Differential oxidase activity of hepatic and pulmonary microsomal cytochrome P-450 isozymes after treatment with cytochrome P-450 inducers.

Phenobarbital, 3-methylcholanthrene, acetone and pyrazole were used as inducers of cytochrome P450 and the NADPH-dependent oxidase activity (O-2 production) of pulmonary and hepatic microsomes was determined. Oxidase activity of microsomes from 3-methylcholanthrene-treated rats was significantly decreased as compared to that of controls when expressed on the basis of cytochrome P450 content (30% decrease for liver, 60% decrease for lung). The oxidase activity of liver microsomes from pyrazole-treated rats showed a significant increase, whereas phenobarbital treated microsomes had average superoxide-generating activity. The contribution of cytochromes CYP 1A, CYP 2B and CYP 2E1 to superoxide-generating activity was investigated using monoclonal antibodies. Monoclonal antibody 1-91-3 against CYP 2E1 inhibited superoxide generation by 58% in liver microsomes from pyrazole-treated rats. Monoclonal antibodies 1-7-1 and 2-66-3 against CYP 1A and CYP2B, respectively, had no effect on superoxide generation. These results indicate that different cytochrome P450 isoforms are mainly responsible for differential superoxide generating activities of microsomes and complement the reconstitution study of Morehouse and Aust. Furthermore, our study indicates that CYP 1A1, induced by 3-MC, demonstrates an unusually low oxidase activity.     

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.     

Polyclonal and monoclonal antibodies as probes of rat hepatic cytochrome P-450 isozymes

Cytochrome P-450 is the terminal oxidase of an electron transport system that is responsible for the oxidative metabolism of a large variety of endogenous and exogenous compounds. This broad substrate selectivity is caused by multiple isozymes of cytochrome P-450 and the wide substrate selectivity of many of these isozymes. We have isolated 11 isozymes of cytochrome P-450 from the livers of rats (cytochromes P-450a-P-450k). We have found both polyclonal and monoclonal antibodies increasingly useful to distinguish among these isozymes and to quantitate enzyme levels in liver microsomal preparations where as many as 15 or more cytochrome P-450 isozymes are present. Several of these isozymes show considerable immunochemical relatedness to each other, and operationally they can be grouped into families of immunochemically related isozymes that include cytochromes P-450b and P-450e in one family, cytochromes P-450c and P-450d in another, and cytochromes P-450f-P-450i, and P-450k in a third family. Immunoquantitation of some of these isozymes has revealed dramatic increases of over 50-fold in the levels of certain of these isozymes when exogenous compounds are administered to rats.     

Interactions of prostaglandins with hepatic microsomal cytochrome P-450

The spectral changes associated with the addition of prostaglandins (PGs) to hepatic microsomes from guinea pigs and rats were examined. PGA₁, PGA₂, PGE₁, PGE₂, PGF_1α and PGF_2α when added to guinea pig liver microsomes exhibited type I spectra. The binding affinities as determined from spectral dissociation constants (K_s) were highest with PGA₁ and PGA₂. With liver microsomes from control or 3-methyl-cholanthrene (MC)-treated rats, PGs did not yield type I spectra; however, in this case a $\text{weak}$ spectrum, designated here as type “II” was at times observed, With microsomes from phenobarbital (Pb)-treated rats only PGA₁ and PGA₂ yielded type I spectra; again in absence of type I spectrum, a weak type “II” was occasionally observed. The addition of PGA₁ and PGA₂ to liver microsomes from Pb-treated rats inhibited the microcomal mediated hydroxylation of hexobarbital. The inhibition by PGA₁ was competitive; the K_i = 8.2 × 10^?4 M was found to be similar in magnitude to the K_s = 7.3 × 10^?4 M of PGA₁ observed with rat liver microsomes. These observations suggested that PGs particularly of the A series interact with the hepatic microsomal cytochrome P-450 monooxygenase system.     

A phenobarbital-inducible hepatic mitochondrial cytochrome P-450 immunochemically related to microsomal P-450b

We have purified and characterized a phenobarbital (PB)-inducible hepatic mitochondrial cytochrome P-450 (P-450), termed P-450mt4, which is distinctly different from the previously characterized mitochondrial isoforms. The level of induction of P-450mt4 by PB in the male livers is nearly 20-fold, as against a marginal induction in the female livers, suggesting that it may be a male predominant isoform. P-450mt4 shows a close resemblance to microsomal P-450b (the major PB-inducible form) with respect to electrophoretic migration (apparent molecular mass of 50 kDa) and immunological cross-reactivity, although it exhibits a distinct isoelectric pH (pI 6.9 vs 6.5 for P-450b), peptide fingerprint pattern, and amino acid composition. Further, the N-terminal sequence analysis shows over 90% positional identity (39 out of 42) between P-450mt4 and P-450b, suggesting that it is a close relative of the P-450 IIB gene family. In vitro reconstitution experiments show that P-450mt4 can metabolize a wide range of substrates such as benzphetamine, (dimethylamino)antipyrine, aflatoxin B1, and vitamin D3, exclusively in the presence of mitochondrial-specific ferredoxin and ferredoxin reductase as electron carriers. P-450mt4 is translated as a 53-kDa precursor, which is transported into mitochondria under in vitro conditions and processed into a mature 50-kDa protein. These results provide conclusive evidence for the occurrence of a male-specific P-450 belonging to the IIB gene family in rat liver mitochondria.     

Metabolism of valproic acid by hepatic microsomal cytochrome P-450

Incubation of valproic acid with rat liver microsomes led to the formation of 3-, 4- and 5-hydroxy-valproic acid. The latter two metabolites, which have been characterized previously from in vivo studies, may be regarded as products of fatty acid ω-1 and ω hydroxylation, respectively. 3-Hydroxy-valproic acid, however, had been thought to derive from the β-oxidation pathway in mitochondria. Conversion of valproic acid to all three metabolites in microsomes required NADPH (NADH was less effective), utilized molecular oxygen, was suppressed by inhibitors of cytochrome P-450 and was stimulated (notably at C-3 and C-4) by phenobarbital pretreatment of the rats. It is concluded that rat liver microsomal cytochrome P-450 catalyzes ω-2 hydroxylation of valproic acid, a reaction not detected previously with fatty acids in mammalian systems, and that the product, 3-hydroxyvalproic acid, should not be used to assess in vivo metabolism of valproate via the β-oxidation pathway.     

Spin state transitions of liver microsomal cytochrome P-450.

Spin state transitions of membrane-bound cytochrome P-450 were investigated by difference spectrophotometry using the 'D'-charge transfer absorbance band at 645 nm as a measure of the amount of hemin iron present in the 5-coordinated state. The magnitude of the 'D'-absorbance band in the absence of exogenous substrates, e.g., the concentration of native high spin cytochrome P-450, was evaluated from the difference in absorbance at 645 nm between ferric cytochrome P-450 and the carbon monoxide derivative of the pigment in its ferrous state. The contribution of the native high spin species to the total cytochrome P-450 content of microsomes was calculated to be between 40% and 65% after induction with phenobarbital and polycyclic hydrocarbons, respectively. Up to 80% of the cytochrome P-450 was found to be present in the high spin state after the addition of exogenous substrates. Further, the steady state concentrations of high spin cytochrome P-450, observed in the presence of reduced pyridine nucleotides, suggest that the rate limiting step for microsomal mixed function oxidation reactions is variable and dependent on the substrate under investigation.     

Magnetic circular dichroism studies of hepatic microsomal cytochrome P-450.

Magnetic circular dichroism (MCD) spectra have been measured for cytochrome P-450 (P-450) purified from phenobarbital-induced rabbit liver microsomes. The temperature dependence of some of the MCD spectra has also been determined. The MCD spectrum of oxidized P-450 seems to suggest that it is in a state intermediate between the ferric low-spin states. Model experiments suggest that this anomaly arises from the coordination of a thiolate anion to the heme. Reduced P-450 shows a very peculiar MCD spectrum; the spectrum as well as its temperature dependence suggest that the heme in reduced P-450 is a "mixture" in terms of redox and/or spin states. The MCD spectrum of the CO complex of reduced P-450 exhibits an apparent Faraday A term around 450 nm which consists of about 50% C term and 50% the other terms, indicating that it is not in a purely ferrous low-spin state. The CO complex of reduced cytochrome P-420 (P-420), on the other hand, shows an MCD spectrum characteristic of a ferrous low-spin heme. It is suggested from model experiments that the thiolate anion coordinates to the heme trans to CO in the P-450-CO complex. The Soret region of the MCD spectrum of the EtNC complex of reduced P-450 is characterized by two apparent A terms around 430 and 455 nm, whereas that of the corresponding complex of P-420 has only one apparent A term around 434 nm.     

Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid

Cytochrome P-450j has been purified to electrophoretic homogeneity from isoniazid-treated adult male rats; and this enzyme appears to be a major protein induced in hepatic microsomes after administration of isoniazid, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hemoprotein has a minimum molecular weight of approximately 51,500, and the ferrous-carbonyl complex of cytochrome P-450j has a Soret maximum at 451-452 nm. The oxidized heme iron appears to be predominately in the high spin state as deduced from the Soret maximum at 395 nm. Ethylisocyanide binds to ferrous cytochrome P-450j to yield spectral maxima at approximately 458 and 430 nm with a resultant 458/430 ratio of 0.7 at pH 7.4. Cytochrome P-450j has no measurable catalytic activity for the metabolism of benzo[a]pyrene (3- and 9-hydroxylation), hexobarbital, testosterone, and 5 alpha-androstane-3 alpha,17 beta-diol-3,17-disulfate. Low, but detectable, catalytic activity is obtained for the metabolism of 7-ethoxycoumarin, benzphetamine, p-nitroanisole, zoxazolamine, and 2-hydroxylation of 17 beta-estradiol. In contrast, cytochrome P-450j effectively catalyzes p-hydroxylation of aniline with a turnover of 12.7 nmol/min/nmol cytochrome P-450j. Hydroxyl radical scavengers, Fe-EDTA, superoxide dismutase, and catalase have no effect on aniline p-hydroxylation catalyzed by cytochrome P-450j. Cytochrome P-450j is distinct from nine other rat hepatic microsomal cytochromes P-450 (P-450a-P-450i) previously purified in this laboratory, as well as different isozymes described by other investigators, based on several parameters including minimum molecular weight, spectral properties, and catalytic activity. In Ouchterlony double diffusion plates, antibodies against cytochromes P-450a-P-450f show no cross-reaction with cytochrome P-450j. Structural differences among cytochromes P-450a-P-450j are apparent from the NH2-terminal sequence of cytochrome P-450j, as well as the electrophoretic profiles of proteolytic digests of the hemoproteins.     

The interaction of vinyl chloride with rat hepatic microsomal cytochrome P-450 in vitro.

In the presence of hepatic microsomes, vinyl chloride produces a ‘type I’ difference spectrum and stimulates carbon monoxide inhibitable NADPH consumption. A comparison of the binding and Michaelis parameters for the interaction of vinyl chloride with uninduced, phenobarbital and 3-methylcholanthrene induced microsomes indicates that the binding and metabolism of vinyl chloride is catalyzed by more than one type P-450 cytochrome, but predominantly by cytochrome P-450. Metabolites of vinyl chloride from this enzyme system decrease the levels of cytochrome P-450 and microsomal heme, but not cytochrome $\text{b}$₅ or NADPH-cytochrome $\text{c}$ reductase $\text{in vitro}$.     

Benzanthrone: a new substrate for hepatic microsomal cytochrome P-450

The metabolism of benzanthrone, a commonly used dy intermediate, by rat hepatic microsomes was investigated using thin layer chromatography (TLC) analysis. Incubation of benzanthrone with hepatic microsomes in the presence of NADPH generating system produced at least seven fluorescent metabolites on TLC plates. TLC spots numbered II, III, IV, V and VI were the major metabolites obtained from hepatic microsomes with the Rf values of 0.53, 0.45, 0.38, 0.33 and 0.26, respectively. Metabolites VII and VIII were faint bands with Rf values of 0.08 and 0.04, respectively. Preincubation of hepatic microsomes with either 1-benzyl-imidazole (10(-4)M) or SKF-525 A (10(-4)M) or metyrapone (10(-3)M) or flushing with carbon monoxide substantially inhibited the benzanthrone metabolism. alpha-Naphtho-flavone (10(-4)M) did not cause any change in hepatic microsomal metabolism of benzanthrone. Oral administration of benzanthrone to animals yielded at least six urinary metabolites. TLC spots numbered II, III, IV, V and VI in the urine were same as those of hepatic microsomal metabolites. However, one of the urinary metabolite numbered IX which stays at the origin of TLC plate with the Rf value of 0.05 may be a conjugate. Our results suggest that benzanthrone acts as a substrate for hepatic heme protein, cytochrome P-450 and that some of the metabolites are excreted in urine.     

Metyrapone - reduced cytochrome P-450 complex: A specific method for the determination of the phenobarbital inducible form of rat hepatic microsomal cytochrome P-450

Using homogeneous cytochrome P-450, we have shown that the well-known metyrapone-dithionite reduced cytochrome P-450 complex is specific for the cytochrome P-450b induced by phenobarbital. A linear relationship was observed between the absorbance of metyrapone-reduced cytochrome P-450 complex and the one of CO-reduced cytochrome P-450 complex, the usual method for the determination of cytochrome P-450. A method has been proposed for the specific determination of the cytochrome P-450b.     

Immobilization of liver microsomal cytochrome P-450

Rabbit liver microsomal cytochrome P-450 was immobilized by entrapment in calcium alginate gel. Aminopyrine demethylation experiments showed that the immobilized enzyme system is highly active and exhibits an unimpaired functional stability as compared with crude microsomes. The alginate entrapped microsomes were employed in a fixed bed recirculation reactor, where aminopyrine was continuously demethylated. Such model enzyme reactor can be a useful tool for studying extracorporeal drug detoxification or preparative substrate conversion with microsomal enzyme systems.     

Inactivation of rat hepatic cytochrome P-450 by spironolactone

Administration of antimineralocorticoid spironolactone (SPL) to rats results in modest destruction of hepatic cytochrome P-450 with parallel loss of heme. This process is accentuated by pretreatment with dexamethasone (DEX), an inducer of cytochrome P-450p and is associated with marked functional loss of cytochrome P-450p-dependent hydroxylases. Cytochrome P-450 destruction may be replicated in vitro when microsomes from DEX-pretreated rats are incubated with SPL and NADPH and is impaired when these rats are given triacetyloleandomycin, an inhibitor of cytochrome P-450p. In vitro SPL-mediated cytochrome P-450 destruction is accompanied by a loss of heme, which appears to be converted to reactive intermediates which covalently bind to microsomes or are converted to polar metabolites.     

Binding of R and S warfarin to hepatic microsomal cytochrome P-450

The R and S enantiomers of the anticoagulant, warfarin, are metabolized to a series of monohydroxylated products by rat hepatic cytochrome P-450. The patterns of metabolites are a function of the warfarin enantiomer used and of the induction of the microsomal enzymes by phenobarbital (PB) and 3-methylcholanthrene (MC). We have studied the binding of R and S warfarin to cytochrome P-450 by difference spectrometry to probe the heterogeneity of cytochrome P-450 and to determine the role of this heterogeneity in the production of the patterns of warfarin metabolites. Uninduced cytochrome P-450 yielded modified type II spectra with R and S warfarin with equivalent binding constants, K_s = 1.50 mM. PB-induced cytochrome P-450 yielded modified type II spectra which varied biphasically with warfarin concentration with K_s(S) = 0.24 and 0.07 mm; K_s(R) = 0.79 and 0.12 mM. MC induction and 2-allyl-2-isopropylacetamide treatment yielded microsomes markedly enriched for cytochrome P-448 which, with R warfarin, yielded a type I spectrum, K_s = 0.24 mM, and with S warfarin a modified type II spectrum with K_s = 0.11 mM. The effects of the type I compound, hexobarbital, the type II compound, imidazole, or the opposite enantiomer to that being studied on the binding spectra of R and S warfarin to the variously induced cytochromes P-450 were investigated as an aid to elucidating the mode of interaction of cytochrome P-450 with warfarin. In all cases, prior binding of R or S warfarin influenced the binding of the opposite enantiomer. We conclude from these results that R and S warfarin bind to two separate forms of PB-induced cytochrome P-450 and two separate forms of MC-induced cytochrome P-448, all of which differ from uninduced cytochrome P-450. The variety of monohydroxylated metabolites of R and S warfarin is probably a consequence of the interactions with these different forms of cytochrome P-450.