[Cytochrome P-450 enzymes].
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Biological Evolution↗
Biomedical subjects
O Pelkonen
Publications and source records attributed to O Pelkonen.
Monoclonal antibody characterization of NADH- and NADPH-dependent hydroxylation of benzo(a)pyrene in liver microsomes from 5,6-benzoflavone-induced C57Bl/6 mice.
Monoclonal antibodies (MAb 1-7-1) directed against the isoenzymes of rat liver cytochrome P-450 induced by methylcholanthrene, inhibited benzo(a)pyrene hydroxylase less strongly at low (0.04 mM) than at high (2 mM) NADH concentration. Inhibition of NADPH dependent hydroxylation was the same irrespective of NADPH concentrations and corresponded to that observed at high NADH concentration. The same was also the h.p.l.c. pattern of the reaction products of benzo(a)pyrene hydroxylation supported by high concentration of both coenzymes. It is postulated that different cytochrome P-450 isoenzymes participate in benzo(a)pyrene hydroxylation, whereas the second one acts at high concentration of both NADH and NADPH.
Animals↗
[Cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria and human placenta: immunochemical properties and structural characteristics].
An immunochemical comparison of components of cholesterol side chain cleavage system from bovine adrenocortical and human placental mitochondria has been carried out. Antibodies against cytochrome P-450scc, adrenodoxin reductase and adrenodoxin from bovine adrenocortical mitochondria were shown to cross-react with corresponding antigens of human placental mitochondria. A highly sensitive immunochemical method for cytochrome P-450scc determination has been developed. Limited proteolysis of cytochrome P-450scc of human placental mitochondria was studied, and the products of trypsinolysis were identified using antibodies against cytochrome P-450scc and fragments of its polypeptide chain: F1, F2 and F3. Immunochemical relatedness of ferredoxins from bovine adrenocortical and human placental mitochondria allowed one to develop a fast and efficient method for cytochrome P-450scc purification from human placental mitochondria by affinity chromatography on adrenodoxin-Sepharose.
Adrenal Cortex↗
Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex.
The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.
Adrenal Glands↗
Comparison of the formation of benzo[a]pyrene diolepoxide-DNA adducts in vitro by rat and human microsomes: evidence for the involvement of P-450IA1 and P-450IA2.
The involvement of cytochrome P-450 isozymes in the activation of benzo[a]pyrene (BP) by human placental and liver microsomes was studied in vitro using monoclonal antibodies (Mab) toward the major 3-methylcholanthrene (MC)-inducible and phenobarbital-inductible rat liver P-450 isozymes (Mab 1-7-1 and Mab 2-66-3, respectively). Microsomes from human placenta and liver and rat liver were incubated with BP and DNA, and BP-diolepoxide-DNA (BPDE-DNA) adducts were measured by synchronous fluorescence spectrophotometry (SFS). The only BP metabolite giving the same fluorescence peak as chemically modified BPDE-DNA was BP-7,8-dihydrodiol. Five (smokers) out of 29 human placentas (smokers and nonsmokers), and five out of nine human livers were able to metabolically activate BP to BPDE-DNA adducts in this system. The Mab 1-7-1 totally inhibited the formation of BPDE-DNA adducts in placental microsomal incubations. Inhibition using rat or human liver microsomes was 50-60% and about 90%, respectively. The Mab 2-66-3 had no effect in any of the microsome types. Adduct formation was inhibited more strongly and at lower concentrations of Mab 1-7-1 compared with the inhibition of AHH activity. This study is a clear indication of the major role of P-450IA1 (P-450c) in human placenta and probably P-450IA2 (P-450d) in human liver in BP activation, while other isozymes also take part in the activation in rat liver. Furthermore, this clearly indicates that AHH activity and BP activation are not necessarily associated.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Benzo[a]pyrene-DNA-adducts and monooxygenase activities in mice treated with benzo[a]pyrene, cigarette smoke or cigarette smoke condensate.
Synchronous fluorescence spectrophotometry (SFS), developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide(BPDE)-DNA, was used to measure the in vivo formation of DNA-adducts in genetically responsive C57BL/6 (B6) and non-responsive DBA/2 (D2) mice. Treatment with cigarette smoke by inhalation for 3-16 days, or i.p. injection of cigarette smoke condensate or neutral fraction did not lead to detectable levels of BPDE-DNA-adducts in either lungs or liver, although aryl hydrocarbon hydroxylase (AHH) activity, an indicator of benzo[a]pyrene (BP) metabolism, was clearly induced in lungs of B6 mouse. A dose-dependent amount of BPDE-DNA-adducts in lung and somewhat less in liver was found after i.p. injection with BP (20-80 mg/kg). Mice treated with vehicle or 4 mg/kg of BP were negative for adducts by SFS. In B6 mice AHH was induced both in lungs and livers while there was no AHH induction in D2 mice although the levels of BPDE-DNA-adducts were somewhat higher than in B6 mice. Thus, no clear correlation seems to exist between AHH activity and the formation of BPDE-DNA-adducts. Also, according to our results SFS can be used to quantitate adduct-formation in in vivo animal studies.
7-Alkoxycoumarin O-Dealkylase↗
Inhibition of hepatic microsomal drug metabolism in rats by five calcium antagonists.
The effects of five calcium antagonists, verapamil, diltiazem, nifedipine, darodipine and isradipine, on rat liver microsomal drug metabolism in vitro and in vivo were studied. All compounds prolonged hexobarbital-induced sleeping time in a dose-dependent manner (doses 3.0 and 30.0 mg/kg, except nifedipine 0.3 and 3.0 mg/kg) and inhibited cytochrome P450-dependent N-demethylation of aminopyrine in vitro in rat liver microsomes. The incubation of all compounds with microsomes resulted in the apparent formation of formaldehyde, suggesting either N- or O-demethylation. Diltiazem, isradipine and darodipine gave rise to a type I spectral change. Nifedipine seemed to produce a type II spectral change. A spectrum of verapamil changed from a type I to a type II as concentration increased. These results indicate that all calcium antagonists studied interact with P450 and are in vitro inhibitors of microsomal drug metabolism in the rat and the inhibition brings out pharmacokinetic drug-drug interactions in vivo.
Animals↗
Preferential inhibition of mouse hepatic coumarin 7-hydroxylase by inhibitors of steroid metabolizing monooxygenases.
Etomidate, metomidate and metyrapone, all potent inhibitors of steroid metabolizing monooxygenases, inhibit preferentially coumarin 7-hydroxylase (COH) amongst several liver microsomal monooxygenase activities from control and pyrazole-treated D2 mice in vitro. SKF-525A, an inhibitor of phenobarbital-inducible monooxygenase activities has a much weaker effect on COH than the other three drugs, even though COH is a phenobarbital-inducible enzyme. Treatment of mice with eto- and metomidate decreases the microsomal COH also in vivo while the other activities remained unchanged (with the exception of 7-ethoxycoumarin O-deethylase (ECDE) in case of metomidate). Despite of the decrease in COH no parallel decrease in the amount of microsomal P450Coh (P450 isoenzyme highly active in the 7-hydroxylation of coumarin) could be found in dot immuno-binding analysis. These data suggest that among several liver microsomal P450 isoenzymes, metyrapone, eto- and metomidate interact preferentially with the P450Coh and that eto- and metomidate may alter selectively the catalytic properties of P450Coh leading to decreased enzyme activity. Two different Ks-values could be found for all three drug in their binding to microsomal cytochrome(s) P450. Based on substrate binding spectra, potassium ferricyanide treatment does not dissociate the complex between reduced P450 and metomidate and does it only partly for etomidate. Furthermore potassium ferricyanide treatment of microsomes does not increase COH after in vivo treatment of mice with eto- and metomidate. These data further suggest that the complex between P450Coh and eto- and metomidate may be particularly strong and independent from the redox state of the haem iron.
Animals↗
[Biochemical study procedures for verifying non-smoking: evaluating the usefulness of biochemical indicators of exposure to cigarette smoke].
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Carboxyhemoglobin↗
Mouse hepatic cytochrome P-450 isozyme induction by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, pyrazole, and phenobarbital.
The effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and pyrazole on mouse hepatic cytochrome P-450 isozyme expression were compared to the P-450 induction pattern elicited by phenobarbital. TCPOBOP and PB administration caused a similar induction profile by increasing microsomal protein and cytochrome P-450 content and the catalytic activities of several monooxygenases in DBA/2N and AKR/J mice. There were, however, several quantitative and some qualitative differences in the induction profile caused by phenobarbital and TCPOBOP. A few strain-related differences were also observed. Immunoblot analysis with polyclonal anti-coumarin hydroxylase (P-450Coh) antibody and epitope-specific monoclonal antibodies 1-7-1 and 2-66-3 showed that both phenobarbital and TCPOBOP increase the amount of P450IIB and P-450Coh. TCPOBOP caused a more pronounced increase in the amount of P-450IIB than phenobarbital, and TCPOBOP also caused an increase in the amount of P-450IA2. These data suggest that in the mouse, TCPOBOP increases mainly the expression of P-450 isozymes responsive to phenobarbital. The effects of pyrazole differed greatly from those caused by TCPOBOP and phenobarbital. In the DBA/2N mice, pyrazole increased coumarin 7-hydroxylation 9.4-fold, whereas in the AKR/J mice the activity was induced only to a level equivalent to the DBA/2N basal level. In immunoblot experiments with anti-P-450Coh antibody, the amount of P-450Coh was considerably higher in DBA/2N mice treated with phenobarbital, TCPOBOP, or pyrazole in comparison with the AKR/J mice, indicating a strain specificity in the inducibility of coumarin 7-hydroxylase by pyrazole.
Animals↗
Inhibition of human hepatic and placental xenobiotic monooxygenases by imidazole antimycotics.
Three imidazole antimycotic drugs, ketoconazole, clotrimazole and miconazole, were studied to characterize the inhibition of aryl hydrocarbon hydroxylase (AHH), 7-ethoxycoumarin O-deethylase (ECDE) and 7-ethoxyresorufin O-deethylase (ERDE) activities in human liver and placenta in vitro in comparison with liver enzymes from control, phenobarbital (PB) and 3-methylcholanthrene (MC) pretreated rats. All three compounds inhibited rat liver enzymes, although MC pretreatment seemed to lead to a resistance of inhibition relative to PB-treated and control animals. There were large differences in the extent of inhibition of human hepatic and placental activities. Furthermore, while the type of inhibition of the hepatic ERDE was competitive or mixed, that of the placental enzyme cannot be described in ordinary terms of inhibition kinetics. Ketoconazole and clotrimazole were relatively potent inhibitors of maternal cigarette smoking-induced placental ECDE activities (IC50 values from 0.5 microM to 5 microM), whereas much less inhibition of the placental AHH activity was obtained with ketoconazole and miconazole (IC50 values from 50 microM to 500 microM). In most cases, hepatic enzymes were less sensitive to antimycotics than placental activities. This was in contrast with results from rat enzyme studies, in which MC pretreatment seemed to decrease the inhibitory response.
7-Alkoxycoumarin O-Dealkylase↗
Immunochemical and catalytical studies on hepatic coumarin 7-hydroxylase in man, rat, and mouse.
The cytochrome P-450-mediated coumarin 7-hydroxylase (COH) was studied in microsomal preparations from Wistar rat, DBA/2N mouse, and human liver. Human liver contained the highest constitutive COH activity of up to about 500 pmol/mg microsomal protein/min. The rat liver contained low levels of COH (about 3-5 pmol/mg protein/min) which could be demonstrated only with high substrate concentrations. Rabbit polyclonal antibody generated against P-450Coh (a P-450 isozyme purified from pyrazole-treated DBA/2N mouse liver showing high activity for coumarin 7-hydroxylation) inhibited COH activity by almost 100% in human liver microsomes and 86-99% in mouse liver microsomes. Also the deethylation of 7-ethoxycoumarin was inhibited somewhat by the antibody, whereas no inhibition was obtained in ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities. None of these enzyme activities was affected by the antibody in the rat liver microsomes. In Ouchterlony immunodiffusion analysis precipitin lines were obtained with human, mouse and rat liver microsomes. Complex coalescence patterns were obtained suggesting full identity between human and pyrazole-treated mouse antigens, partial identity between mouse and rat antigens, and no identity between human and rat antigens. Western blot analysis with the anti-P-450Coh antibody revealed a distinct 48-kDa protein in all four human samples tested. A 50-kDa protein comigrating exactly with P-450Coh was observed in microsomes from PB and pyrazole-treated mouse liver microsomes. No distinct protein bands appeared in rat liver samples. These data suggest that despite slightly differing molecular masses, the human and mouse P-450s supporting COH are structurally conserved at their active centers. The corresponding rat P-450 appears to differ from that of mouse and man.
Animals↗
Pyrazole is different from acetone and ethanol as an inducer of the polysubstrate monooxygenase system in mice: evidence that pyrazole-inducible P450Coh is distinct from acetone-inducible P450ac.
The induction of liver microsomal monooxygenase activities elicited by pyrazole, ethanol, and acetone, all shown to be inducers of rat P450j and rabbit P450LM3a, has been compared in inbred strains of DBA/2N, AKR/J, and Balb/c mouse. Pyrazole strongly increases coumarin 7-hydroxylase (COH) activity in DBA/2N but much less in other strains. The effect of pyrazole on aniline p-hydroxylase and ethanol oxidase activities is also strain dependent: an increase was seen only in the DBA/2N strain. Ethanol and acetone were unable to induce COH, whereas aniline p-hydroxylase and ethanol oxidase were elevated about 1.4- to 3.3-fold in all strains. No strain difference could be detected in aniline p-hydroxylase or ethanol oxidase inducibility. There was a strong correlation between aniline p-hydroxylase and ethanol oxidase activities in every strain, whereas no positive correlation could be found between COH and aniline p-hydroxylase activities. Immunoinhibition experiments showed that a polyclonal antibody against purified pyrazole-inducible COH (P450Coh) blocked about 90% of COH activity, but only about 10% of aniline p-hydroxylase or ethanol oxidase in mouse liver microsomes. Monoclonal antibody 1-91-3 (raised against rat acetone-inducible P450ac) did not inhibit COH, whereas aniline p-hydroxylase was blocked 46-76% and ethanol oxidase 25-70%, depending on the source of microsomes. In immunoblots, anti-P450Coh recognized only its own antigen but not the P450ac, whereas monoclonal antibody 1-98-1 against P450ac detected P450ac and a corresponding form in the D2 mouse liver, but not the P450Coh. The purified P450ac and P450Coh had molecular masses of 52 and 50 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These antigens were expressed differentially in response to pyrazole, ethanol, and acetone: P450Coh was increased only after pyrazole treatment, but 1-98-1-detectable protein was elevated in D2 mouse liver microsomes by ethanol and acetone, but not by pyrazole. We conclude that mouse P450Coh and rat P450ac are not corresponding forms of the same isozyme, and that a P450ac-like protein, responsible for most of aniline p-hydroxylation and ethanol oxidation, is present in the D2 mouse liver. These two P450 isozymes are also dissimilarly expressed in the mouse liver in response to inducer administration.
Acetone↗
Polymorphism of debrisoquine hydroxylation among Finns and Lapps.
Debrisoquine hydroxylation polymorphism was studied in 155 Finns and 70 Lapps. The frequency of the poor metabolizer phenotype was 3.2% among Finns (95% confidence interval 0.4-6.0%) and 8.6% among Lapps (95% confidence interval 2.0-15.1%).
Debrisoquin↗
Inhibitor panel studies of human hepatic and placental cytochrome P-450-associated monooxygenase activities.
1. A panel of nine inhibitors displaying some P-450 isozyme specificity was used to characterize aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin 0-deethylase (ERDE) activities in human liver and placenta in vitro in comparison with liver enzymes from control, phenobarbital (PB) and 3-methylcholanthrene (MC) treated rats. 2. SKF 525A and cimetidine inhibited more potently hepatic AHH than the placental enzyme. 7,8-Benzoflavone inhibited more efficiently placental AHH than the hepatic enzyme, whereas ERDE was inhibited at the same level in both tissues. Quinine, quinidine, SKF 525A and metyrapone inhibited ERDE almost to the same extent in both tissues, but the variability was larger with the liver enzyme. Aminoglutethimide, debrisoquine or tetrahydrofuran did not inhibit AHH or ERDE significantly in either tissue. 3. When compared with inhibition profiles obtained with rat liver microsomes, the human hepatic and placental ERDE resembled most that of MC-treated rat liver enzyme. Inhibition profile of placental AHH activity was also similar, but the inhibition characteristics of hepatic AHH activity resembled more closely control or PB-induced rat liver. It also seems that isozymes for alcohol induction or debrisoquine hydroxylation do not contribute significantly to hepatic or placental AHH or ERDE. 4. The inhibitor panel selected on the basis of known pretreatment and isozyme specificity might be useful in the characterization of enzymes and metabolic biotransformations participating in the metabolism of new substrates.
Aminoglutethimide↗
[Medical treatment of the fetus].
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Drug-Related Side Effects and Adverse Reactions↗
Synchronous fluorescence spectrophotometry of benzo[a]pyrene diol epoxide-DNA adducts: a tool for detection of in-vitro and in-vivo DNA damage by exposure to benzo[a]pyrene.
We have applied synchronous fluorescence spectrophotometry (SFS) to study benzo[a]pyrene diol epoxide (BPDE)-DNA adducts in biological samples. Adducts are measured as benzo[a]pyrene (BP) tetrols after acid hydrolysis, and give a peak at 374 nm of emission with a 34-nm wavelength difference. In vitro and in animal studies, there is a positive correlation between the amount of adducts and the BP dose. In cell culture studies, the amount of adducts is increased by increasing both the dose of BP and the time of culture with BP. In preliminary human studies, BPDE-DNA has been found by SFS in placental DNA from some but not all smoking mothers and in blood cell DNA from some individuals in occupationally exposed groups.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Aryl hydrocarbon hydroxylase in lymphocytes and lung tissue from lung cancer patients and controls.
The inducibility of aryl hydrocarbon hydroxylase (AHH) activity in peripheral mitogen-treated lymphocytes, and of AHH and other monoxygenase activities in lung samples, was studied in 41 patients--34 with pulmonary carcinoma, 4 with a benign lung tumour and 3 with chronic obstructive pulmonary disease. Lymphocyte AHH induction alone was studied in 43 non-smoking and 37 smoking surgical patients. Absolute induced and non-induced AHH activities were at about the same level in the lymphocytes from the lung cancer patients as in those from the non-smoking controls, whereas the activities in smoking controls were about 100% higher. The mean inducibility ratios were very similar in all groups, ranging from 4.4 in the benign tumour patients to 5.4 in both control groups. Thymidine incorporation was on average about 40% lower in the lymphocytes from the lung cancer patients. AHH activity was detectable in all the peripheral lung samples, both normal or tumorous tissue, and its inter-individual variation was more than 67-fold. ECDE activity was also detectable in all the samples studied and its correlation with AHH activity was statistically significant (r = 0.888), suggesting that the same enzyme metabolizes both substrates. ERDE was detectable only in the samples with the highest AHH and ECDE activities. There was no correlation between basal or induced lymphocyte AHH activities and lung tissue AHH activity, but there were statistically significant correlations between lung AHH activity and the inducibility ratio with (r = 0.618) or without correction by thymidine incorporation (r = 0.442). These correlations suggest that there are common regulatory factors for AHH inducibility in different tissues. No significant difference in any drug metabolism parameter measured was observed between the lung cancer patients and the controls.
Animals↗