Characterization of pirenzepine interaction with cytochrome P-450

Pirenzepine interacts with the haem iron of cytochrome P-450 from rat-and pig-liver microsomes, to give absorption spectra with max. at 424-429 nm, and min. at 391-399 nm. Binding to cytochrome P-450 was not detected with human-liver microsomes. Inhibition of 7-ethoxycoumarin dealkylation by pirenzepine using rat-liver microsomes gave values of I50 = 5 mM and Kis = 0.53 mM. E.p.r. spectra showed that pirenzepine probably interacts with the haem iron through the pirenzepine N-4(1) tertiary amine group.     

Cimetidine and ranitidine: their interaction with human and pig liver microsomes and with purified cytochrome P-450

Cimetidine and ranitidine interact with microsomes from human and pig liver and with purified cytochrome P-450 in the ligand-type manner. The affinity for cimetidine is about 10 times as high as that for ranitidine. Accordingly amplitudes of the specta are much higher for cimetidine. These results are in accordance with those obtained earlier with rat liver microsomes. The inhibitory potency of either compound with regard to dealkylation of 7-ethoxycoumarin appears to be less in the human preparation.     

Characterization of cimetidine, ranitidine, and related structures' interaction with cytochrome P-450

Cimetidine (I) interacts with the hemin iron of cytochrome P-450 from rat liver microsomes, with its imidazole and cyano coordinating groups. Ranitidine (II) interacts through its nitronic acid oxygen and its amine nitrogen, as shown by optical difference and ESR-spectra. I, N-cyano-N'[2-[[[5-(dimethylamino)-methyl-2-furanyl]methyl] thio]-ethyl]-N"-methyl guanidine (IV), 4(5)-hydroxymethyl-5(4)-methyl imidazole (VII), 4(5)-methyl-5(4)-[(2-aminoethyl)-thiomethyl]-imidazole hydrochloride (IX), 2-[[[(5-dimethylamino)-methyl-2-furanyl]-methyl]-thio]ethene amine dihydrochloride (X) and imidazole (XI) inhibit 7-ethoxycoumarin dealkylation competitively. In I both imidazole and cyano groups contribute to the inhibitory activity, the latter group being more effective according to electron spin resonance. Mixed type inhibition was observed with II, desmethylranitidine (VIII) and N-[[2-(5-methylimidazol-4-yl)methylthio]-ethyl]-N'-methyl-2-nitro-1, 1-ethenediamine (III). These compounds inhibited the reaction to a small extent; ranitidine S-oxide (VI) did not interact at all with microsomes from phenobarbital-pretreated rats. Using microsomes from 3-methylcholanthrene-pretreated rats, the affinities of interaction and the amplitudes of optical difference spectra were higher with VIII than with its parent, compound II.     

Metabolism of tetraorganolead compounds by rat-liver microsomal mono-oxygenase. II. Enzymic dealkylation of tetraethyl lead

The biological degradation of tetraethyl lead to the triethyl lead cation by rat-liver microsomes from untreated, phenobarbital-pretreated and methylcholanthrene-pretreated rats has been studied; NADPH and oxygen are essential. The reaction is inhibited by CO and can be reactivated in the presence of O2 by irradiation with u.v. light with a max. at 450 nm. Substrate binding to cytochrome P-450 is of type 1. Apparent Km values for triethyl lead formation in microsomes were determined. The highest activities (i.e. about 2 nmol triethyl lead per nmol cytochrome P-450 per min) and the lowest apparent Km values (i.e. 7 X 10(-6) M) are found in microsomes from methylcholanthrene-pretreated rats. In microsomes from control and phenobarbital-pretreated rats Ks values from substrate-binding studies (about 2 X 10(-6) M) are one order of magnitude lower than the apparent Km values (3 X 10(-5) M).     

Differential effects of proteinase K on the components of the liver microsomal cytochrome P-450 mixed function oxidase system.

Mild proteolysis of rat liver microsomes with increasing concentrations of proteinase K caused a marked decrease in the levels of microsomal cytochrome P-450 reductase (Fp) without having any significant effect on the cytochrome P-450s. About 20% of the microsomal cytochrome b5 was susceptible to proteolysis at low concentrations of proteinase K, while the remaining 80% was resistant to proteolysis, even at significantly higher proteinase K concentrations. Low concentrations of the proteases released about 30% of Fp from microsomes isolated from both uninduced and phenobarbital-induced rats, but did not affect the rates of benzphetamine bital-induced rats, but did not affect the rates of benzphetamine demethylation significantly. Further depletion of microsomal Fp at higher concentrations of proteinase K resulted in reductions of the rates of benzphetamine demethylation. However, even at higher protease concentrations, the decrease in the rate of the demethylation reaction was significantly less than the loss of Fp. Similar results were observed for the metabolism of two other substrates, 7-ethoxycoumarin and p-nitroanisole, suggesting that the P-450s, not the Fp, were the rate-limiting components in the metabolism of these xenobiotics by microsomes. It is clear that the decreases in the P-450-dependent oxidations were due to depletion of the NADPH-cytochrome P-450 reductase since reconstituting the protease-treated microsomes with native Fp restored the oxidation reactions. The amount of Fp required to completely restore the oxidation of benzphetamine only partially restored the oxidation of 7-ethoxycoumarin and p-nitroanisole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymic and electrophoretic characterization of the hexachlorobenzene-induced cytochrome P-450 in the liver of rat

1. Hexachlorobenzene induces the cytochtome P-450 system in rat liver microsomes. The catalytic activity of the enzyme towards various substrates (dealkylation of 7-ethoxycoumarin, hydroxylation of biphenyl and NADPH-dependent reduction of cytochrome c) corresponds to that obtained in animals treated with a mixture of phenobarbital and benzpyrene.

2. Electrophoretic separation of the partly purified hexachlorobenzene-induced cytochrome P-450 in sodium dodecyl sulphate polyacrylamide gels exhibits a protein pattern similar to that found in microsomes from rats treated with phenobarbital plus benzpyrene.     

Inhibition by SKF 525-A of 7-ethoxycoumarin O-deethylation in microsomal and the reconstituted monooxygenase systems from PCB-treated rat livers

Addition of diethylaminoethyl 2,2-diphenylvalerate-HCl (SKF 525-A) to the incubation mixture containing liver microsomes or purified cytochrome P-450 (PCB P-450) from PCB (KC-500)-treated rats resulted in non-competitive inhibition of 7-ethoxycoumarin O-deethylation activity whereas the addition to the incubation mixture containing purified cytochrome P-448 (PCB P-448) showed a competitive inhibition. Fortification of PCB-induced microsomes with purified NADPH-cytochrome P-450 reductase enhanced the O-deethylation activity. With the reductase-fortified microsomes, SKF 525-A inhibited the O-deethylation in a competitive manner. Based on these results, we confirmed that SKF 525-A inhibits non-competitively and competitively, depending on the species of cytochrome P-450. Our results also support the view that in microsomes from PCB-treated rats, PCB P-450 rather than PCB P-448 is mainly involved in the O-deethylation reaction, presumably due to the presence of a limited amount of NADPH-cytochrome P-450 reductase in microsomes.     

Cimetidine interaction with liver microsomes in vitro and in vivo. Involvement of an activated complex with cytochrome P-450

The O-deethylation of 7-ethoxycoumarin was inhibited in a mixed type manner by cimetidine in vitro and in microsomes isolated from rats treated with cimetidine in vivo. It was found that the inhibition was even greater if cimetidine was preincubated with the microsomal suspension in the presence of an NADPH-generating system prior to the addition of substrate. In vitro the decrease in activity was accompanied by a decrease in cytochrome P-450 content. This decrease was unaffected by the addition of EDTA to the microsomal suspensions, eliminating the possibility that free radical production was responsible for the decrease in cytochrome P-450. The decrease in activity and cytochrome P-450 content following preincubation of microsomal suspensions with cimetidine could be attenuated if potassium ferricyanide was added to the suspensions. The deethylation activity and cytochrome P-450 content of liver microsomes prepared from cimetidine-treated rats was decreased compared to control animals. The activity and cytochrome P-450 content of microsomes from cimetidine-treated rats could also be restored if microsomes were washed with potassium ferricyanide prior to incubation with substrate. It is proposed that an intermediate complex of cimetidine and cytochrome P-450 could be involved in the inhibition of microsomal metabolism by cimetidine.     

The interaction of styrene oxide with hepatic cytochrome P-450 in vitro and effects of styrene oxide inhalation on xenobiotic biotransformation in mouse liver and kidney.

Female mice (strain CB-20) were killed 0.5 hr, 18 hr and 5 days after inhaling styrene oxide for 6 hr per day for 3 consecutive days at concentrations of 200, 200 and 100 p.p.m., respectively. Acute intoxication was manifest, both clinically and as a depression of non-protein sulfhydryl content in liver and kidney. During the recovery period of 5 days a transient rise in microsomal 7-ethoxycoumarin O-deethylase activity in both tissues paralleled changes in cytochrome P-450 content. The activity of microsomal epoxide hydratase (measured with styrene oxide as the substrate) was not affected by the treatment, neither was the UDP-glucuronosyltransferase activity. In the presence of hepatic microsomes from phenobarbital-treated mice, styrene oxide produced a characteristic Type I difference spectrum. A comparison of the binding parameters for the interaction of styrene oxide with uninduced and phenobarbital- and 3-methylcholanthrene-induced microsomes indicates that the binding of styrene oxide is catalyzed by more than one type of P-450 hemoprotein. but predominantly by phenobarbital-induced cytochrome P-450. In addition, in phenobarbital-pretreated microsomes styrene oxide had two spectral dissociation constants (K_s), 0.05 mM and 0.4 mM.     

Clotrimazole as an inhibitor of benzo[a]pyrene metabolite-DNA adduct formation in vitro and of microsomal mono-oxygenase activity

The fungistatic drug clotrimazole (1-[(o-chlorophenyl)diphenylmethyl]imidazole) in concentrations of 5 or 50 microM completely prevented the formation of benzo[a]pyrene metabolite-DNA adducts in vitro catalyzed by liver microsomes from phenobarbital-or 3-methylcholanthrene-treated rats, respectively. Microsomal 7-ethoxycoumarin de-ethylase and aryl hydrocarbon hydroxylase were effectively inhibited by clotrimazole and three clotrimazole derivatives in all induction states tested, with I50 values down to 7 x 10(-8) M. The mechanism of inhibition was noncompetitive in phenobarbital-stimulated microsomes. Microsomal epoxide hydratase in vitro was enhanced up to 450% by clotrimazole and one of the analogues in concentrations between 5 and 500 microM. Clotrimazole spectrally interacted with reduced cytochrome P-450, exhibiting a double-banded Soret region with peaks at 427 and 446 nm, and partially prevented cytochrome P-450-CO complex formation. When administered in vivo, clotrimazole effectively induced cytochrome P-450 content, mono-oxygenase activity and epoxide hydratase activity in rat liver microsomes. The induction pattern was similar to that obtained with phenobarbital. The analogues were less potent inducers.     

Inhibition of human liver cytochrome P-450 by omeprazole.

The effects of omeprazole on cytochrome P-450 mediated 7-ethoxycoumarin deethylation were studied in human liver microsomes. Omeprazole inhibited both the high and low affinity components of deethylation, with an estimated Ki of 0.03 mM for the high affinity component. The results are further evidence that the previously reported prolongation of the half-life of diazepam by omeprazole in vivo is due to inhibition of cytochrome P-450 monooxygenases.     

Inhibition of hepatic mixed-function oxidase activity in vitro and in vivo by various thiono-sulfur-containing compounds.

Ten thiono-sulfur-containing compounds of varying structure were administered by intraperitoneal injection to untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated adult male rats. Six hr later, the concentration of hepatic cytochrome P-450 and the ability of the hepatic microsomes to metabolize benzphetamine were examined. In the untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated groups, two, four and four compounds, respectively, significantly decreased the concentration of cytochrome P-450 in the hepatic microsomes. A similar effect on benzphetamine metabolism was also seen. When examined 48 hr after the administration of the ten thiono-sulfurcontaining compounds, four, five and seven of the compounds decreased both the levels of hepatic cytochrome P-450 and the rate of benzphetamine metabolism in the untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated animals respectively. Eight of the thiono-sulfur-containing compounds were incubated in the presence of NADPH with hepatic microsomes isolated from untreated, phenobarbital-pretreated or 3-methylcholanthrene-pretreated animals. All of the compounds examined significantly decreased the concentration of cytochrome P-450 in the microsomes from each treatment group. Similar reductions in benzphetamine metabolism were also seen. When these same compounds were incubated with microsomes in the absence of NADPH, no significant reduction of cytochrome P-450 or benzphetamine metabolism was seen. When the oxygen analogs of six of the thiono-sulfur compounds were administered in vivo or incubated with hepatic microsomes either in the presence or absence of NADPH, no significant reduction of cytochrome P-450 or benzphetamine metabolism was seen.     

A novel form of cytochrome P-450 in beagle dogs. P-450-D3 is a low spin form of cytochrome P-450 but with catalytic and structural properties similar to P-450d

A form of cytochrome P-450, P-450-D3, cross reactive with antibodies to rat P-450d was purified from liver microsomes of polychlorinated biphenyl (PCB)-treated female Beagle dogs to an electrophoretic homogeneity. Judging from the result of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of P-450-D3 was estimated to be 54,000. The oxidized form of P-450-D3 showed a peak at 416 nm indicating that the cytochrome is mostly in a low spin state. The carbon monoxide bound reduced form of P-450-D3 showed a peak at 448 nm. In a reconstituted system, P-450-D3 catalyzed drug oxidations including benzphetamine and aminopyrine N-demethylations, 7-ethoxycoumarin and p-propoxyaniline O-dealkylations, and aniline and benzo(a)pyrene hydroxylations. The rate of aniline hydroxylation catalyzed by P-450-D3 was similar to that catalyzed by P-450c which is a low spin form of cytochrome P-450 purified from liver microsomes of PCB-treated rats, whereas the catalytic activities of P-450-D3 for 7-ethoxycoumarin O-deethylation and benzo(a)pyrene hydroxylation were considerably lower than those of P-450c. The amino terminal portion of P-450-D3 was found to be highly similar to those of P-450d, human P3-450 and P3-450 when four amino acid deletions were tentatively inserted between fifth and sixth amino acids from the N-terminal, but not that of P-450c which is a low spin form of cytochrome P-448 purified from rat liver microsomes. These results indicate that Beagle dogs possess a low spin form of cytochrome P-450 with spectral properties similar to P-450c but with catalytic and structural properties similar to P-450d.     

Metabolism of tetraorganolead compounds by rat-liver microsomal mono-oxygenase: II. Enzymic dealkylation of tetraethyl lead

1. The biological degradation of tetraethyl lead to the triethyl lead cation by rat-liver microsomes from untreated, phenobarbital-pretreated and methylcholanthrene-pretreated rats has been studied; NADPH and oxygen are essential.

2. The reaction is inhibited by CO and can be reactivated in the presence of O₂ by irradiation with u.v. light with a max. at 450nm.

3. Substrate binding to cytochrome P-450 is of type 1.

4. Apparent K_m values for triethyl lead formation in microsomes were determined. The highest activities (i.e. about 2 nmol triethyl lead per nmol cytochrome P-450 per min) and the lowest apparent K_m values (i.e. 7 × 10^-6 M) are found in microsomes from methylcholanthrene-pretreated rats.

5. In microsomes from control and phenobarbital-pretreated rats K_s values from substrate-binding studies (about 2 × 10^-6M) are one order of magnitude lower than the apparent K_m values (3 × 10^-5 M).     

Purification and properties of cytochrome P-450 and NADPH-cytochrome c (P-450) reductase from human liver microsomes.

Cytochrome P-450 and NADPH-cytochrome c (P-450) reductase were purified to 10.6 nmoles per mg of protein and 19.9 units per mg of protein, respectively, from human liver microsomes. The purified cytochrome was assumed to be in a low spin state as judged by the absolute spectrum. n-Octylamine and aniline produced type II difference spectra and SKF 525-A and benzphetamine type I spectra when bound to the purified cytochrome P-450. The purified human cytochrome P-450 catalyzed laurate oxidation as determined by NADPH oxidation but not aniline hydroxylation, benzphetamine N-demethylation and 7-ethoxycoumarin O-deethylation when reconstituted with the reductases purified from human and rat liver microsomes. The human cytochrome P-450, however, catalyzed drug oxidations when cumene hydroperoxide was used as the oxygen source. The purified human NADPH-cytochrome c (P-450) reductase contained FAD and FMN at a ratio of 1:0.76. The reductase was capable of supporting 7-ethoxycoumarin O-deethylation activity of cytochrome P-448 purified from 3-methylcholanthrene-treated rat liver microsomes.     

Interaction of miconazole, ketoconazole and itraconazole with rat-liver microsomes

The interaction of the antimycotics miconazole, ketoconazole and itraconazole with liver microsomes from untreated rats or from rats pretreated with phenobarbital or 3-methylcholanthrene, gave rise to type II difference spectra. The interactions of the antimycotics with control, phenobarbital-induced or 3-methylcholanthrene-induced microsomes were biphasic, except for the monophasic binding of ketoconazole to phenobarbital-induced microsomes. The N-demethylation of N,N-dimethylaniline, the O-demethylation of p-nitroanisole and the hydroxylation of aniline in microsomes from untreated and inducer-treated rats were lowered by miconazole and ketoconazole, the former being the more potent inhibitor. Control microsomes were less sensitive than induced microsomes. Itraconazole was almost devoid of inhibitory properties. The three antimycotics were non-competitive (mixed) inhibitors of enzyme activities in phenobarbital-induced microsomes. The Ki values were of the same order of magnitude as the Ks values, except for itraconazole. For the latter drug, Ki values were much greater than could be expected from the spectral studies. It is concluded that the antimycotics affect microsomal enzyme activities via a direct interaction of an azole-nitrogen with the haem group of cytochrome P-450. The interaction with mammalian cytochrome P-450 decreases from miconazole greater than ketoconazole much greater than itraconazole and is much weaker than the interaction of the antimycotics with yeast cytochrome P-450.     

Self-catalyzed inactivation of cytochrome P-450 during microsomal metabolism of cannabidiol

When cannabidiol (CBD) was incubated with hepatic microsomes of mice in the presence of an NADPH-generating system, a significant decrease of cytochrome P-450 content was observed by measuring its carbon monoxide difference spectra. The decrease of cytochrome P-450 by CBD required NADPH and molecular oxygen. The effect was partially inhibited by SKF 525-A but not by various scavengers of active oxygen species, superoxide anion, hydroxyl radical and singlet oxygen. The incubation of CBD with hepatic microsomes did not affect total heme but decreased significantly free sulfhydryl contents in the microsomes. The derivatives of CBD modified in the resorcinol moiety, CBD-monomethyl- and dimethylethers, almost lost the effect on cytochrome P-450, whereas those modified in the terpene moiety, 8,9-dihydro- and 1,2,8,9-tetrahydro-CBDs exhibited some potency to inactivate cytochrome P-450. The inactivation of cytochrome P-450 by CBD and related compounds led to the inhibition of hepatic microsomal p-nitroanisole O-demethylase and aniline hydroxylase activities. These results suggest that the resorcinol moiety of CBD plays some role in the inactivation of cytochrome P-450 by the cannabinoid.     

Stimulatory and inhibitory effects of dimethyl sulfoxide on microsomal aniline hydroxylase activity

Dimethyl sulfoxide (DMSO) at a single dose of 3 ml/kg body wt, administered i.p. to male rats, caused a significant increase in the hepatic microsomal aniline hydroxylase activity. However, the level of cytochrome P-450, the activities of NADPH-cytochrome c reductase, benzphetamine N-demethylase and aminopyrine N-demethylase were unchanged at 24 h post-treatment. DMSO interacted with control rat liver microsomes in vitro and produced a type II spectral change (peak at 420 nm and trough at 392 nm). On the other hand, liver microsomes from DMSO-treated rats gave qualitatively similar spectra, but with a higher magnitude of binding. Liver microsomes from DMSO-treated rats showed a 3.4-fold increase in Vmax for aniline hydroxylase, while Km was found to be the same when compared with control rat liver microsomes. In vitro addition of 6 mM DMSO to microsomal incubations from control and DMSO-treated rats caused a 9-fold and a 25-fold increase in Km, respectively, while Vmax values for aniline hydroxylase were unchanged. When DMSO (6 mM) was incubated with rat liver microsomes in the presence of NADPH, there was formation of formaldehyde. The results suggest an interaction of DMSO with microsomal cytochrome P-450.     

In vivo effect of ozone inhalation on xenobiotic metabolism of lung and liver of rats

Male Jcl:Wistar rats were exposed to 0.4 and 0.8 ppm O3 daily for 7 h for 14 d to examine the effect of O3 on xenobiotic metabolism of lung and liver microsomes. An exposure to 0.4 ppm O3 did not affect the microsomal xenobiotic metabolism of either lung or liver. On the other hand, 0.8 ppm O3 increased significantly the NADPH-cytochrome P-450 reductase activity and the cytochrome P-450 content of lung microsomes. The activities of lung benzo[a]pyrene hydroxylase and 7-ethoxycoumarin O-deethylase also increased significantly on d 7, and remained at a higher level by the d 14. These results show that exposures to 0.8 ppm O3 induce the xenobiotic metabolizing systems in the lung. In the liver, after the first day of exposure to 0.8 ppm O3, a significant reduction occurred in all components of the electron-transport systems examined as well as in the microsomal protein. A significant decrease was also observed in benzo[a]pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and aniline hydroxylation. The decreased activities recovered in the following period of exposure. In contrast, the p-nitroanisole N-demethylase activity was not altered during the 14-d exposures. These results suggest that some isozymes of the hepatic cytochrome P-450 are sensitive to O3 inhalation but other(s) are resistant.     

Comparative effects of ethanol administration on hepatic monooxygenases in rats and mice

The inducing effects of chronic ethanol ingestion on hepatic monooxygenases in Sprague-Dawley and Long-Evans rats, and A/J and C57BL/6J mice, were studied. Cytochrome P-450 content was significantly increased in livers of all animals receiving the experimental ethanol-containing liquid diet. The CO-difference spectra of microsomes from ethanol-treated animals showed a shift in the absorbance maximum to 451–452 nm, compared to the absorbance maximum of 450 nm observed with microsomes from control animals. Ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase activities in livers of ethanol-treated animals were minimally affected. The shift in the absorbance maxima to longer wavelengths in the CO-difference spectrum and the minimal effects on the metabolism of ethylmorphine and benzo[a]pyrene demonstrate that ethanol differs in its inducing properties, when compared to the properties of the two widely used hepatic microsomal enzyme inducers, phenobarbital and 3-methylcholantrhene. In contrast to the minimal effects observed on the metabolism of ethylmorphine and benzo[a]pyrene, several fold increases were observed in hepatic 7-ethoxycoumarin 0-deethylase and aniline hydroxylase activities in the treated animals. Polyacrylamide gel electrophoresis of hepatic microsomes from those animals receiving ethanol revealed protein band(s) in the cytochrome P-450 molecular weight region, the intensities of which were markedly increased relative to that from control animals. The heme-associated peroxidase activity was also increased in the same molecular weight region. The results of the present spectral, catalytic, and electrophoretic studies demonstrate that in mice, as in rats, chronic ethanol treatment causes the induction of specific cytochrome(s) P-450 with preferential activity toward aniline and 7-ethoxycoumarin.