12-hydroxy derivatives of both arachidonic acid and eicosapentaenoic acid by the action of a lipoxygenase that can be selectively inhibited by baicalein. Since arachidonic acid and eicosapentaenoic acid are available to cerebral blood vessels in certain pathological settings, these 12-hydroxy acid lipoxygenase products may mediate some of the cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.
The binding of the antipsychotic agent 3H-remoxipride to membranes of rat liver and brain (whole brain and cerebellum) was studied with filtration technique. Saturable high affinity binding was obtained for all three types of tissue preparations. Heterogenous binding sites were found since various types of compounds inhibited the binding of 3H-remoxipride with shallow dose-response curves. In the liver preparation it was possible to differentiate between two different binding sites. One site, called rem1, was defined with 100 nM alaproclate and bound sigma 2 ligands with high affinity, e.g. haloperidol, GBR 12909, DTG and (+)-3-PPP. High correlation (r = 0.93) was obtained between the inhibition of the binding of 3H-remoxipride to the rem1 site and the inhibition of the binding of the sigma ligand 3H-DTG reported previously (Ross 1991), indicating that the rem1 site is identical to a sigma 2-like binding site. The other 3H-remoxipride binding site in the rat liver, rem2 appears to be identical to the site binding alaproclate, proadifen and cocaine with high affinity. Cd2+ and Zn2+ were potent inhibitors of both binding sites, Cd2+ particularly of rem2 binding and Zn2+ preferably of rem1 binding. The apparent Bmax (pmol/g original tissue) and KD (nM) values for rem1 binding were 441 +/- 43 and 80 +/- 14, and for rem2 binding 727 +/- 116 and 95 +/- 6. The binding of 3H-remoxipride to the brain preparations was also inhibited with shallow dose-response curves.(ABSTRACT TRUNCATED AT 250 WORDS)
In rats with inflammation induced by turpentine injection, changes in drug disposition occur in-vivo and in the perfused isolated liver. Therefore the biotransformation of a low extraction drug, antipyrine, and of two high extraction drugs, lignocaine and propranolol, has been evaluated in the 9000g supernatant fraction of the liver of turpentine-treated rats. Aminopyrine N-demethylase activity and cytochrome P450 content were also measured. Turpentine treatment significantly reduced the in-vitro breakdown of the three drugs; aminopyrine N-demethylase activity and cytochrome P450 content were also decreased. Similar results were found in the proadifen-treated rats, except that in those, the cytochrome P450 content was slightly increased. The changes in drug disposition seen after turpentine-induced inflammation, could therefore be due in part to a change in hepatic enzymatic activity.
The capacity of human liver microsomes to N-oxidize guanethidine from 25 subjects has been assessed. Guanethidine N-oxidation was optimal at pH 8.5 and proceeded at only 16% of the maximal rate at pH 7.4. The mean rates of guanethidine N-oxidation at pH 8.5 and 7.4 were 2.46 +/- 0.89 (mean +/- s.d., n = 25) and 0.38 +/- 0.22 (mean +/- s.d., n = 22), respectively. Interindividual differences in the rate of guanethidine N-oxidation at pH 8.5 and 7.4 were 17- and 11-fold, respectively. The cytochrome P450 inhibitors, proadifen and 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), at both pH 8.5 and 7.4 caused less than 20% reduction in the rate of guanethidine N-oxidation by human liver microsomes. These data indicate that guanethidine N-oxidation can be used as a measure of flavin-containing monooxygenase activity in human liver.
A simple fluorometric assay method as well as a sensitive HPLC method for determination of bunitrolol, a beta-adrenoreceptor blocking drug, and its 4-hydroxylated metabolite is described. More than 90% of bunitrolol metabolized was accounted for by the formation of 4-hydroxybunitrolol in rat hepatic microsomes. Bunitrolol 4-hydroxylation required NADPH, and was inhibited by CO, proadifen and metyrapone, indicating that this reaction is mediated by cytochrome P450. This reaction was also inhibited by cimetidine competitively, and the inhibition constant, Ki, was 40 +/- 11.5 microM (mean +/- s.e. n = 3).