Temporary decrease in renal quinone reductase activity induced by chronic administration of estradiol to male Syrian hamsters. Increased superoxide formation by redox cycling of estrogen

Cytochrome P-450-mediated redox cycling between the synthetic estrogen diethylstilbestrol (DES) and diethylstilbestrol-4',4"-quinone (DES Q) has previously been demonstrated. Cytochrome P-450 reductase catalyzes the reduction of DES Q presumably via a semiquinone formed by one-electron reduction. A reducing action of NAD(P)H quinone reductase (EC 1.6.99.2) mediating two-electron reduction of DES Q has been investigated in the present work. Quinone reductase catalyzed the conversion in the presence of NADH or NADPH of DES Q to 53-65% Z-DES, a marker product of reduction. Dicumarol (15 microM), a known specific inhibitor of quinone reductase, inhibited this reduction almost completely. Using microsomes from Syrian hamster kidney, a target organ of estrogen-induced carcinogenesis, the reduction of DES Q was only partially inhibited by dicumarol. Apparent Km values of quinone reductase and cytochrome P-450 reductase were 17.25 and 11.9 microM, respectively. These data demonstrate that in hamster kidney, quinone reductase and cytochrome P-450 reductase compete for the reduction of DES Q. Microsomal 02-. radical generation was stimulated 10-fold over base levels by the addition of 100 microM DES Q. The formation of 02-. radicals was inhibited by addition of superoxide dismutase (0.2 mg/ml) or by 2'-AMP or NADP, known inhibitors of cytochrome P-450 reductase. In contrast, dicumarol enhanced microsome-mediated 02-. formation. It is concluded that cytochrome P-450 reductase in hamster kidney microsomes mediates one-electron reduction of estrogen quinones to free radicals (semiquinones), which may subsequently enter redox cycling with molecular oxygen to form 02-.. Moreover, quinone reductase reduces DES Q directly to E- and Z-DES, and thus may prevent the formation of toxic intermediates during redox cycling of estrogens. Measurements of quinone reductase activity in liver and kidney of hamsters treated with estrogen for various lengths of time revealed a temporary decrease in activity by 80% specifically in the kidney after 1 month of chronic treatment with estradiol. Thus, a temporary decrease in quinone reductase activity, which occurred specifically in estrogen-exposed hamster kidney, may enhance the formation of free radical intermediates generated during biotransformation of estrogens.     

The reductive activation of the antitumor drug RH1 to its semiquinone free radical by NADPH cytochrome P450 reductase and by HCT116 human colon cancer cells

RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), which is currently in clinical trials, is a diaziridinyl benzoquinone bioreductive anticancer drug that was designed to be activated by the obligate two-electron reductive enzyme NAD(P)H quinone oxidoreductase 1 (NQO1). In this electron paramagnetic resonance (EPR) study we showed that RH1 was reductively activated by the one-electron reductive enzyme NADPH cytochrome P450 reductase and by a suspension of HCT116 human colon cancer cells to yield a semiquinone free radical. As shown by EPR spin trapping experiments RH1 was reductively activated by cytochrome P450 reductase and underwent redox cycling to produce damaging hydroxyl radicals in reactions that were both H₂O₂- and iron-dependent. Thus, reductive activation by cytochrome P450 reductase or other reductases to produce a semiquinone that can redox cycle to produce damaging hydroxyl radicals and/or DNA-reactive alkylating species may contribute to the potent cell growth inhibitory effects of RH1. These results also suggest that selection of patients for treatment with RH1 based on their expression levels of NQO1 may be problematic.     

The reductive activation of the antitumor drug RH1 to its semiquinone free radical by NADPH cytochrome P450 reductase and by HCT116 human colon cancer cells

RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), which is currently in clinical trials, is a diaziridinyl benzoquinone bioreductive anticancer drug that was designed to be activated by the obligate two-electron reductive enzyme NAD(P)H quinone oxidoreductase 1 (NQO1). In this electron paramagnetic resonance (EPR) study we showed that RH1 was reductively activated by the one-electron reductive enzyme NADPH cytochrome P450 reductase and by a suspension of HCT116 human colon cancer cells to yield a semiquinone free radical. As shown by EPR spin trapping experiments RH1 was reductively activated by cytochrome P450 reductase and underwent redox cycling to produce damaging hydroxyl radicals in reactions that were both H₂O₂- and iron-dependent. Thus, reductive activation by cytochrome P450 reductase or other reductases to produce a semiquinone that can redox cycle to produce damaging hydroxyl radicals and/or DNA-reactive alkylating species may contribute to the potent cell growth inhibitory effects of RH1. These results also suggest that selection of patients for treatment with RH1 based on their expression levels of NQO1 may be problematic.     

Mechanism(s) for the metabolism of mitoxantrone: electron spin resonance and electrochemical studies

Mitoxantrone has been reported to lack certain properties that characterize quinone containing antitumor agents that undergo enzymatic reduction. These properties are the stimulation of NADPH oxidation, the stimulation of oxygen consumption by microsomes and reductases and, the absence of oxygen free radicals during these reactions. Having these properties implies the presence of a futile redox cycle that requires the generation and the oxidation of a semiquinone free radical. It would follow that if mitoxantrone does not redox cycle in the presence of reductases, then the semiquinone free radical is not produced or, if it is formed, it reacts quickly to form diamagnetic products. However, using liver microsomes, there are reports of the formation of the mitoxantrone free radial anion. In this paper we investigated the mitoxantrone free radical anion generated electrochemically and found that in the presence of oxygen it behaved like other semiquinones. That is, it is oxidized to the parent compound (presumably generating oxygen free radicals), indicating the ability to redox cycle. The reduction potential to generate such free radical in aqueous medium is very high (-0.79 V) when compared to diaziquone (-0.36 V) and Adriamycin (-0.6 V). This suggests that mitoxantrone may not be a substrate for reductases. Under reductive conditions with purified NADPH cytochrome P-450 reductase which very easily reduces diaziquone and Adriamycin, mitoxantrone was not reduced. However, under the same conditions, mitoxantrone was oxidized by the prototype oxidase horseradish peroxidase with the production of a mitoxantrone free radical. This oxidation was accompanied by a drastic change in color and the formation of a dark precipitate. Because microsomes contain a variety of enzymes, we suggest that the previously observed free radical in microsomes is probably due to the oxidation of mitoxantrone. In this theory, this product is probably a polymer which would not require oxygen to be formed. Thus, under oxidative conditions, the mitoxantrone free radical cation will also display impaired redox activity.     

Association and redox properties of the putidaredoxin reductase-nicotinamide adenine dinucleotide complex

Putidaredoxin reductase (PdR) is the flavin protein that carries out the first electron transfer involved in the cytochrome P450cam catalytic cycle. In PdR, the flavin adenine dinucleotide (FAD/FADH2) redox center acts as a transformer by accepting two electrons from soluble nicotinamide adenine dinucleotide (NAD+/NADH) and donating them in two separate, one-electron-transfer steps to the iron-sulfur protein putidaredoxin (Pdx). PdR, like the two more intensively studied monoflavin reductases, adrenodoxin reductase (AdR) and ferredoxin-NADP+ reductase (FNR), has no other active redox moieties (e.g., sulfhydryl groups) and can exist in three different oxidation states: (i) oxidized quinone, (ii) one-electron reduced semiquinone (stable neutral species (blue) or unstable radical anion (red)), and (iii) two-electron fully reduced hydroquinone. Here, we present reduction potential measurements for PdR in support of a thermodynamic model for the modulation of equilibria among the redox components in this initial electron-transfer step of the P450 cycle. A spectroelectrochemical technique was used to measure the midpoint oxidation-reduction potential of PdR that had been carefully purified of all residual NAD+, E0' = -369 +/- 10 mV at pH 7.6, which is more negative than previously reported and more negative than the pyridine nucleotide NADH/NAD+ (-330 mV). After addition of NAD+, the formation of the oxidized reductase-oxidized pyridine nucleotide complex was followed by the two-electron-transfer redox reaction, PdRox:NAD+ + 2e- --> PdRrd:NAD+, when the electrode potential was lowered. The midpoint potential was a hyperbolic function of increasing NAD+ concentration, such that at concentrations of pyridine nucleotide typically found in an intracellular environment, the midpoint potential would be E0' = -230 +/- 10 mV, thereby providing the thermodynamically favorable redox equilibria that enables electron transfer from NADH. This thermodynamic control of electron transfer is a shared mechanistic feature with the adrenodoxin P450 and photosynthetic electron-transfer systems but is different from the kinetic control mechanisms in the microsomal P450 systems where multiple reaction pathways draw on reducing power held by NADPH-cytochrome P450 reductase. The redox measurements were combined with protein fluorescence quenching of NAD+ binding to oxidized PdR to establish that the PdRox:NAD+ complex (KD = 230 microM) is about 5 orders of magnitude weaker than PdRrd:NAD+ binding. These results are integrated with known structural and kinetic information for PdR, as well as for AdR and FNR, in support of a compulsory ordered pathway to describe the electron-transfer processes catalyzed by all three reductases.     

Studies on the microsomal mixed function oxidase system: redox properties of detergent-solubilized NADPH-cytochrome P-450 reductase.

Hepatic microsomal NADPH-cytochrome P-450 reductase was solubilized from rabbit liver microsomes in the presence of detergents and purified to homogeneity by column chromatography. The purified reductase had a molecular weight of 78 000 and contained 1 mol each of FAD and FMN per mol of enzyme. On reduction with NADPH in the presence of molecular oxygen, an 02-stable semiquinone containing one flavin free radical per two flavins was formed, in agreement with previous work on purified trypsin-solubilized reductase. The reduction of oxidized enzyme by NADPH, and autoxidation of NADPH-reduced enzyme by air, proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The reductase reduced cytochrome P-450 (from phenobarbital-treated rabbits) and cytochrome P-448 (from 3-methylcholanthrene-treated rabbits). The rate of reduction of cytochrome P-450 increased in the presence of a substrate, benzphetamine, but that of cytochrome P-448 did not.     

Superoxide generation mediated by 8-nitroguanosine, a highly redox-active nucleic acid derivative

Formation of 8-nitroguanosine may be characterized as nucleic acid modification induced by nitric oxide (NO). We show here that 8-nitroguanosine is a highly redox-active nucleic acid derivative that strongly stimulated superoxide generation from various NADPH-dependent reductases, including cytochrome P450 reductase and all isoforms of NO synthase. This reaction involves these reductases in a redox cycling reaction via single-electron reduction of 8-nitroguanosine to form 8-nitroguanosine anion radical. One electron is then transferred from this radical to molecular oxygen. 8-Nitroguanosine formed in vivo may function as a potent redox cofactor that intensifies oxyradical generation by various NADPH/reductase-like enzymes and thus participates in diverse physiological and pathological events.     

Flavoenzyme-catalyzed redox cycling of hydroxylamino- and amino metabolites of 2,4,6-trinitrotoluene: implications for their cytotoxicity

The toxicity of 2,4,6-trinitrotoluene (TNT), a widespread environmental contaminant, is exerted through its enzymatic redox cycling and/or covalent binding of its reduction products to proteins and DNA. In this study, we examined the possibility of another cytotoxicity mechanism of the amino- and hydroxylamino metabolites of TNT, their flavoenzyme-catalyzed redox cycling. The above compounds acted as redox-cycling substrates for single-electron transferring NADPH:cytochrome P-450 reductase (P-450R) and ferredoxin:NADP(+) reductase (FNR), as well as substrates for the two-electron transferring flavoenzymes rat liver NAD(P)H:quinone oxidoreductase (NQO1) and Enterobacter cloacae NAD(P)H:nitroreductase (NR). Their reactivity in P-450R-, FNR-, and NR-catalyzed reactions increased with an increase in their single-electron reduction potential (E(1)(7)) or the decrease in the enthalpy of free radical formation. The cytotoxicity of the amino- and hydroxylamino metabolites of TNT towards bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was partly prevented by the antioxidant N,N'-diphenyl-p-phenylene diamine and desferrioxamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea, thus pointing to the involvement of oxidative stress. In general, their cytotoxicity increased with an increase in their electron accepting properties, or their reactivity towards the single-electron transferring FNR and P-450R. Thus, our data imply that the flavoenzyme-catalyzed redox cycling of amino and hydroxylamino metabolites of TNT may be an important factor in their cytotoxicity.     

Prevention of lipid peroxidation by NAD(P)H in rat liver submitochondrial particles

In rat liver submitochondrial particles both NADH and NADPH inhibit lipid peroxidation induced by cumene hydroperoxide. Concomitantly with the inhibition of lipid peroxidation, NADH and NADPH strongly stimulate the peroxidase activity of rat liver submitochondrial particles. Rotenone slightly prevents both the protective effect on malondialdehyde formation and peroxidase activity. The peroxidase activity of rat liver submitochondrial particles was attributed to the NAD(P)H-mediated reduction of mitochondrial cytochrome P-450 which can act upon hydroperoxides, by decomposing them to alcohols.     

Mechanism of phospholipid peroxidation induced by ferric ion-ADP-adriamycin-co-ordination complex

A system, which contains NADPH, purified cytochrome P-450 reductase and adriamycin, produces H2O2, O-2 and adriamycin semiquinone radical with O2 consumption and NADPH oxidation. This system, however, does not promote a peroxidation cleavage of unsaturated phospholipid. On the other hand, ferric ion-ADP-adriamycin-co-ordination complex, which may convert to a perferryl ion-co-ordination complex by an intramolecular electron transfer mechanism in air, acts as a powerful initiator for lipid peroxidation. A similar perferryl ion-co-ordination complex could also be produced from ferric ion-ADP-adriamycin-co-ordination complex after reducing it by NADPH-dependent cytochrome P-450 reductase in air.     

Mechanisms of hydroxyl radical formation and ethanol oxidation by ethanol-inducible and other forms of rabbit liver microsomal cytochromes P-450

The hydroxyl radical-mediated oxidation of 5,5-dimethyl-1-pyrroline N-oxide, benzene, ketomethiolbutyric acid, deoxyribose, and ethanol, as well as superoxide anion and hydrogen peroxide formation was quantitated in reconstituted membrane vesicle systems containing purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochromes P-450 LM2, P-450 LMeb , or P-450 LM4, and in vesicle systems devoid of cytochrome P-450. The presence of cytochrome P-450 in the membranes resulted in 4-8-fold higher rates of O-2, H2O2, and hydroxyl radical production, indicating that the oxycytochrome P-450 complex constitutes the major source for superoxide anions liberated in the system, giving as a consequence hydrogen peroxide and also, subsequently, hydroxyl radicals formed in an iron-catalyzed Haber-Weiss reaction. Depletion of contaminating iron in the incubation systems resulted in small or negligible rates of cytochrome P-450-dependent ethanol oxidation. However, small amounts (1 microM) of chelated iron (e.g. Fe3+-EDTA) enhanced ethanol oxidation specifically when membranes containing the ethanol and benzene-inducible form of cytochrome P-450 (cytochrome P-450 LMeb ) were used. Introduction of the Fe-EDTA complex into P-450 LMeb -containing incubation systems caused a decrease in hydrogen peroxide formation and a concomitant 6-fold increase in acetaldehyde production; consequently, the rate of NADPH consumption was not affected. In iron-depleted systems containing cytochrome P-450 LM2 or cytochrome P-450 LMeb , an appropriate stoichiometry was attained between the NADPH consumed and the sum of hydrogen peroxide and acetaldehyde produced. Horseradish peroxidase and scavengers of hydroxyl radicals inhibited the cytochrome P-450 LMeb -dependent ethanol oxidation both in the presence and in the absence of Fe-EDTA. The results are not consistent with a specific mechanism for cytochrome P-450-dependent ethanol oxidation and indicate that hydroxyl radicals, formed in an iron-catalyzed Haber-Weiss reaction and in a Fenton reaction, constitute the active oxygen species. Cytochrome P-450-dependent ethanol oxidation under in vivo conditions would, according to this concept, require the presence of non-heme iron and endogenous iron chelators.     

One-electron oxidation-reduction properties of hepatic NADH-cytochrome b5 reductase

The one-electron oxidation-reduction properties of flavin in hepatic NADH-cytochrome b5 reductase were investigated by optical absorption spectroscopy, electron paramagnetic resonance (EPR), and potentiometric titration. An intermediate with a peak at 375 nm previously described by Iyanagi (1977) [ Iyanagi , T. (1977) Biochemistry 16, 2725-2730] was confirmed to be a red anionic semiquinone. The NAD+-bound reduced enzyme was oxidized by cytochrome b5 via the semiquinone intermediate. This indicates that electron transfer from flavin to cytochrome b5 proceeds in two successive one-electron steps. Autoxidation of the NAD+-bound reduced enzyme was slower than that of the NAD+-free reduced enzyme and was accompanied by the appearance of an EPR signal. Midpoint redox potentials of the consecutive one-electron-transfer steps in the presence of excess NAD+ were Em,1 = -88 mV and Em,2 = 147 mV at pH 7.0. This corresponds to a semiquinone formation constant of 8. The values of Em,1 and Em,2 were also studied as a function of pH. A mechanism for electron transfer from NADH to cytochrome b5 is discussed on the basis of the one-electron redox potentials of the enzyme and is compared with the electron-transfer mechanism of NADPH-cytochrome P-450 reductase.     

Comparison of highly purified sheep liver and lung NADPH-cytochrome P-450 reductases by the analysis of kinetic and catalytic properties

1. Reductase was purified to electrophoretic homogeneity from sheep liver and lung microsomes. The specific activity of both enzymes ranged from 55 to 66 mumol cytochrome c reduced/min/mg protein. 2. Liver and lung reductases appeared to have similar kinetic and spectral properties. Km (NADPH) and Km (cytochrome c) values were calculated to be 14.3 +/- 1.23 microM and 22.2 +/- 2.78 microM for liver and 11.1 +/- 0.70 microM and 20.0 +/- 2.15 microM for lung reductase, respectively. Kinetic studies showed that cytochrome c can bind the oxidized form of the enzyme as well as its reduced form and both reductases operated through a ping-pong type mechanism. 3. These reductases cannot be distinguished on the basis of monomer molecular weights (Mr 78,000) except that the liver reductase was found to be more susceptible to proteolytic attack. 4. Both reductases supported aniline 4-hydroxylation and ethylmorphine N-demethylation reactions to the same extent in the reconstituted systems. However, sheep lung reductase appeared only 36.5 and 14.8% as effective in catalyzing benzo[a]pyrene reaction as an equivalent amount of reductase from liver in the presence of liver cytochrome P-450 and 3MC-treated rat liver cytochrome P-448, respectively.     

Comparative study of the C27-steroid-hydroxylating system from bovine liver mitochondria

A method for purification of C27-steroid hydroxylating cytochrome P-450 (cytochrome P-450(27)) from bovine liver mitochondria was developed. The purification procedure included enzyme extraction from submitochondrial particles with sodium cholate, ammonium sulfate fractionation and biospecific chromatography on cholate-Sepharose and adrenodoxin-Sepharose. The resulting enzyme preparation (317-fold purification, 16% yield) was not electrophoretically homogeneous but did not contain hemoprotein admixtures. The kinetic parameters of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation in a reconstituted system containing hepatoredoxin reductase, hepatoredoxin and cytochrome P-450(27) (Km = 23 microM, kcat = 0.3 s-1 at 25 degrees C) were determined. A reciprocal functional equivalency of hepatoredoxin reductase and adrenodoxin reductase as well as of hepatoredoxin and adrenodoxin in reconstituted systems of steroid 27-hydroxylation (liver) and cholesterol side chain cleavage (adrenal cortex) was established. This equivalency was thought to be due to the similarity in essential physico-chemical properties of reductase components which was especially well-pronounced in the case of hepatoredoxin and adrenodoxin. Estimation of the functional role of lysine, dicarboxylic acid and histidine residues in ferredoxin molecules by the chemical modification method revealed the similarity of the structural organization of their protein globules: the polar residues were shown to be essential for the maintenance of native conformation; dicarboxylic acid residues formed a binding domain for the interaction with electron transport proteins, whereas histidine residues seem to participate in electron transport. At the same time, cytochrome P-450(27) and cytochrome P-450 which split the side chain of cholesterol differ in their substrate specificity, immunochemical and catalytic properties.     

Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems

The antimycin-sensitive ubisemiquinone radical (QC) of the ubiquinol-cytochrome c oxidoreductase of submitochondrial particles and chromatophores of Rhodopseudomonas sphaeroides Ga has been studied by a combination of redox potentiometry and EPR spectroscopy. This g = 2.005 radical signal appears at physiological pH values and increases in intensity with increasing pH up to pH 7.6 in submitochondrial particles and pH 9.0 in R. sphaeroides after which its intensity remains unchanged. The Em7 (ubiquinone/quinol) of the signal, estimated from redox titration data is 80 mV for submitochondrial particles, and 150 mV in chromatophores. Each of these values is higher than that of the quinone pool by 20 mV in submitochondrial particles and 60 mV in R. sphaeroides. This indicates that the quinone at the binding site is out of equilibrium with the pool, and that binding site preferentially binds quinol over quinone. Analysis of the shapes of the semiquinone titration curves, taken together with the midpoint elevation, indicates a quinone-binding site: cytochrome c1 stoichiometry of 1:1 in both submitochondrial particles and chromatophores. At its maximal intensity, the semiquinone concentration at the binding site is 0.26 in submitochondrial particles (greater than pH 7.6) and 0.4 in chromatophores (greater than pH 9.0). In both systems, the midpoint of the ubiquinone/ubisemiquinone couple is constant as the pH is raised up to the pH of maximal semiquinone formation whereafter it becomes more negative at the rate of -60 mV/pH unit. The midpoint of the ubisemiquinone/quinol couple, on the other hand, varies by -120 mV/pH unit at pH values up to the transition pH, after which it, too, changes by -60 mV/pH unit. This seemingly anomalous behavior may be explained by invoking a protonated group at or near the quinone-binding site whose pK corresponds to the pH transition point in the quinone/semiquinone/quinol redox chemistry when the site is free or when quinone or quinol occupies the site. This pK is elevated to at least pH 9.0 in submitochondrial particles and 10.5 in R. sphaeroides when semiquinone is bound to the site.     

Reductase and oxidase activity of rat liver cytochrome P450 with 2,3,5,6-tetramethylbenzoquinone as substrate.

The main objective of the present study was to investigate the proposed role of cytochrome P450 in the reductive metabolism of quinones as well as in the formation of reduced oxygen species in liver microsomes from phenobarbital (PB-microsomes) and beta-naphthoflavone (beta NF-microsomes) pretreated rats. In the present study, 2,3,5,6-tetramethylbenzoquinone (TMQ) was chosen as a model quinone. Anaerobic one-electron reduction of TMQ by PB-microsomes showed relatively strong electron spin resonance (ESR) signals of the oxygen-centered semiquinone free radical (TMSQ), whereas these signals were hardly detectable with beta NF-microsomes. Under aerobic conditions TMSQ formation was diminished and concomitant reduction of molecular oxygen occurred in PB-microsomes. Interestingly, TMQ-induced superoxide anion radicals, measured by ESR (using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide), and hydrogen peroxide generation was found to occur with beta NF-microsomes as well. Furthermore, SK&F 525-A (a type I ligand inhibitor of cytochrome P450) inhibited TMQ-induced hydrogen peroxide formation in both PB- and beta NF-microsomes. However, metyrapone and imidazole (type II ligand inhibitors of cytochrome P450) inhibited molecular oxygen reduction in beta NF-microsomes and not in PB-microsomes. The present study indicates that cytochrome P450-mediated one-electron reduction of TMQ to TMSQ and subsequent redox cycling of TMSQ with molecular oxygen constitutes the major source for superoxide anion radical and hydrogen peroxide generation in PB-microsomes (i.e. from the reductase activity of cytochrome P450). However, most of the superoxide anion radical formed upon aerobic incubation of TMQ with beta NF-microsomes originates directly from the dioxyanion-ferri-cytochrome P450 complex (i.e. from the oxidase activity of cytochrome P450). In conclusion, both the one-electron reduction of TMQ and molecular oxygen were found to be cytochrome P450 dependent. Apparently, both the reductase and oxidase activities of cytochrome P450 may be involved in the reductive cytotoxicity of chemotherapeutic agents containing the quinoid moiety.     

Purification of rat liver microsomal cytochrome P-450b without the use of nonionic detergent

Sodium cholate, Emulgen 911, and (3-[(-cholamidopropyl)-dimethyl- ammonio]-1-propanesulfonate) (CHAPS) were selected to examine the effects of ionic, nonionic, and zwitterionic detergents on testosterone hydroxylation catalyzed by four purified isozymes of rat liver microsomal cytochrome P-450, namely P-450a, P-450b, P-450c, and P-450h, in reconstituted systems containing optimal amounts of dilauroylphosphatidylcholine and saturating amounts of NADPH- cytochrome P-450 reductase (reductase). The major phenobarbital-inducible form of rat liver microsomal cytochrome P-450, designated P-450b, was extremely sensitive to the inhibitory effects of Emulgen 911, which is used in several procedures to purify this and other forms of cytochrome P-450. In contrast, sodium cholate and CHAPS had little effect on the catalytic activity of cytochrome P-450b, even at ten times the concentration of Emulgen 911 effecting 50% inhibition (IC-50). By substituting the zwitterionic detergent CHAPS for Emulgen 911, we purified cytochrome P-450b without the use of nonionic detergent. The protein is designated cytochrome P-450b^* to distinguish it from cytochrome P-450b purified with the use of Emulgen 911. NADPH-cytochrome P-450 reductase was also purified both with and without the use of nonionic detergent. The absolute spectra of cytochrome P-450b and P-450b^* were indistinguishable, as were the carbon monoxide (CO)- and metyrapone-difference spectra of the dithionite-reduced hemoproteins. When reconstituted with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine, cytochromes P-450b and P-450b^* catalyzed the N-demethylation of benzphetamine and aminopyrine, the 4-hydroxylation of aniline, the O-dealkylation of 7-ethoxycoumarin, the 3-hydroxylation of hexobarbital, and the 6-hydroxylation of zoxazolamine. Both hemo-proteins catalyzed the 16α- and 16β-hydroxylation of testosterone, as well as the 17-oxidation of testosterone to androstenedione. Both hemoproteins were poor catalysts of erythromycin demethylation and benzo[a]pyrene 3-/9-hydroxylation. The rate of biotransformation catalyzed by cytochrome P-450b^* was up to 50% greater than the rate catalyzed by cytochrome P-450b when reconstituted with either reductase or reductase^*. The activity of cytochrome P-450b and P-450b^* increased up to 50% when reconstituted with reductase^* instead of reductase. In addition to establishing the feasibility of purifying an isozyme of rat liver microsomal cytochrome P-450 without the use of nonionic detergent, these results indicate that the catalytic activity of cytochrome P-450 is not unduly compromised by residual contamination with the nonionic detergent Emulgen 911.     

The drug metabolism systems of liver and liver tumors: A comparison of activities and characteristics

Summary Transplantable rat liver tumors 5123 t.c., 7288 ct.c., 5123 t.c.(H) and the Novikoff hepatoma have active mixed function oxidase systems capable of metabolizing a variety of drug and polycyclic hydrocarbon substrates. The tumor drug metabolism systems are at best 20% as active as rat liver. The tumor drug metabolism activities are induced by pretreatment with phenobarbital or -naphthoflavone and can be inhibited with specific inhibitors such as carbon monoxide or 7,8-benzoflavone. Tumor drug metabolism systems appear to consist of cytochrome P-450 and cytochrome P-450 reductase. The properties of the two protein components from tumors are highly similar to the corresponding components of the liver drug metabolism system.Cytochrome P-450 reductase has been at least partially purified from the Novikoff hepatoma and hepatoma 5123 t.c.(H). The kinetic and physical properties of the tumor reductases are similar to those of the liver reductase except that the K_m of hepatoma 5123 t.c.(H)     

Protein rotation study of cytochrome P-450 in submitochondrial particles: effect of KCl and intermolecular interactions with redox partners

The rotational diffusion of cytochrome P-450 in submitochondrial particles (SMP) of bovine adrenocortical mitochondria was measured by detecting the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate the effect of KCl on intermolecular interactions involving cytochrome P-450 and to investigate the interactions of cytochrome P-450 with other redox partners. The rotational diffusion of cytochrome P-450 was significantly dependent on KCl concentration. When the KCl concentration was increased from 0 to 1,000 mM, the mobile population of cytochrome P-450 was increased from 33 to 82%. After removing the KCl, the mobile population of cytochrome P-450 returned to the original 33%. These results suggest that nonspecific protein aggregates are dissociated by the presence of KCl, possibly due to the change in electrostatic interactions, resulting in mobilization of cytochrome P-450. SMP were observed to be nearly free from adrenodoxin and adrenodoxin reductase. The addition of adrenodoxin to SMP increased the mobile population of cytochrome P-450 from 35 to 54%. Further addition of adrenodoxin reductase to SMP containing adrenodoxin immobilized cytochrome P-450 by 6%. The addition of only adrenodoxin reductase to SMP, however, did not immobilize cytochrome P-450. The present results are consistent with our previous observations [Ohta, Y., Mitani, F., Ishimura, Y., Yanagibashi, K., Kawamura, M., & Kawato, S. (1990) J. Biochem. 107, 97-104] that cholesterol-bearing P-450SCC forms a transient ternary association with adrenodoxin and adrenodoxin reductase.     

A form of rabbit liver cytochrome P-450 that catalyzes the 7 alpha-hydroxylation of cholesterol

A form of cytochrome P-450 which comigrates with cytochrome P-450LM4 (molecular weight, 55 000) on SDS-polyacrylamide gel was purified from liver microsomes of cholestyramine-treated rabbits. This form of cytochrome P-450 catalyzed the 7 alpha-hydroxylation of cholesterol with an activity of 37.5 pmol/min per nmol cytochrome P-450 in the reconstituted enzyme system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The substrate specificity of this form of cytochrome P-450 was compared with cytochrome P-450LM4 isolated from phenobarbital- and beta-naphthoflavone-treated rabbit liver microsomes. The latter two isoenzymes do not catalyze 7 alpha-hydroxylation of cholesterol, but are more active in O-deethylation of 7-ethoxycoumarin and p-nitrophenetole. Ouchterlony double diffusion revealed cross-reactivity between anti-P-450LM4 (phenobarbital) IgG and cytochrome P-450 isolated from cholestyramine- or beta-naphthoflavone-treated rabbit liver microsomes. A two-dimensional iodinated tryptic peptide fingerprint indicated only minor structural differences among these three cytochrome P-450LM4 preparations.