Cytochrome P-450-mediated oxidation of 2-hydroxyestrogens to reactive intermediates

2-Hydroxyestradiol, 2-hydroxyestrone and 2-hydroxy-17α-ethynylestradiol, oxidation products of naturally occurring estrogens and synthetic estrogens in some oral contraceptives were found to be converted by rat liver microsomes to reactive metabolites that become irreversibly bound to microsomal protein. The irreversible binding required microsomes, oxygen and NADPH. The NADPH could be replaced by a xanthine-xanthine oxidase system which is known to generate superoxide anions. The irreversible binding was substantially inhibited by superoxide dismutase, 30% in those incubations containing NADPH and 98% in those incubations containing the xanthine-xanthine oxidase system. Further studies with 2-hydroxyestradiol showed that microsomal cytochrome P-450 was rate limiting in the NADPH-dependent irreversible binding, because the binding was inhibited 62% by an antibody against NADPH-cytochrome $\text{c}$ reductase and 70% in an atmosphere of CO:O₂ (9:1) when compared to an atmosphere of N₂:O₂ (9:1). Phenobarbital, a known inducer of cytochrome P-450, had no effect on the irreversible binding of 2-hydroxyestradiol, whereas another inducer of P-450, pregnenolone-16α-carbonitrile, markedly increased the irreversible binding. In contrast, cobaltous chloride, an inhibitor of the synthesis of cytochrome P-450, decreased both P-450 and the irreversible binding. These results are consistent with a mechanism for irreversible binding of estrogens and 2-hydroxyestrogens to microsomes that requires oxidation of the catechol nucleus by cytochrome P-450-generated superoxide anion.     

Activation and irreversible binding of regiospecifically labeled catechol estrogen by rat liver microsomes: evidence for differential cytochrome P-450 catalyzed oxidations

Estradiol and 2-hydroxyestradiol labeled with 3H at different positions in rings A or B were incubated with male rat liver microsomes, and their oxidative transformation was followed by the transfer of 3H into 3H2O. 14C-labeled estrogen or catechol estrogen was used to determine the fraction that becomes bound covalently to microsomal protein. The further metabolism of 2-hydroxyestradiol involves activation of the steroid at C-4 and, to a much lesser extent at C-1, by a cytochrome P-450 mediated reaction as indicated by the effects of NADPH, spermine, SKF-525A, and CO in the microsomal system. Glutathione promoted the loss of 3H from C-4 of either estradiol or 2-hydroxyestradiol but had less effect on this reaction at C-1 and inhibited it at C-6,7. It also abolished the irreversible binding of 14C-labeled estradiol and 2-hydroxyestradiol to microsomal protein. NADPH was needed specifically for glutathione to exert its effect both on the transfer of 3H into 3H2O and on the formation of water-soluble products from catechol estrogen by rat liver microsomes. It could not be replaced by NADP, NAD, or NADH. Ascorbic acid inhibited these enzymatic reactions but did not affect significantly the initial 2-hydroxylation of estradiol. Evidence is also provided for the further hydroxylation of 2-hydroxyestradiol at C-6 (or C-7). These results indicate that cytochrome P-450 activates catechol estrogens by an electron abstraction process.     

Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: role of cytochrome P-450.

In order to define the site of bioactivation of CCl₄, CHCl₃ and CBrCl₃ in the NADPH cytochrome $\text{c}$ reductase-cytochrome P-450 coupled systems of liver microsomes, the ¹⁴C-labeled hepatotoxins were incubated $\text{in}$$\text{vitro}$ with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O₂ (8:2) and addition of a cytochrome $\text{c}$ reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome $\text{c}$ reductase activity was unchanged, the covalent binding of CCl₄, CHCl₃, and CBrCl₃ was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N₂ enhanced the binding of CCl₄, inhibited the binding of CHCl₃ and did not influence the binding of CBrCl₃. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome $\text{c}$ reductase and that CCl₄ bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl₃ activation proceeds by cytochrome P-450 dependent oxidative pathways.     

Solubilization and partial purification of cytochrome P-450 from rat lung microsomes

Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino-$\text{n}$-octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome $\text{c}$ reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found.     

The effect of enzyme induction on the irreversible binding of ethynyloestradiol to guinea pig liver microsomes

The effect of pretreatment with phenobarbitone, rifampicin, β-naphthoflavone, antipyrine and spironolactone on the irreversible binding of ethynyloestradiol to guinea pig liver microsomes has been examined and the corresponding changes in microsomal P-450 content and cytochrome $\text{c}$ reductase activity measured. Rifampicin produced the greatest increase (220%) in irreversible binding while phenobarbitone produced the greatest increase in both microsomal P-450 content (172%) and cytochrome $\text{c}$ reductase activity (210%). There was no correlation of irreversible binding with either microsomal P-450 content or with cytochrome $\text{c}$ reductase activity.     

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 $\text{in vitro}$.     

NADPH:cytochrome P-450(c) reductase: Biochemical characterization in rat brain and cultured neurons and evolution of activity during development

NADPH:cytochrome P-450 (c) reductase is a microsomal enzyme which is involved in the cytochrome P-450-dependent biotransformation of many exogenous agents as well as of some endogenous molecules. Using cytochromec as a substrate, the kinetic parameters of this enzyme were determined in brain microsomes. The comparison of the NADPH:cytochrome P-450 reductase's V_max values and cytochrome P-450 contents in both fractions, suggests a role of cerebral NADPH:cytochrome P-450 reductase in cytochrome P-450 independent pathways. This is also supported by the different developmental pattern of brain enzyme as compared to the liver enzyme, and by the presence of a relatively high NADPH:cytochrome P-450 reductase activity in immature rat brain and neuronal cultures, while cytochrome P-450 was hardly detectable in these preparations. The enzyme activity was not induced by a phenobarbital chronic treatment neither in the adult brain nor in cultured neurons, suggesting a different regulation of the brain enzyme expression.     

NADPH-dependent reductase solubilized from microsomes by peroxidation and its activity

Isolated rat liver microsomes were subjected to enzymatic or non-enzymatic lipid peroxidation $\text{in vitro}$. NADPH-dependent cytochrome $\text{c}$ reductase activity was released from the microsomes into the media during peroxidation. This activity could be recovered from the media by DEAE-cellulose chromatography. The recovered enzyme retained high activity for the reduction of cytochrome $\text{c}$ and a lower level of activity for the reduction of cytochrome P-450. The active fractions were capable of enzymatically supporting the peroxidation of isolated mitochondria in the presence of organically complexed Fe⁺³ and NADPH, and in this respect the specific activity was found to be about ten times higher than in microsomes.     

Response of the mixed function oxidase system of rat hepatic microsomes to parathion and malathion and their oxygenated analogs

$\text{In}$$\text{vitro}$ inhibition of ethylmorphine metabolism in rat hepatic microsomes by parathion, malathion, malaoxon and paraoxon was not well correlated with their effects on NADPH oxidation, cytochrome C reduction or the reduction of cytochrome P-450. A parallel relationship was observed between inhibition of ethylmorphine metabolism by parathion, malathion and malaoxon and the binding affinity of these agents to microsomal cytochrome P-450 obtained from rats pretreated with an anticholinesterase agent, Bis-[?-nitrophenol] phosphate.     

Cytochrome P-450 purified to apparent homogeneity from phenobarbital-induced rabbit liver microsomes: catalytic activity and other properties

Cytochrome P-450 was purified to a content of over 17 nmoles per mg of protein from liver microsomes of phenobarbital-treated rabbits by fractionation with polyethylene glycol 6000, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography in the presence of Renex 690, a nonionic detergent. The purified preparation exhibited a single polypeptide band (molecular weight, 49,000 daltons) when submitted to SDS-polyacrylamide gel electrophoresis. Cytochromes P-420 and $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase were absent. The reconstituted system containing purified cytochrome P-450, reductase, and phosphatidylcholine catalyzed the hydroxylation of benzphetamine, cyclohexane, aniline, and laurate.     

Effects of anti-NADPH-cytochrome c reductase and anti-cytochrome b5 antibodies on the hepatic and pulmonary microsomal metabolism and covalent binding of the pulmonary toxin 4-ipomeanol.

An antibody prepared against purified rat liver NADPH-cytochrome $\text{c}$ reductase inhibited both the pulmonary and hepatic microsomal covalent binding of 4-ipomeanol as well as the respective NADPH-cytochrome $\text{c}$ reductase activities, findings which are consistent with previous studies which indicated the participation of cytochrome P450 in the metabolic activation of the toxin. An antibody prepared against purified rat liver cytochrome b₅, which strongly inhibited both the rat hepatic and pulmonary NADH-dependent cytochrome $\text{c}$ reductases, and was inactive against the respective NADPH-dependent cytochrome $\text{c}$ reductases, had little effect on metabolic activation of 4-ipomeanol by hepatic microsomes, but strongly inhibited both the NADH-supported and the NADPH-supported pulmonary microsomal metabolism and covalent binding of the compound. These results suggest that metabolic activation of 4-ipomeanol involves a two-electron transfer in which transfer of the second electron via cytochrome b₅ is rate-limiting in lung microsomes.     

Purification of cytochrome P-450 from liver microsomes of phenobarbital-treated guinea pigs

Cytochrome P-450 was purified from phenobarbital-treated guinea pigs to a specific content of 19.8 nmoles per mg of protein, and was free of cytochrome b₅ and NADPH-cytochrome $\text{c}$ reductase. The purified cytochrome P-450 gave a single protein band on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 49,000 was estimated. Benzphetamine N-demethylation activity could be reconstituted by mixing the purified cytochrome, NADPH-cytochrome $\text{c}$ reductase and phosphatidylcholine.     

Synthesis of a complementary DNA to rat liver albumin mRNA.

NADPH reduces both liver microsomal cytochrome P-450 and cytochrome $\text{b}{}_5$. In the presence of CO, ferrous cytochrome P-450 can slowly transfer electrons to amaranth, an azo dye. This reaction is followed by the reoxidation of cytochrome $\text{b}{}_5$ which proceeds at essentially the same rate as does cytochrome P-450 oxidation. It is suggested that cytochrome $\text{b}{}_5$ directly reduces cytochrome P-450 in rat liver microsomes.     

Induction of polysubstrate monooxygenase and aflatoxin production by phenobarbitone in Aspergillus parasiticus NRRL 3240

The presence of the components of polysubstrate monooxygenase (PSMO) activity, viz., cytochrome P-450 and NADPH cytochrome P-450 reductase has been established for the first time in the microsomes of $\text{Aspergillus parasiticus}$. The microsomes were able to metabolize benzphetamine. NADPH cytochrome P-450 reductase, benzphetamine metabolism and aflatoxin production was increased by the presence of phenobarbitone (PB, 2mg/ml) in the medium. These results demonstrate that induction of PSMO activity could be a prerequisite for increased production of aflatoxins, since hydroxylation of intermediates is an obligatory step in aflatoxin biosynthesis.     

Purification of a substrate complex of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-treated rabbits.

Cytochrome P-450 was purified as a 3-methylcholanthrene complex from liver microsomes of 3-methylcholanthrene-treated rabbits to a specific content of 17 to 18 nmoles per mg of protein with a yield of about 10 %. The purified protein gave only a single protein band on sodium dodecylsulfate-urea-poly-acrylamide gel electrophoresis, and its apparent molecular weight was estimated to be about 54,000, a value which is higher than that for cytochrome P-450 from phenobarbital-treated rabbits by about 4,000. The reconstituted system containing the purified cytochrome and NADPH-cytochrome $\text{c}$ reductase was active in NADPH-dependent hydroxylation of benzo[α]pyrene.     

Membrane effects on drug monoxygenation activity in hepatic microsomes

The temperature dependence of drug monooxygenation in phenobarbital-induced rat liver microsomes has been investigated. With 7-ethoxycoumarin as a substrate the activity of the microsomes could be measured down to 0°C by the increase in fluorescence of the dealkylated reaction product 7-hydroxycoumarin (umbelliferone).Arrhenius plots of the activities at various temperatures between 0°C and 45°C showed a break in the activation energy around 20°C.Addition of deoxycholate or high concentrations of glycerol, known to solubilize membrane-bound enzymes, abolished the break of the activation energy. Cholesterol, incorporated into the microsomal membrane in amounts equimolar to the microsomal phospholipid content led to a decrease of the activation energy at low temperatures and to an increase at higher temperatures, resulting in a loss of the break.The activity of microsomal NADPH-cytochrome $\text{c}$ reductase with the water-soluble electron acceptor dichlorophenolindophenol showed no discontinuity in the Arrhenius plot. In addition the cumene hydroperoxide-mediated and cytochrome $\text{P-450-}\text{dependent}$ O-dealkylation of 7-ethoxycoumarin proceeded without a break in the activation energy.It is concluded that phospholipid phase transitions affect the electron transfer from the reductase to cytochrome $\text{P-450}$.     

Selective induction of cytochrome b5 and NADH cytochrome b5 reductase by propylthiouracil

Both the cytochrome b₅ level and NADH cytochrome b₅ reductase activity in rat liver microsomes were increased 2-fold by repeated i.p. administration of 1.5 mmol/kg propylthiouracil (PTU) for 2 weeks, but neither the cytochrome P-450 level nor NADPH cytochrome P-450 reductase activity were affected by the treatment. Liver microsomes from PTU-treated rats showed a significant decrease in aminopyrine N-demethylation, but not in benzphetamine N-demethylation, aniline hydroxylation or 7-ethoxycoumarin O-deethylation. A single administration of the compound had no effect on any components of the system. $\text{In vitro}$, drug hydroxylation activities were not affected by PTU up to 1.0 mM. From the above evidence, repeated administration of PTU selectively induced cytochrome b₅ and NADH cytochrome b₅ reductase in rat liver microsomes.     

Purification of the major cytochrome P-450 of liver microsomes from rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Cytochrome P-450 was isolated in highly purified form from liver microsomes of adult male rabbits treated with 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin (TCDD). Preparations average 17.8 ± 0.8 nmoles cytochrome P-450 per mg protein and have an estimated molecular weight of 54,500. The visible absorption spectrum of the purified cytochrome displays absorption spectral maxima characteristic of high spin forms of cytochrome P-450. When reconstituted with highly purified NADPH-cytochrome P-450 reductase, this cytochrome catalyzes the hydroxylation of acetanilide and the O-deethylation of 7-ethoxyresorufin, two activities induced by TCDD.     

Immunohistochemical localization of NADPH-cytochrome c reductase in rat liver.

NADPH-cytochrome $\text{c}$ reductase (NADPH-cytochrome reductase, EC 1.6.2.4), the flavoprotein which is responsible for the NADPH-dependent reduction of cytochromes P-450 in hepatic microsomes, has been localized immunohistochemically at the light microscopic level in rat liver. Localization was achieved through the use of sheep antiserum to rat hepatic microsomal NADPH-cytochrome $\text{c}$ reductase in an unlabeled antibody peroxidase-antiperoxidase technique. Parenchymal cells throughout the liver lobule were found to be stained positively for NADPH-cytochrome $\text{c}$ reductase, although the intensity of immunostaining was slightly greater in the centrilobular regions. Immunostaining for NADPH-cytochrome $\text{c}$ reductase was not detected in Kupffer cells, connective tissue cells, or in cells of the hepatic vasculature.     

A gel-electrophoretically homogeneous preparation of cytochrome P-450 from liver microsomes of phenobarbital-pretreated rabbits

Cytochrome P-450 was purified from liver microsomes of phenobarbital-pretreated rabbits to a specific content of 16 to 17 nmoles per mg of protein with a yield of about 10 %. The purified cytochrome yielded only a single protein band on sodium dodecylsulfate-urea-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 45,000 was estimated for the protein. The preparation was free of cytochrome $\text{b}{}_5$, NADH-cytochrome $\text{b}{}_5$ reductase, and NADPH-cytochrome $\text{c}$ reductase activities. Aniline hydroxylase and ethylmorphine N-demethylase activities could be reconstituted upon mixing the purified cytochrome with an NADPH-cytochrome $\text{c}$ reductase preparation (purified by a detergent method) and phosphatidyl choline.