Cytochrome P-450-mediated denitrification of 2-nitropropane in mouse liver microsomes

Enzymatic denitrification of 2-nitropropane (2NP) was investigated in an NADPH-dependent hepatic microsomal system from male CD1 mice. The involvement of cytochrome P-450 (P-450) as the catalyst in 2NP denitrification was revealed by the induction of nitrite-releasing activity following phenobarbital (PB) pretreatment, by a decrease in activity with carbon tetrachloride pretreatment, by the inhibition of the reaction with classical P-450 inhibitors, and by the observation of a type I binding spectrum. Under optimal conditions, two pH-dependent peaks of activity were observed at pH 7.6 and pH 8.8, each with its own optimal substrate concentration. Inhibition of the reaction by metyrapone and carbon monoxide (CO) (among others) produced differential responses dependent on pH. These results, along with two pH optima and two substrate optima, suggested the involvement of multiple P-450 isozymes. Average specific activities were 8.05 nmoles of nitrite released per minute per milligram microsomal protein at pH 7.6 and 6.44 nmoles of nitrite released per minute per milligram microsomal protein at pH 8.8. Acetone was identified as the second product of the reaction by gas chromatography/mass spectrometry (GC/MS). Stoichiometry studies indicated that the acetone production was slightly less than expected (about 70%) from nitrite release. Up to 25% residual activity was observed under anaerobic conditions. These results suggested that though the predominant reaction mechanism was oxidative, oxygen-independent metabolism of 2NP also occurred to some extent. In contrast to the reported lack of activity in untreated rat, the observed denitrification in uninduced mouse liver microsomes was significant and suggested that major species-specific differences exist in the in vitro metabolism of 2NP.     

Drug metabolism components in liver microsomes from Hypostomus punctatus, a Brazilian benthic fish (Cascudo)

1. The major components of hepatic drug biotransformation system were identified in a Brazilian freshwater benthic fish. 2. Cytochrome P-450 difference spectra were obtained adding 0.02 mM phenazine ethosulphate and 2 mM ascorbate to microsomal suspensions. Basal levels of P-450 were high (0.9 nmol/mg of microsomal protein) and were not induced by 3-MC. 3. Microsomal NADPH-cytochrome C reductase activity was determined in presence of 1.3 x 10(-4) M NADPH, 3.3 x 10(-5) M cytochrome C, 1.0 x 10(-4) M EDTA, 66 micrograms of microsomal protein per ml in a 0.3 M Tris-HCl buffer, pH 8.6. Basal levels of NADPH-cytochrome C were 152.7 nmoles/min/mg of microsomal protein.     

Cumene hydroperoxide-supported denitrification of 2-nitropropane in uninduced mouse liver microsomes

Cumene hydroperoxide supported oxidative denitrification of 2-nitropropane was investigated in uninduced mouse liver microsomes. The cytochrome P-450 peroxygenase catalyzed reaction resulted in the production of nitrite and acetone. Several lines of evidence suggested the involvement of multiple forms of cytochrome P-450. Acetone production was at least two times greater than nitrite release possibly due to sequestration of nitrite in the reaction mixtures.     

Dose-related effects of phenobarbital on hepatic microsomal enzymes

To study the relationship between the dose of phenobarbital (PB) and the magnitude of its effects on microsomal enzymes, cytochrome P-450, UDP-glucuronyl transferase (UDPGT), and glucose-6-phosphatase (G6P) activities were determined in liver homogenate and microsome preparations from control rats and rats treated for 6 days with PB at doses ranging from 1 to 125 mg/kg/day. Both P-450 and UDPGT activities were enhanced by PB in a dose-related fashion. However, while the lowest dose of the drug to produce significant induction of both enzymes was the same (3 mg/kg), maximal induction of P-450 (214%) and UDPGT (285%) was obtained with different doses of PB, namely 75 and 125 mg/kg, respectively. UDPGT induction could equally be demonstrated regardless of whether "native" enzyme or enzyme activated by UDP-N-acetyl glucosamine, digitonin or deoxycholate was employed. In contrast to these inducing effects of the drug on P-450 and UDPGT, PB treatment resulted in a dose-related inhibition of G6P activity. The inhibitory effect was observed with both "native" and deoxycholate-activated enzymes, and could be demonstrated whether the data were expressed as enzyme specific activity (nanomoles per minute per milligram microsomal protein) or as total G6P activity (micromoles per minute per 100 g body weight). These results indicate that: (I) enzyme induction by PB is dose-related; (ii) induction of both P-450 and UDPGT is obtained in the rat with doses of the drug similar to those given to man; and (iii) observed inhibition of G6P activity by PB does not solely reflect an enzymatic dilution secondary to the proliferated endoplasmic reticulum.     

Cytochrome P-450 and parathion metabolism in the fetal and adult gonads of the horse.

Mixed function oxidase-catalyzed dearylation of parathion was determined in microsomal fractions of gonadal and liver tissues of fetal and adult horse. When parathion metabolism was calculated on a basis of milligram of protein, the activity was found to be similar between fetal and adult gonads but not liver. However, on a basis of nmoles of cytochrome P-450, parathion metabolism was similar between fetal and adult liver but not gonadal tissues; the metabolism being 20 times higher in the adult than the fetal gonad.     

Hydroxylase regulation in Candida tropicalis grown on alkanes.

Candida tropicalis synthesizes a hydroxylase (3 to 5 nmol of product formed per minute per milligram of protein) and a cytochrome P-450 (0.10 to 0.13 nmol per milligram of protein) during growth on n-tetradecane. A three- to four-fold increase in the level of NADPH cytochrome c reductase is also observed in those cells as compared to the level of cells grown on glycerol. The most efficient inducers of the hydroxylase and of cytochrome P-450 are straight-chain alkanes having at least 10 carbon atoms. Alkenes and higher alcohols are also good inducers. There is little or no growth on ramified hydrocarbons such as pristane and on long-chain aldehydes and fatty acids. The partial inhibition of growth on decane is probably due to the denaturation of the microsomal electron carrier systems by the fatty acid formed by hydroxylation of the decane in the yeast.     

Denitrification by the fungus Cylindrocarpon tonkinense: anaerobic cell growth and two isozyme forms of cytochrome P-450nor.

We examined the denitrification system of the fungus Cylindrocapon tonkinense and found several properties distinct from those of the denitrification system of Fusarium oxysporum. C. tonkinense could form N2O from nitrite under restricted aeration but could not reduce nitrate by dissimilatory metabolism. Nitrite-dependent N2O formation and/or cell growth during the anaerobic culture was not affected by further addition of ammonium ions but was suppressed by respiration inhibitors such as rotenone or antimycin, suggesting that denitrification plays a physiological role in respiration. Dissimilatory nitrite reductase and nitric oxide reductase (Nor) activities could not be detected in cell extracts of the denitrifying cells. The Nor activity was purified and found to depend upon two isoenzymes of Cytochrome P-450nor (P-450nor), which were designated P-450nor1 and P-450nor2. These isozymes differed in the N-terminal amino acid sequence, isoelectric point, specificity to the reduced pyridine nucleotide (NADH or NADPH), and the reactivity to the antibody to P-450nor of F. oxysporum. the difference between the specificities to NADH and NADPH suggests that P-450nor1 and P-450nor2 play different roles in anaerobic energy acquisition.     

Denitrification by the fungus Fusarium oxysporum and involvement of cytochrome P-450 in the respiratory nitrite reduction

From conditions for production in Fusarium oxysporum of the unique nitrate/nitrite-inducible cytochrome P-450, tentatively called P-450dNIR, it was expected that the fungus is capable of metabolizing nitrate dissimilatively. Here we report that F. oxysporum exhibits a distinct denitrifying ability which results in the anaerobic evolution of nitrous oxide (N2O) from nitrate or nitrite. Comparison of the cell growth during denitrification indicated that the dissimilatory reduction of nitrate to nitrite is an energetically favorable process in F. oxysporum; however, further reduction of nitrite to N2O might be energy-exhausting and may function as a detoxification mechanism. A potent nitrite reductase activity to form N2O could be reconstituted by combination of the cell-free extract prepared from the denitrifying cells and an NADH-phenadinemethosulfate-dependent reducing system. The activity was strongly inhibited by carbon monoxide, cyanide, oxygen (O2), and the antibody against P-450dNIR. The results, along with those concerning inducing conditions of P-450dNIR, were highly indicative that the cytochrome is involved in the denitrifying nitrite reduction. This work has thus presented not only the first demonstration that a eukaryote exhibits a marked denitrifying ability, but also the first instance of a cytochrome P-450 that is involved in a reducing reaction with a distinct physiological significance against a hydrophilic, inorganic substrate.     

Pronounced and differential effects of ionic strength and pH on testosterone oxidation by membrane-bound and purified forms of rat liver microsomal cytochrome P-450

The aim of this study was to determine the effects of ionic strength and pH on the different pathways of testosterone oxidation catalyzed by rat liver microsomes. The catalytic activity of cytochromes P-450a (IIA1), P-450b (IIB1), P-450h (IIC11) and P-450p (IIIA1) was measured in liver microsomes from mature male rats and phenobarbital-treated rats as testosterone 7 alpha-, 16 beta-, 2 alpha- and 6 beta-hydroxylase activity, respectively. An increase in the concentration of potassium phosphate (from 25 to 250 mM) caused a marked decrease in the catalytic activity of cytochromes P-450a (to 8%), P-450b (to 22%) and P-450h (to 23%), but caused a pronounced increase in the catalytic activity of cytochrome P-450p (up to 4.2-fold). These effects were attributed to changes in ionic strength, because similar but less pronounced effects were observed with Tris-HCl (which has approximately 1/3 the ionic strength of phosphate buffer at pH 7.4). Testosterone oxidation by microsomal cytochromes P-450a, P-450b, P-450h and P-450p was also differentially affected by pH (over the range 6.8-8.0). The pH optima ranged from 7.1 (for P-450a and P-450h) to 8.0 (for P-450p), with an intermediate value of 7.4 for cytochrome P-450b. Increasing the pH from 6.8 to 8.0 unexpectedly altered the relative amounts of the 3 major metabolites produced by cytochrome P-450h. The decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h that accompanied an increase in ionic strength or pH could be duplicated in reconstitution systems containing purified P-450a, P-450b or P-450h, equimolar amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. This result indicated that the decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h was a direct effect of ionic strength and pH on these enzymes, rather than a secondary effect related to the increase in testosterone oxidation by cytochrome P-450p. Similar studies with purified cytochrome P-450p were complicated by the atypical conditions needed to reconstitute this enzyme. However, studies on the conversion of digitoxin to digitoxigenin bisdigitoxoside by liver microsomes, which is catalyzed specifically by cytochrome P-450p, provided indirect evidence that the increase in catalytic activity of cytochrome P-450p was also a direct effect of ionic strength and pH on this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic regulation of coumarin hydroxylase activity in mice. Biochemical characterization of the enzyme from two inbred strains and their F1 hybrid.

Hydroxylation of coumarin to 7-hydroxycoumarin by liver microsomes from control or phenobarbital-pretreated mice is 5- to 10-fold higher in the DBA/2J strain compared to the AKR/J strain, while activities of nine other cytochrome P-450 mediated oxidations show only minor differences. Mixing experiments with whole liver homogenates and subcellular fractionations do not reveal the presence of enzyme activators or inhibitors or competing enzyme reactions in either strain. Comparisons of pH optima (pH 7.6), heat stability at 52 degrees C (6 to 8 min for 50% inactivation), and Km values (0.45 to 0.50 microM coumarin) for coumarin hydroxylase show no significant differences in the two strains of mice or their F1 hybrid. Similarly, only minor differences in inhibition of coumarin hydroxylase by carbon monoxide, SKF-525A, menadione, and several other inhibitors of microsomal mixed function oxidase reactions are observed in the two strains. In contrast to these data, aniline and metyrapone, two compounds which bind to the heme iron of cytochrome P-450 to form ferrihemochromes, show differential and opposite patterns of inhibition of enzyme activity in the DBA/2J and AKR/J mouse strains. This latter observation suggests that a structurally different cytochrome P-450 may hydroxylate coumarin in these two inbred mouse strains.     

Analysis of debromination of 1,2-dibromoethane by cytochrome P-450-linked hydroxylation systems as observed by bromide electrode

Debromination of 1,2-dibromoethane (DBE) by a rabbit liver microsomal preparation and a reconstituted cytochrome P-450 enzyme system was investigated. The reaction was performed in our newly constructed reaction vessel, in which a bromide electrode was installed. During the reaction, the liberated bromide ion was continuously measured by the bromide electrode, and the amount was recorded. In the microsomal preparation, the DBE-debromination rate per nmol cytochrome P-450 was enhanced by phenobarbital-pretreatment of rabbits compared with the untreated microsomes, whereas it was diminished by 3-methylcholanthrene-pretreatment. The debromination reaction was reconstituted in a purified enzyme system containing phenobarbital-inducible rabbit liver microsomal cytochrome P-450 (P-450PB), NADPH-cytochrome P-450 reductase, and NADPH. The optimum conditions required the presence of dilauroylphosphatidylcholine and cytochrome b5. Cytochrome b5 was found not to be an obligatory component for the DBE-debromination in the reconstituted system, but it stimulated the activity about 3.4-fold. Preincubation of the reconstituted mixture with guinea pig anti-cytochrome P-450PB antiserum markedly inhibited the debromination reaction.     

Properties of the System for the Mixed Function Oxidation of Kaurene and Kaurene Derivatives in Microsomes of the Immature Seed of Marah macrocarpus: Electron Transfer Components

Cytochrome P-450 and cytochrome b₅ at levels of approximately 0.10 and 0.60 nanomole per milligram of microsomal protein were detected by spectral measurements in microsomes prepared from endosperm tissue of immature Marah macrocarpus seeds. TPNH-cytochrome c reductase, DPNH-cytochrome c reductase, andDPNH-cytochrome b₅ reductase activities were also present in these microsomes at levels of approximately 0.060, 0.22, and 0.52 unit per milligram of microsomal protein, respectively. (One unit of reductase is the amount of enzyme catalyzing the reduction of 1 micromole of electron acceptor per minute.) Treatments of microsomes with steapsin or trypsin were not effective in solubilizing any of these electron transport components in detectable form. However, treatment of a microsomal suspension in 25% glycerol with 1% sodium deoxycholate led to the release of about 60% of the protein and each of the above hemoproteins and electron transfer activities to the fraction which was not pelleted after centrifugation for 2 hours at 105,000g. Some ent-kaur-16-ene oxidase activity could be detected in the solubilized fraction after removal of the detergent. Cytochrome b₅ and DPNH-cytochrome b₅ reductase activity were largely separated from one another and from an overlapping mixture of TPNH-cytochrome c reductase and DPNH-cytochrome c reductase when the sodium deoxycholate-solubilized fraction was chromatographed on a DEAE-cellulose column. No cytochrome P-450 or cytochrome P-420 was detected in the column fractions and no ent-kaur-16-ene oxidase activity was detected when the column fractions were tested singly or in combination.     

Monooxygenase activity of rat liver microsomes immobilized by entrapment in a crosslinked prepolymerized polyacrylamide hydrazide

Rat liver microsomes were immobilized by entrapment in a chemically crosslinked synthetic gel obtained by crosslinking prepolymerized polyacrylamide-hydrazide with glyoxal. Approximately 88% of the microsomal fraction was entrapped in the gel. The specific rate of O-demethylation of p-nitroanisole was used to assay the microsomal cytochrome P-450 activity of the immobilized microsomal preparations. The gel entrapped microsomes showed monooxygenase activity at 37 degrees C of Vmax = 2.3 nmol p-nitrophenol/min per nmol cytochrome P-450, similar to that of microsomes in suspension. The Km value for the p-nitroanisole-immobilized microsomal cytochrome P-450 system (1.2 X 10(-5) M) was rather close to that of microsomes in suspension (0.8 X 10(-5) M). Under the experimental conditions used the pH activity curve of the immobilized preparation was shifted towards more alkaline values by approx. 0.5 pH unit in comparison with microsomes in suspension. The rate of cytochrome c reduction by the immobilized microsomal system (11.7 nmol/min per mg protein) at 25 degrees C was considerably lower than that of the control (microsomes in suspension, 78 nmol/min per mg protein). Enzyme activity in both preparations showed the same temperature dependence at the temperature range of 10 to 37 degrees C. The immobilized microsomal monooxygenase system could be operated continuously for several hours at 37 degrees C provided that adequate amounts of an NADPH-generating system were added periodically. Under similar conditions a control microsomal suspension lost its enzymic activity within 90 min.     

Cytochrome P-450 and NADPH cytochrome c reductase in rat brain: formation of catechols and reactive catechol metabolites.

Microsomes isolated from whole rat brain were found to contain cytochreme P-450 (0.025 to 0.051 nmoles/mg) and NADPH cytochrome $\text{c}$ reductase activity (26.0 to 55.0 nmoles/mg/min). The oxidation of estradiol to a reactive metabolite that became covalently bound to rat brain microsomal protein was inhibited 63% by an atmosphere of CO:O₂ (9:1), indicating the involvement of a cytochrome P-450 oxygenase. In contrast, this atmosphere had no effect on the binding of either the catechol estrogen, 2-hydroxyestradiol, or several catecholamines to rat brain microsomes. An antibody prepared against NADPH cytochrome $\text{c}$ reductase was found to decrease significantly both the formation of 2-hydroxyestradiol from estradiol by rat brain microsomes and the covalent binding of the catechol estrogen and catecholamines to rat brain microsomal protein.     

Effect of mesitylene on ethanol metabolism in rat liver microsomes.

Increased catalase activity was observed in the liver microsomal fraction of ethanol-treated rats (10% v/v aqueous ethanol solution per os for 5 weeks). In contrast, cytochrome P-450 concentration and specific activity of NADPH-cytochrome c reductase remained at the same level as in the liver of control rats (drinking water). The ratio of microsomal H2O2-generation to catalase activity was lower in the "ethanol" group than in the control one. This phenomenon seems to be related to the increased contribution of the "peroxidatic" reaction (increased rate of ethanol oxidation). Administration of mesitylene (1,3,5-trimethylbenzene) by gastric tube for 3 days (5 mmoles per kg daily) increased cytochrome P-450 concentration, specific activity of NADPH-cytochrome c reductase and ethanol metabolism.     

Participation of two structurally related enzymes in rat hepatic microsomal androstenedione 7 alpha-hydroxylation.

Rat hepatic cytochrome P-450 form 3 (testosterone 7 alpha-hydroxylase; P-450 gene IIA1) and P-450 form RLM2 (testosterone 15 alpha-hydroxylase; P-450 gene IIA2) are 88% identical in primary structure, yet they hydroxylate testosterone with distinct and apparently unrelated regioselectivities. In this study, androstenedione and progesterone were used to assess the regioselectivity and stereospecificity of these two P-450 enzymes towards other steroid substrates. Although P-450 RLM2 exhibited low 7 alpha-hydroxylase activity with testosterone or progesterone as substrate (turnover number less than or equal to 1-2 nmol of metabolite/min per nmol of P-450), it did catalyse androstenedione 7 alpha-hydroxylation at a high rate (21 min-1) which exceeded that of P-450 3 (7 min-1). However, whereas P-450 3 exhibited a high specificity for hydroxylation of these steroids at the 7 alpha position (95-97% of total activity), P-450 RLM2 actively metabolized these compounds at four or more major sites including the nearby C-15 position, which dominated in the case of testosterone and progesterone. The observation that androstenedione is actively 7 alpha-hydroxylated by purified P-450 RLM2 suggested that this P-450 enzyme might make significant contributions to microsomal androstenedione 7 alpha-hydroxylation, an activity that was previously reported to be associated with immunoreactive P-450 3. Antibody inhibition experiments were therefore carried out in liver microsomes using polyclonal anti-(P-450 3) antibodies which cross-react with P-450 RLM2, and using a monoclonal antibody that is reactive with and inhibitory towards P-450 3 but not P-450 RLM2. P-450 3 was thus shown to catalyse only around 35% of the total androstenedione 7 alpha-hydroxylase activity in uninduced adult male rat liver microsomes, with the balance attributed to P-450 RLM2. The P-450-3-dependent 7 alpha-hydroxylase activity was increased to approximately 65% of the total in phenobarbital-induced adult male microsomes, and to greater than 90% of the total in untreated adult female rat liver microsomes. These observations are consistent with the inducibility of P-450 3 by phenobarbital and with the absence of P-450 RLM2 from adult female rat liver respectively. These findings establish that P-450 RLM2 and P-450 3 can both contribute significantly to microsomal androstenedione 7 alpha-hydroxylation, thus demonstrating that the 7 alpha-hydroxylation of this androgen does not serve as a specific catalytic monitor for microsomal P-450 3.     

Microsomal flavonoid 3'-monooxygenase from maize seedlings

Identification of flavonoid 3′-monooxygenase establishes another reaction in the biosynthesis of flavonoid compounds in maize (Zea mays L.). The flavonoid 3′-hydroxylase was obtained as a microsomal enzyme preparation by buffer extraction of 5 day old maize seedlings and ultracentrifugation. Seedlings were exposed to light 24 hours prior to enzyme extraction. The extraction buffer required the addition of sucrose or glycerin and dithiothreitol to obtain an active hydroxylase that retained its activity on storage at −70°C. Enzymic activity required O₂ and NADPH, was optimum at pH 8.5 and 30°C, and could be inhibited 79% by carbon monoxide. Carbon monoxide inhibition could be reduced to 21% by irradiation of the samples with 450 nanometer light during incubation. Kaempferol, a flavonol; naringenin, a flavanone; and apigenin, a flavone, all served as substrates for the hydroxylase. Treatment of the microsomal enzyme preparation, previously reduced with sodium dithionite, with carbon monoxide gave a 455 nanometer absorption peak which disappeared on oxidation of the preparation with the formation of a 420 nanometer peak. These results suggest a cytochrome P-450 type monooxygenase enzyme. The concentration of cytochrome P-450 was 0.21 nanomoles per milligram protein. Identification of the monooxygenase provides further biochemical information about a biosynthetic sequence for which the genetics have been studied intensely.     

Soluble, nitrate/nitrite-inducible cytochrome P-450 of the fungus, Fusarium oxysporum

Both soluble and microsomal fractions of Fusarium oxysporum contain cytochrome P-450(P-450). We report here that the P-450 in the soluble fraction was induced only when nitrate or nitrite was added to the growth medium, whereas the microsomal P-450 was synthesized regardless of the medium compositions. The reduced-CO complex of the soluble P-450 exhibited an absorption spectrum that is different from that of the microsomal counterpart. These results indicate that the soluble P-450 is distinct from the microsomal species and suggest a novel function for the former P-450.     

A spectrophotometric method to monitor the catalytic activity of microsomal cytochrome P-450 IIB1/2: comparison with fluorometric assay

A simple spectrophotometric method to monitor the catalytic activity of microsomal cytochrome P-450 IIB1/2 has been developed. The method employs measurement of utilization of NADPH, consumption of the substrate, pentoxyresorufin (PRF) and formation of the product, resorufin (RF) in the same reaction mixture containing hepatic microsomes from phenobarbital treated rats. The velocity of NADPH utilization (16.36 nmole/min/nmole P-450), PRF consumption (1.58 nmole/min/nmole P-450) and RF formation (1.57 nmole/min/nmole P-450) suggested a stoichiometry of 1:1 between the substrate and the product alongwith utilization of 10 molecules of NADPH. However, the Km for the enzyme activity (nmole RF formed/min/nmole P-450) using varying concentrations of PRF and NADPH as substrates were found to be 11.6 and 20.2 microM, respectively. The spectrophotometric method was compared with fluorometric method in terms of linearity with time, P-450 content and Vmax, Km values observed for the reaction. Inhibition studies with metyrapone and SKF 525A in the utilization of NADPH, consumption of PRF and formation of RF suggested that the method could be useful in monitoring the effect of various inhibitors on the P-450 IIB1/2 reaction.