Alteration in surface structure of Clara cells and pulmonary cytochrome P-450b level in rats exposed to ozone.

Changes in the surface structure of Clara cells in the terminal bronchioles following exposure of rats to 0.4 ppm ozone (O3) for 14 days were evaluated and compared to the content of pulmonary cytochrome P-450, an enzyme active in xenobiotic metabolism. Exposure to O3 caused a striking alteration of Clara cells in the terminal bronchiole. After 6 h exposure apical protrusions of Clara cells enlarged and these Clara cells formed clusters. However after 24 h exposure, the Clara cells decreased in number and flattened. They increased in number and enlarged again during the subsequent period of exposure. By the 14th day of O3 exposure the number of Clara cells had increased significantly. The content of cytochrome P-450b (IIB1), a main isozyme of pulmonary cytochromes P-450 of rats, was determined by an immuno-blotting method using anti-cytochrome P-450b antibody. The cytochrome P-450b in the rats exposed to O3 increased significantly to 1.37- and 1.81-times that of the control on the 7th and 14th days, respectively. Immuno-electron microscopy demonstrated that cytochrome P-450b was localized abundantly in endoplasmic reticulum of Clara cells. Morphological alterations in Clara cells appear to be closely related with changes in the cytochrome P-450b content of the lung.     

Cytochrome P-450 isozyme selectivity in the oxidation of acetaminophen

Highly purified isozymes of cytochrome P-450 catalyzed the formation of 3-glutathion-S-ylacetaminophen (GS-APAP) and 3-hydroxyacetaminophen (3-OH-APAP) from acetaminophen (APAP). A major isozyme from untreated male rats (P-450UT-A) catalyzed the formation of ca. 2.0 nmol/nmol of P-450/10 min of 3-OH-APAP and approximately 7.2 nmol of GS-APAP/nmol of P-450/10 min. Antibodies specific for cytochrome P-450UT-A caused a decrease in the amounts of both metabolites produced in microsomal incubations. In contrast to these results, two other constitutive P-450 isozymes from rat liver, cytochrome P-450UT-F and the female specific isozyme P-450UT-I, produced less of both oxidative metabolites. Moreover, they produced significantly more of the catechol metabolite than the glutathione conjugate. These results are in accord with the observation that male rats are more susceptible to acetaminophen hepatotoxicity than female rats. Isozymes induced by phenobarbital also produced more of the catechol than the glutathione conjugate. Conversely, the major isozyme induced by beta-naphthoflavone, cytochrome P-450 beta NF-B, produced a significantly greater amount of GS-APAP than 3-OH-APAP. When comparison was made to a major phenobarbital inducible form (cytochrome P-450PB-B) a definite isozyme specificity for the formation of the two metabolites was seen. The catechol was formed at rates of 2.21 and 0.53 nmol/nmol of P-450/10 min by cytochromes P-450PB-B and P-450 beta NF-B, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isozyme selectivity of the inhibition of rat liver cytochromes P-450 by chloramphenicol in vivo

The isozyme-selectivity of chloramphenicol as an inhibitor of rat liver cytochromes P-450 has been investigated. Untreated rats and rats treated with the inducers phenobarbital, beta-naphthoflavone, pregnenolone 16 alpha-carbonitrile, and clofibrate have been injected intraperitoneally with chloramphenicol, and inhibition of specific cytochrome P-450 isozymes has been assessed by monitoring the metabolism of warfarin, testosterone, isosafrole, or lauric acid in subsequently prepared hepatic microsomal preparations. Of eight major cytochrome P-450 isozymes which could be monitored in this fashion, three were inhibited by more than 50% by a dose of chloramphenicol of 300 mg/kg, whereas no evidence of inhibition of the remaining isozymes was obtained. P-450PB-C, an isozyme which is present in significant amounts in untreated rats and which is induced approximately 2-fold by phenobarbital, was the most susceptible cytochrome P-450 to inhibition by chloramphenicol both in vivo and in vitro. P-450PB-B, the major phenobarbital-inducible isozyme, and P-450UT-A, a male-specific testosterone 2 alpha- and 16 alpha-hydroxylase, were intermediate in their susceptibility to chloramphenicol. In contrast, the major isozymes induced by beta-naphthoflavone, pregnenolone 16 alpha-carbonitrile, and clofibrate, as well as a constitutive testosterone 7 alpha-hydroxylase, were not inhibited by chloramphenicol.     

Pulmonary cytochromes P-450 from rabbits treated with phenobarbital

Pretreatment of rabbits with phenobarbital caused significant increases in total pulmonary cytochrome P-450 content, benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase activities and to a lesser extent in benzphetamine N-demethylase activity in lung microsomes. However, 7-ethoxyresorufin O-deethylase activity was not significantly altered. In addition, the pulmonary concentration of the cytochrome P-450-metyrapone complex was increased significantly. Column chromatography of the pulmonary monooxygenases demonstrated that in untreated and phenobarbital-treated rabbits, cytochromes P-450I and P-450II constituted the major forms of cytochrome P-450 isozymes. In addition, the chromatographic studies showed that pretreatment with phenobarbital caused an increase in the content of cytochrome P-450I, but not of cytochrome P-450II. These observations were confirmed by subjecting the pulmonary cytochromes to gel electrophoresis, and staining of the gels for protein and heme.     

Modulation of the induction of rat hepatic cytochromes P-450 by selenium deficiency

The induction by phenobarbital of liver microsomal cytochrome P-450 has been demonstrated to be impaired in rats fed a selenium-deficient diet. Cytochrome P-450 isozyme specific immunologic and molecular techniques were used in the present study to better define the role of selenium in the induction of cytochrome P-450 by phenobarbital. Phenobarbital treatment of the selenium-deficient rats resulted in an increase in the level of total cytochrome P-450 50% of that observed with control rats and in a 10-fold increase in microsomal heme oxygenase. Quantitative immunoblot analyses demonstrated that the levels of cytochromes P-450b + e and P-450p in the phenobarbital-treated selenium-deficient rats were approximately 50% of those found in the phenobarbital-treated control rats. Finally, RNA hybridization studies using cDNA probes to cytochromes P-450b + e or P-450p demonstrated that the accumulations of the RNAs encoding these cytochromes P-450 were unaffected by the selenium status of the rats. These studies suggest that the impaired phenobarbital induction of the cytochromes P-450 in the selenium-deficient rats is the result of an increase in the degradation of the cytochromes P-450 or a decrease in the translation of the mRNAs coding for them.     

Identification of the enzymes catalyzing metabolism of methoxyflurane

The hepatic microsomal metabolism of methoxyflurane in rabbits is markedly stimulated by treatment with phenobarbital. However, the increased rate of metabolism cannot be completely accounted for by the activity of the purified phenobarbital-inducible cytochrome P-450 isozyme 2, even in the presence of cytochrome b5. The discovery of a second hepatic phenobarbital-inducible cytochrome P-450, isozyme 5, led us to undertake experiments to determine in hepatic and pulmonary preparations the portion of microsomal metabolism of methoxyflurane catalyzed by cytochrome P-450 isozymes 2 and 5. We report herein that isozyme 2 accounts for 25% and 29%, respectively, of the O-demethylation of methoxyflurane in hepatic microsomes from untreated and phenobarbital-treated rabbits, and for 25% of the methoxyflurane metabolism in pulmonary microsomes. Results for isozyme 5 indicate that it catalyzes 19% and 27% of methoxyflurane metabolism in control and phenobarbital-induced liver, and 47% of O-demethylation in the lung. In summary, we demonstrate that methoxyflurane O-demethylation in lung, phenobarbital-induced liver, and control liver microsomes is catalyzed by cytochrome P-450 isozymes 2 and 5. Results with purified cytochrome P-450 isozyme 5 are consistent with those obtained using microsomal preparations. Furthermore, metabolism of methoxyflurane by purified isozyme 5 is markedly stimulated by cytochrome b5. A role for cytochrome b5 in cytochrome P-450 isozyme 5-catalyzed metabolism of methoxyflurane was also demonstrated in microsomes. Antibody to isozyme 5 was unable to inhibit methoxyflurane metabolism in the presence of maximally inhibiting concentrations of cytochrome b5 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective inactivation of four rat liver microsomal androstenedione hydroxylases by chloramphenicol analogs

The steroid androstenedione has been shown to be a valuable tool for the study of the selective inactivation of cytochrome P-450 isozymes in intact rat liver microsomes. The validity of this approach was investigated using microsomes, purified cytochrome P-450 isozymes, antibodies to particular cytochromes P-450, and the known mechanism-based inactivator chloramphenicol. Enzyme inactivation and antibody inhibition studies show that microsomes from both phenobarbital- and non-phenobarbital-treated rats are needed to accurately monitor the inactivation of the major phenobarbital-inducible cytochrome P-450 isozyme (PB-B) and of the major constitutive androstenedione 16 alpha-hydroxylase (UT-A). Similar experiments indicate that, although isozyme P-450g does catalyze the 6 beta-hydroxylation of androstenedione in a reconstituted system, this cytochrome appears to make only a minimal contribution to microsomal 6 beta-hydroxylase activity, which reflects instead the activity of pregnenolone-16 alpha-carbonitrile-induced isozymes. With these parameters investigated, initial enzyme inactivation studies showed that the antibiotic chloramphenicol caused different rates of NADPH-dependent enzyme inactivation among the four androstenedione hydroxylases monitored (16 beta greater than 6 beta greater than 16 alpha greater than 7 alpha). Based on these data, 12 chloramphenicol analogs were examined, and the results with these compounds show that their selectivity as cytochrome P-450 inactivators is a function of at least three structural features: 1) the number of halogen atoms, 2) the presence of a para-nitro group on the phenyl ring, and 3) substitutions on the ethyl side chain. For example, the compound N-(2-phenethyl)dichloroacetamide was shown to reversibly inhibit but not inactivate the cytochrome(s) P-450 responsible for androstenedione 6 beta-hydroxylase activity, whereas N-(2-p-nitrophenethyl) and N-(1,2-diphenethyl)dichloroacetamide rapidly inactivated the 6 beta-hydroxylase. The ability to monitor the activity of multiple isozymes with a single substrate should allow the development of a systematic approach to the design of selective inactivators of rat liver cytochromes P-450.     

Comparison of monooxygenase activities and cytochrome P-450 isozyme concentrations in human liver microsomes

Concentrations of three human liver microsomal cytochrome P-450 isozymes and 20 different monooxygenase activities were determined in human liver microsomal preparations. The results of correlation analysis suggest that: there are important variations in the amounts of the three cytochrome P-450 isozymes measured, particularly P-450(8) and P-450(9); aldrin epoxidase, d-benzphetamine N-demethylase, and S-warfarin 4-hydroxylase activities are linked to cytochrome P-450(5); aryl hydrocarbon (benzo(a)pyrene) hydroxylase and 4-nitroanisole-O-demethylase activities are linked to P-450(8); hydroxylations at the 4'-, 6-, 7-, and 8-positions of R-warfarin are closely linked to each other but are not correlated with other measured monooxygenase activities or P-450 isozyme levels; and P-450(9) is not related to any of the catalytic activities tested. Thus, certain monooxygenase activities can be attributed to specific cytochrome P-450 isozymes. This approach should be useful in suggesting the roles of different cytochromes P-450 in drug metabolism in man which can be further examined using in vitro and in vivo methods.     

Distribution of microsomal monooxygenases in the rabbit respiratory tract

The distribution of microsomal cytochrome P-450 isozymes 2, 4, 5, and 6 and the pulmonary FAD-containing monooxygenase was determined in 10 different anatomical regions of the respiratory tract using immunoblot analysis and enzymatic assays. Cytochrome P-450 isozymes 2 and 5 and the FAD-containing monooxygenase were detected by immunoblotting in all of the pulmonary and nasal samples, although levels in nasal tissues were generally much lower than those levels found in the lung. Cytochrome P-450 isozyme 4, which is generally not present in extrahepatic tissues, was detected in nasal ethmoturbinates and mucosa. Different isozymes of cytochrome P-450 appear to be responsible for the N-demethylation of benzphetamine in lung as compared with nasal tissues. Isozyme 2 is responsible for the N-demethylation of benzphetamine in the lung, whereas another isozyme, possibly isozyme 3a is responsible for N-demethylation in nasal tissues. The presence of isozyme 3a in nasal samples was indicated by the presence of high rates of aniline hydroxylation.     

Identification of a human liver cytochrome P-450 homologous to the major isosafrole-inducible cytochrome P-450 in the rat

The rat 3-methylcholanthrene-inducible family of liver cytochromes P-450 contains two proteins (P-450c and P-450d) that are immunochemically related, possess 68% total sequence homology, and are induced by a number of toxic or carcinogenic compounds. To determine whether equivalent isozymes of hepatic cytochrome P-450 are expressed in humans, as they are in several mammalian species, we performed immunoblot analyses on microsomes prepared from 14 human liver specimens and found that each one contained a 52.5-kDa protein (termed HLd) that reacted with antibodies specific for rat P-450d. In addition, one specimen contained a 54-kDa protein (termed HLc) that reacted with antibodies specific for rat P-450c. HLd was purified through the use of immunoaffinity chromatography and was found to be 56% homologous to rat P-450d and 61% homologous to the equivalent isozyme in the rabbit (P-450 LM4) through their first 18 NH2-terminal amino acids. Finally, levels of immunoreactive HLd varied more than 10-fold among these patients but were unrelated to the patients' drug treatments, smoking habits, or amount of immunoreactive HLp, a human liver cytochrome P-450 related to the glucocorticoid-inducible family of rat cytochromes P-450. We conclude that, in man, there is a cytochrome P-450 family composed of two isozymes (HLc and HLd) that are immunochemically and structurally related to the 3-methylcholanthrene-inducible family observed in several other species.     

Dichloromethyl compounds as mechanism-based inactivators of rat liver cytochromes P-450 in vitro

Twenty dichloromethyl compounds have been tested as potential mechanism-based inactivators of the major phenobarbital-inducible isozyme of rat liver cytochrome P-450 (PB-B) in a reconstituted system. With the exception of dichloromethane and dichloroacetamide, all the compounds decreased the ethoxycoumarin deethylase activity of the enzyme in a time- and NADPH-dependent manner. The inhibitory compounds could be divided into two classes according to whether the loss of monooxygenase activity was accompanied by a decrease in spectrally detectable cytochrome P-450. N-Monosubstituted dichloroacetamides in which the side-chain consisted of a phenyl or n-octyl group were able to mimic the action of chloramphenicol and inactivate the PB-B without destroying the heme moiety. In contrast, dichloroacetamides containing an n-hexyl, n-butyl, or methyl substituent caused a significant loss of heme, as did the five non-amides tested: 1,1,2,2-tetrachloroethane, 1,1-dichloroacetone, methyl dichloroacetate, alpha,alpha-dichlorotoluene, and alpha,alpha-dichloroacetophenone. Representative compounds were also examined as inactivators of the major beta-naphthoflavone-inducible isozyme of rat liver cytochrome P-450 (BNF-B), using a reconstituted system, as well as of constitutive cytochromes P-450, using intact liver microsomes from untreated rats. These studies suggested a marked difference in isozyme selectivity between certain of the compounds. For example, of the isozymes monitored, only the PB-B was affected by alpha,alpha-dichlorotoluene in an NADPH-dependent manner, whereas N-octyl dichloroacetamide inactivated not only the PB-B and BNF-B, but also certain constitutive cytochromes, as evidenced by decreases in microsomal S-warfarin hydroxylase activities. These studies help delineate the structural requirements for the use of dichloromethyl compounds as probes of cytochrome P-450 function and as potential isozyme-selective inhibitors.     

Co-induction of cytochrome P-450 isozymes in rat liver by 2,4,5,2',4',5'-hexachlorobiphenyl or 3-methoxy-4-aminoazobenzene

A multitude of xenobiotics have been demonstrated to co-induce either cytochromes P-450c and P-450d or cytochromes P-450b and P-450e in rat hepatic microsomes. Recently, the compounds 2,4,5,2',4',5'-hexachlorobiphenyl (HCB) and 3-methoxy-4-aminoazobenzene (3-MeO-AAB) have been suggested as selective inducers of cytochrome P-450b (Eur. J. Biochem. 151:67 (1985)) and P-450d (Biochem. Biophys. Res. Commun. 133:1072 (1985)), respectively. Since the identification of inducers with such unique characteristics would have implications with regard to the mechanism of induction of all four isozymes, we have examined the induction of cytochromes P-450b and P-450e by HCB and cytochromes P-450c and P-450d by 3-MeO-AAB in liver microsomes from adult male rats. Immunoblot analysis with monoclonal antibodies directed against cytochromes P-450b and P-450e indicate that HCB induces both isozymic species at the three dosage levels examined (10, 90, and 180 mg/kg). Similarly, 3-MeO-AAB does not appear to represent a unique inducer. Immunoblots of hepatic microsomes from animals treated with three different dosage regimens of 3-MeO-AAB demonstrate that, even at the lowest dosage level (50 mg/kg), both cytochromes P-450c and P-450d are induced. Moreover, immunoinhibition of 7-ethoxyresorufin O-deethylase (EROD) activity by monospecific antibody against either cytochrome P-450c or P-450d confirms this result. 3-MeO-AAB increases this enzyme activity 10-fold; approximately one-third of this induced activity is inhibited with monospecific anti-P-450c, while two-thirds is inhibited with monospecific anti-P-450d. This study also demonstrates that hepatic EROD activity is not an accurate estimate of cytochrome P-450c content since the majority of this enzyme activity in control and 3-MeO-AAB-treated rats is inhibited with monospecific anti-P-450d but not with monospecific anti-P-450c.     

Participation of cytochrome P-450 isozymes in N-demethylation, N-hydroxylation and aromatic hydroxylation of methamphetamine

1. Five isozymes of cytochrome P-450 were purified from liver microsomes of phenobarbital-pretreated (P-450-SD-I and -II), 3-methylcholanthrene-pretreated (P-450-SD-III) and untreated rats (P-450-SD-IV and -V) to determine their catalytic activities in metabolic reactions of methamphetamine. 2. All the isozymes except P-450-SD-III showed considerably high N-hydroxylating activity of methamphetamine. The cytochromes P-450 initiate N-demethylation of this drug by two metabolic pathways, C-hydroxylation and N-hydroxylation. 3. Both N-demethylation and N-hydroxylation of methamphetamine were efficiently catalysed by the phenobarbital-inducible forms P-450-SD-I and -II and constitutive forms P-450-SD-IV and -V. 4. The constitutive forms P-450-SD-IV and -V revealed high catalytic activities of p-hydroxylation of methamphetamine, but phenobarbital- and 3-methylcholanthrene-inducible isozymes showed only low activities. 5. The present results indicate that the different extents of the metabolic intermediate complex formation with cytochrome P-450 (455 nm complex) in the microsomes from phenobarbital-, 3-methylcholanthrene-pretreated, and untreated rats is not attributable to the activities of the respective isozymes of cytochrome P-450 to form the precursor of the complex, N-hydroxymethamphetamine.     

Dose response for induction of two cytochrome P-450 isozymes and their mRNAs by 3,4,5,3'4'5'-hexachlorobiphenyl indicating coordinate regulation in rat liver

The present study compares the time course and dose-response curves for induction of the two major 3-methylcholanthrene (3-MC)-inducible isozymes of cytochrome P-450 and their mRNAs in livers of male rats after administration of 3,4,5,3'4'5'-hexachlorobiphenyl (HCB). Isozyme concentrations were measured by radioimmunoassay. The corresponding translatable mRNAs were measured by translation of polysomes in a cell-free translational system followed by immunoprecipitation and electrophoretic analysis of the translational products. The time course for induction of the two isozymes by HCB indicated that cytochrome P-448MC (P-450c) peaked sooner than P-448HCB (P450d). However, the time course for induction of the two mRNAs was identical. The dose-response curves for induction of the two isozymes and their mRNAs demonstrated that the ED50 for induction of P-448MC was identical to that of P-448HCB, suggesting that the two proteins are induced coordinately by this compound in liver. HCB did not induce P-450PB (the major phenobarbital-inducible isozyme) or affect mRNA levels for this isozyme. Although cytochrome P-448HCB is the predominant cytochrome in liver microsomes from HCB-induced rats, the magnitude of the induction of this isozyme (40-fold) is lower than that of P-448MC (600-fold), because cytochrome P-448HCB is present in higher concentrations in livers of untreated rats than P-448MC (90 versus 3 pmol/mg). Polysomes from control rats also contain more translationally active P-448HCB mRNA than P-448MC mRNA (0.009 versus 0.003% of the total translational products). The increase in the translatable mRNAs (12-fold for P-448HCB mRNA and 40-fold for P-448MC mRNA) was less than the increase in the isozymes. The discrepancy between the magnitude of the induction of the isozymes and their respective mRNAs suggests that factors other than an increase in mRNA influence the magnitude of the increase of the isozymes by HCB. However, HCB did not affect translational efficiency of total mRNA as measured in vitro in the present study. Differences in half-lives of the proteins or effects of HCB on the stability of the proteins might account for the magnitude of the increase in the isozymes after HCB treatment.     

In vivo effects of erythromycin, oleandomycin and erythralosamine derivatives on hepatic cytochrome P-450

Rats have been treated with several derivatives of the erythromycin, erythralosamine or oleandomycin series, in order to compare their ability to induce cytochrome P-450 and to form stable 456 nm-absorbing cytochrome P-450 metabolite complexes. The data obtained confirm that the cytochromes P-450 induced in rats by various macrolides are similar to that induced by pregnenolone 16 alpha-carbonitrile: the cytochrome P-450 IIIA1 isozyme. It showed that: (i) formation of a stable inhibitory 456 nm-absorbing cytochrome P-450 complex is not a prerequisite for cytochrome P-450 induction but enhances induction by stabilization of the IIIA isozyme. Therefore, the best inducers lead also to the maximal in vivo amounts of cytochrome P-450 metabolite complex (except for 2'MBEM); (ii) affinity for cytochrome P-450 IIIA1 is not directly involved for induction; and (iii) hydrophobicity favors induction and formation of complexes. Structural factors are also involved.     

Structural comparison of monoclonal antibody immunopurified pulmonary and hepatic cytochrome P-450 from 3-methylcholanthrene-treated rats

A pulmonary cytochrome P-450 was purified from lung microsomes of 3-methylcholanthrene (MC)-treated rats by immunoaffinity chromatography using a monoclonal antibody to MC-induced rat liver cytochrome P-450. The isolated pulmonary cytochrome P-450 was MC-inducible and had an apparent molecular weight of 57 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight, as well as the NH2-terminal sequence of the first nine amino acids of the pulmonary cytochrome P-450, was identical to that of an epitopically related MC-induced rat liver cytochrome P-450. In addition, partial proteolysis of both cytochromes P-450 yielded indistinguishable peptide patterns on SDS-Page. Treatment of rats with MC, therefore, induces a pulmonary cytochrome P-450 which is structurally identical to the MC-induced hepatic enzyme by several criteria.     

Analogues of chloramphenicol as mechanism-based inactivators of rat liver cytochrome P-450: modifications of the propanediol side chain, the p-nitro group, and the dichloromethyl moiety

The importance of the p-nitro group, the propanediol side chain, and the dichloromethyl moiety of chloramphenicol in regulating its effectiveness and selectivity as a mechanism-based inactivator of rat liver cytochromes P-450 has been examined. 1-p-Nitrophenyl-2-dichloroacetamidoethane, 1-p-nitrophenyl-2-dibromoacetamidoethane, and 1-phenyl-2-dichloroacetamidoethane were as effective as chloramphenicol at inactivating the major phenobarbital-inducible isozyme of rat liver cytochrome P-450, whereas 1-p-nitrophenyl-2-difluoroacetamidoethane caused no enzyme inactivation. Unlike chloramphenicol, 1-p-nitrophenyl-2-dichloroacetamidoethane and 1-phenyl-2-dichloroacetamidoethane also inactivated the major beta-naphthoflavone-inducible isozyme of rat liver cytochrome P-450. Alkaline hydrolysis of the adducts formed upon in vitro incubation of liver microsomes from phenobarbital- and beta-naphthoflavone-induced rats with [14C]-1-p-nitrophenyl-2-dichloroacetamidoethane resulted in the release of 4-nitro-1-phenethyl-1,2-dicarboxylic acid amide and oxalic acid. Enzymatic digests of the radio-labeled protein produced by incubation of a reconstituted system containing the major isozymes induced by beta-naphthoflavone or phenobarbital with [14C]-1-p-nitrophenyl-2-dichloroacetamidoethane led to the release of 4-nitro-1-phenethyl-1,2-dicarboxylic acid amide and 4-nitro-1-phenethyl oxamyl lysine. These results suggest that a single oxamyl chloride intermediate is responsible for the covalent modification and, hence, inactivation of both isozymes by 1-p-nitrophenyl-2-dichloroacetamidoethane.     

The cytochrome P-450 monooxygenase system of rabbit lung enzyme components, activities, and induction in the nonciliated bronchiolar epithelial (Clara) cell, alveolar type II cell, and alveolar macrophage

Enzyme components and activities of the cytochrome P-450 monooxygenase system in microsomal preparations from the Clara cell, alveolar type II cell, and alveolar macrophage fractions isolated from lungs of untreated rabbits and rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin were examined. Results are compared to those obtained with microsomal preparations from whole lung. Concentrations of cytochrome P-450 isozymes 2 and 5 and NADPH-cytochrome P-450 reductase activities were higher in preparations from Clara cell fractions than in preparations from type II cell fractions or whole lung. For the most part, however, differences among these preparations were 2-fold or less. Microsomal preparations from the macrophage fraction contained low or undetectable levels of cytochrome P-450 isozymes but relatively high levels of cytochrome P-450 reductase activity. The concentration of cytochrome P-450 isozyme 6, in contrast to those of isozymes 2 and 5, was found to be highest in microsomal preparations from whole lung. Treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin increased the concentrations of isozyme 6 in preparations from the Clara and type II cell fractions and from whole lung about 20-fold. In contrast, the content of isozyme 6 in preparations from the macrophage fraction increased greater than 90-fold. In all cases, induction of isozyme 6 resulted in substantial increases in the O-deethylation of 7-ethoxyresorufin and only minor increases in the hydroxylation of benzo(a)pyrene. Activities per unit of isozyme 6, following induction, were similar in all preparations, and we estimate that less than 20% of the potential activity of isozyme 6 is expressed with benzo(a)pyrene and greater than 40% with 7-ethoxyresorufin. These similarities exist in spite of significant differences among the preparations from different fractions in the ratios of isozyme 6 to NADPH-cytochrome P-450 reductase.     

Purification and characterization of a mouse liver cytochrome P-450 induced by cannabidiol

A cytochrome P-450 isozyme (Mr = 51,600) was purified to apparent homogeneity from hepatic microsomes of mice pretreated with cannabidiol (CBD), a major constituent of marijuana. The isozyme exhibited high pentoxyresorufin O-dealkylase, hexobarbital hydroxylase, and 16 alpha- and 16 beta-testosterone hydroxylase activities and formed a Fe+2-metyrapone complex, properties characteristic of the major hepatic cytochrome P-450s previously purified from phenobarbital (PB)-pretreated animals. In addition, the CBD-induced cytochrome P-450 was immunoreactive with an antibody raised against the major rat hepatic PB-inducible cytochrome P-450 and exhibited an NH2-terminal amino acid sequence greater than 90% homologous with that of the PB-inducible rat liver isozyme. Because of the many similarities between the CBD-induced isozyme and certain other isozymes previously purified from PB-pretreated animals, a cytochrome P-450 isozyme was purified from PB-pretreated mice by a chromatographic procedure similar to that employed for purification of the CBD-induced isozyme. The PB-inducible isozyme was indistinguishable from the CBD-inducible cytochrome P-450 on the bases of apparent molecular weight, absorption spectra, NH2-terminal amino acid sequence, peptide mapping, immunoreactivity, and catalytic activity. Although the CBD- and PB-inducible P-450 isozymes appear to be qualitatively very similar, PB appears to be a quantitatively better inducer of the isozyme. Thus, CBD exposure results in the induction of an isozyme that is refractory to CBD-mediated inactivation, thereby apparently altering the cytochrome P-450 isozymal composition of mouse hepatic microsomes.     

Effects of a series of 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine on the major inducible cytochrome P-450 isozymes of rat liver

Various 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) cause mechanism-based inactivation of cytochrome P-450 (P-450) by destroying the heme prosthetic group. We have examined the isozyme selectivity of representative DDC analogues with respect to the major inducible P-450 isozymes of rat liver. Hepatic microsomes from untreated, phenobarbital (PB)-treated, beta-naphthoflavone (beta NF)-treated, and dexamethasone (DEX)-treated rats were incubated with a DDC analogue and NADPH and were subsequently analyzed for P-450 and heme content, P-450 isozyme immunoreactivity, and enzyme activity. Compared with the uninduced state, 4-isopropyl-DDC caused slightly less P-450 destruction following beta NF induction and much greater destruction following DEX pretreatment. Also, 4-hexyl-DDC was found to cause less P-450 destruction following PB or DEX pretreatment, compared with results obtained with untreated rats. These results suggest that DDC analogues possess different isozyme selectivity profiles. Monoclonal antibodies (MAbs) directed against the major inducible isozymes of P-450 were used to probe Western blots of microsomal protein following DDC analogue treatment. The formation of lower molecular mass (45-55 kDa) immunoreactive proteins in microsomes from beta NF-treated rats following DDC analogue treatment was revealed by two MAbs (1-31-2 and 1-36-1), suggesting that the apoprotein of the major beta NF-inducible isozyme, P-450c, is subject to alteration by DDC analogues. In microsomes from DEX-treated rats, DDC analogues caused the formation of higher molecular mass (80, 94, and 115 kDa) proteins showing immunoreactivity with MAb 2-13-1, directed against a major DEX-inducible isozyme belonging to the P-450p family. These immunochemical findings are supported by the demonstration that DDC analogues also caused mechanism-based inhibition of the catalytic activity of P-450c (7-ethoxyresorufin O-deethylase) and P-450p (erythromycin N-demethylase) but not that of the major PB-inducible isozyme, P-450b (7-pentoxyresorufin O-dealkylase). The combined immunochemical and enzymic studies indicate that rat liver P-450 c and p are targets for mechanism-based inactivation by DDC analogues.