Generation of free radicals during the reductive metabolism of nilutamide by lung microsomes: possible role in the development of lung lesions in patients treated with this anti-androgen.

The pulmonary metabolism of nilutamide, a nitroaromatic anti-androgen drug leading to pulmonary lesions in a few recipients, has been investigated in rats. Incubation of nilutamide (1 mM) with rat lung microsomes and NADPH under anaerobic conditions led to the formation of the nitro anion free radical, as indicated by ESR spectroscopy. The steady state concentration of this radical was not decreased by CO or SKF 525-A (two inhibitors of cytochrome P450), but was decreased by NADP+ (10 mM) or p-chloromercuribenzoate (0.47 mM) (two inhibitors of NADPH-cytochrome P450 reductase activity). Anaerobic incubations of [3H]nilutamide (0.1 mM) with rat lung microsomes and a NADPH-generating system resulted in the in vivo covalent binding of [3H]nilutamide metabolites to microsomal proteins; covalent binding required NADPH; it was decreased in the presence of NADP+ (10 mM), or in the presence of the nucleophile glutathione (10 mM), but was unchanged in the presence of carbon monoxide. Under aerobic conditions, in contrast, the nitro anion free radical was reoxidized by oxygen, and its ESR signal was not detected. Covalent binding was essentially suppressed. Instead, there was consumption of NADPH and oxygen, and production of superoxide anion and hydogen peroxide. We conclude that nilutamide is reduced by rat lung microsomes NADPH-cytochrome P450 reductase into a nitro anion free radical. In anaerobiosis, the radical is reduced further to covalent binding species. In the presence of oxygen, in contrast, this nitro anion free radical undergoes redox cycling, with the generation of reactive oxygen species.     

Trichloroethylene: Its interaction with hepatic microsomal cytochrome P-450 in vitro

The effects of inducing agents on the binding and metabolism of trichloroethylene by hepatic microsomal cytochrome P-450 are reported. The binding constant (K_s) for the interaction of trichloroethylene with hepatic microsomal cytochrome P-450 was not altered by induction with phenobarbital, 3-methylcholanthrene or spironolactone, while the maximum extent of binding (ΔA_max) was increased only following phenobarbital induction. The K_s values (ca. 1 mM) obtained for the binding of trichloroethylene to cytochrome P-450 were similar whether the enzyme was partially purified or an integral part of hepatic microsomes. The Michaelis constant (K_m) for the production of chloral hydrate from trichloroethylene by hepatic microsomal cytochrome P-450 was not altered by induction of different forms of cyfochrome P-450. V_max for the production of chloral hydrate and the rate of hepatic microsomal NADPH oxidation in the presence of excess trichloroethylene were increased by phenobarbital induction, but not by spironolactone or 3-methylcholanthrene induction. The artificial electron donors NaClO₂ and H₂O₂, but not NaIO₄, supported the metabolism of trichloroethylene by partially purified cytochrome P-450 from phenobarbital-induced rat liver microsomes. Incubation of hepatic microsomes with NADPH and trichloroethylene resulted in decreased levels of cytochrome P-450 and heme, but did not alter the levels of NADPH-cytochrome c reductase, cytochrome b₅ or glucose-6-phosphatase. The degradation of the heme moiety of cytochrome P-450 by trichloroethylene was not supported by NADH and was not inhibited by reduced glutathione (GSH). The inhibitors of cytochrome P-450—SKF 525-A, metyrapone and CO—inhibited the binding and metabolism of trichloroethylene and the trichloroethylenemediated degradation of cytochrome P-450. It is concluded that the form of cytochrome P-450 which is induced by phenobarbital, binds and metabolizes trichloroethylene, whereas other forms of the enzyme, such as cytochrome P-448, do not. Trichloroethylene appears to be activated by the phenobarbital-induced form of cytochrome P-450 to a reactive species which can then chemically alter the heme moiety of cytochrome P-450.     

Enflurane and methoxyflurane: their interaction with hepatic microsomal stearate desaturase

The effects of the volatile anesthetic agents enflurane (CClFHCF₂OCF₂H) and methoxyflurane (CCl₂HCF₂OCH₃) on hepatic microsomal electron transfer components and stearate desaturase are reported. Both enflurane and methoxyflurane stimulated electron now from NADH and NADPH through hepatic microsomal cytochrome b₅. The stimulation of electron flow from cytochrome b₅ by the anesthetic agents was not inhibited by metyrapone or CO, but was inhibited by 0.5 mM KCN. The effects of enflurane and methoxyflurane were influenced by the diet and pretreatment of the rat prior to death. A high-carbohydrate diet enhanced the effects, while fasting with or without phenobarbitone treatment diminished them. The anesthetic agents did not affect the rate constant for the autoxidation of purified trypsin-cleaved cytochrome b₅ or the activity of hepatic microsomal NADH- and NADPH-cytochrome c reductase, except that enflurane slightly increased the activity of NADH-cytochrome c reductase. The values of the equilibrium constants (K_eq) for the stimulation of the oxidation of hepatic microsomal cytochrome b₅ by enflurane and methoxyflurane were determined to be 1.2 and 0.5 mM, respectively. The K_eq for enflurane differed from the K_s and K_m values for the interaction of this anesthetic agent with cytochrome P-450, whereas the K_eq for methoxyflurane differed from the K_m for NADPH oxidation by cytochrome P-450, but not from the K_s for binding to cytochrome P-450 or the K_m for fiuoride ion production from this anesthetic agent by cytochrome P-450. The K_i values of 0.08 and 0.11 mM obtained for cyanide inhibition of the enhancement of the oxidation of cytochrome b₅ by enflurane and methoxyflurane, respectively, are within experimental error of the K_i for cyanide inhibition of stearate desaturase. Enflurane and methoxyflurane, however, did not inhibit the conversion of stearoyl CoA to oleate by hepatic microsomal stearate desaturase. It is concluded that enflurane and methoxyflurane stimulate hepatic microsomal electron flow from NADH and NADPH through cytochrome b₅in vitro, apparently by interacting with stearate desaturase.     

One-electron reduction of mitomycin c by rat liver: Role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H₂O₂ from redox cycling of the reduced mitomycin c in the presence of O₂ and the alkylation of ρ-nitrobenzylpyridine (NBP) in the absence of O₂ were taken as parameters.

2. With liver microsomes from both 3-methylcholanthrene (MC)- and phenobarbital (PB)-pretreated rats, reverse type I difference spectra were observed, indicative of a weak interaction between mitomycin c and the substrate binding site of cytochrome P-450. Mitomycin c inhibited the oxidative dealkylation of aminopyrine and ethoxyresorufin in both microsomal systems.

3. Under aerobic conditions the H₂O₂ production in the microsomal systems was dependent on NADPH, O₂ and mitomycin c, and was inhibited by the cytochrome P-450 inhibitors, metyrapone and SKF-525A.

4. Although purified NADPH-cytochrome P-450 reductase was also effective in reduction of mitomycin c and the concomitant reduction of O₂, complete microsomal systems and fully reconstituted systems of cytochrome P-450b or P-450c and the reductase were much more efficient.

5. Under anaerobic conditions in the microsomal systems both reduction of mitomycin c (measured as the rate of substrate disappearance) and the reductive alkylation of NBP were dependent on cytochrome P-450.

6. The relative rate of reduction of mitomycin c by purified NADPH-cytochrome P-450 reductase was lower than that by a complete microsomal system containing both cytochrome P-450 and a similar amount of NADPH-cytochrome P-450 reductase.

7. It is concluded that although NADPH-cytochrome P-450 reductase is active in the one-electron reduction of mitomycin c, the actual metabolic locus for the reduction of this compound in liver microsomes under a relatively low O₂ tension is more likely the haem site of cytochrome P-450.     

One-electron reductive bioactivation of 2,3,5,6-tetramethylbenzoquinone by cytochrome P450.

Bioreductive activation of quinones in mammalian liver has generally been attributed to NADPH-cytochrome P450 reductase. However, in view of the 20-30-fold molar excess of cytochrome P450 over NADPH-cytochrome P450 reductase on the endoplasmic reticulum of the rat liver cell and the capability of cytochrome P450 to bind and reduce xenobiotics, it was considered of interest to investigate the possible role of cytochrome P450 in the bioreduction of quinones. In the present study, 2,3,5,6-tetramethyl-1,4-benzoquinone (TMQ) was chosen as a model quinone. First, TMQ was found to bind at the metabolic active site of phenobarbital (PB)-inducible cytochrome P450s of rat liver microsomes, indicating that TMQ is a potential substrate for cytochrome P450-mediated biotransformation. Second, with electron spin resonance, one-electron reduction of TMQ to a semiquinone free radical (TMSQ) was found to occur in these microsomal fractions. SK&F 525-A, a well-known inhibitor of cytochrome P450, strongly inhibited TMSQ formation in these subcellular fractions without affecting NADPH-cytochrome P450 reductase activity. One-electron reductive bioactivation of TMQ was further investigated with purified NADPH-cytochrome P450 reductase alone and in reconstituted systems of purified cytochrome P450-IIB1 and NADPH-cytochrome P450 reductase. As measured by ESR, purified cytochrome P450-IIB1 in the presence of NADPH-cytochrome P450 reductase was able to reduce TMQ to TMSQ at a much greater rate than in the presence of NADPH-cytochrome P450 reductase alone. Reduction of TMQ was also investigated by measuring the initial rate of NADPH oxidation by TMQ under anaerobic conditions. Inhibitors of cytochrome P450, namely SK&F 525-A and antibodies against PB-inducible cytochrome P450s, caused a substantial decrease in reductive metabolism in PB-treated microsomes. These antibodies were also effective in the inhibition of TMQ-induced NADPH oxidation in a complete reconstituted system of equimolar concentrations of cytochrome P450-IIB1 and NADPH-cytochrome P450 reductase, indicating that the reaction was specific for cytochrome P450-IIB1. Finally, initial rates of NADPH oxidation were determined in reconstituted systems containing varying amounts of NADPH-cytochrome P450 reductase and cytochrome P450-IIB1 to determine the contribution of either enzyme in the reduction of TMQ. As expected, NADPH-cytochrome P450 reductase was able to reduce TMQ to a small extent. However, reconstitution in the presence of increasing amounts of cytochrome P450-IIB1 (relative to NADPH-cytochrome P450 reductase) resulted in increasing rates of TMQ-induced NADPH oxidation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hepatic and extrahepatic microsomal electron transport components and mixed-function oxygenases in the marine fish Stenotomus versicolor.

NADPH-cytochrome c reductase, benzo[a]pyrene hydroxylase and aminopyrine demethylase activities in hepatic microsomes from the marine fish scup (Stenotomus versicolor) were characterized according to dependence of Ph, temperature, ionic strength and Mg²⁺. The kinetic properties of benzo[a] pyrene hydroxylase were variable, depending on protein and substrate concentration, with measured K_m values for benzo[a]pyrene between 4 × 10^?7 M and 4 × 10^?5 M. The K_m for aminopyrine was 7 × 10^?4 M, and NADPH-cytochrome c reductase had K_m values of 2.1 × 10^?5 M and 1.3 × 10^?5 M for cytochrome c and NADPH. respectively. NADH supported benzo[a]pyrene hydroxylation at 10 per cent of the rate seen with NADPH, and no synergism was observed. Aminopyrine demethylation proceeded at least as well with NADH as with NADPH, and there was synergism when combined. NADPH- and NADH-cytochrome c reductases were detected in “microsomes” from fourteen extrahepatic tissues, including kidney, testis, foregut, gill, heart, red muscle, hindgut, buccal epidermis, pyloric caecum, spleen, brain, lens, ovary and white muscle. Benzo[a]pyrene hydroxylase was detected in all but white muscle, while cytochrome P-450 and aminopyrine demethylase were detectable in fewer tissues. Reduced, CO-ligated absorption maxima in the Soret region were 450 nm for all those but liver (occasionally 449 nm) and heart (about 447 nm). The estimated turnover numbers for benzo[a]pyrene hydroxylase and aminopyrine demethylase, and the influence of 7,8-benzoflavone in vitro on benzo[a]pyrene hydroxylase indicate that the cytochromes P-450 in different fish tissues are not catalytically equivalent.     

Perinatal development of hepatic microsomal mixed function oxidase activity in swine

Hepatic microsomal cytochrome P-450, cytochrome b₅, NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase levels were measured in fetal (107-days gestation), newborn and 1-, 2-, 3-, 4- and 6-week-old swine. Cytochrome P-450 levels and NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase activities increased in near parallel with ethylmorphine demethylase (V_max) activity between the first and the sixth postnatal week. The activities or levels of all parameters measured appeared to plateau between the fourth and sixth week post-partum. The only qualitative change observed after 1 week of age was a slight increase in the K_m for ethylmorphine demethylation. NADPH-cytochrome c reductase activity of fetal liver was relatively high, being approximately 40 per cent of the values attained at 6 weeks of age. This was in contrast to very low levels of NADPH-cytochrome P-450 reductase activity and cytochrome P-450 content of fetal liver. Clearly the activity of the flavoprotein NADPH-cytochrome c reductase does not limit the rate of reduction of cytochrome P-450 in the microsomal fraction of fetal liver. The possibility that cytochrome P-450 exists in a different form, or ratio of forms, in fetal liver could not be ascertained from carbon monoxide (CO) or ethylisocyanide (EtCN) difference spectra of fetal microsomal preparations. However, the dithionite difference CO spectra of cytochrome P-450 did not change with age.     

Relationship of the single-electron reduction potential of quinones to their reduction by flavoproteins

The aerobic and anaerobic metabolism of a series of quinones of known single-electron reduction potential has been studied using flavoenzymes catalyzing single-electron reduction. Metabolism was more closely related to single-electron reduction potential than to structural features or lipid solubility of the quinones studied. The pattern of quinone reduction with purified NADPH-cytochrome P-450 reductase was similar to that seen with NADH: ubiquinone oxidoreductase with NADPH as the cofactor; the lower limit for reduction was a quinone single-electron reduction potential of ?240 mV. The lower limit for quinone reduction with purified NADH-cytochrome b₅, reductase and NADH: ubiquinone oxidoreductase with NADH as the cofactor was a single-electron reduction potential of ?170 mV. With all three enzymes there was a decreased quinone metabolism at higher single-electron reduction potentials. The same pattern of quinone metabolism was seen using purified or microsomal NADPH-cytochrome P-450 reductase and purified or microsomal NADH-cytochrome b₅, reductase respectively. Microsomal quinone metabolism under aerobic conditions showed an increased V_max and an unchanged K_m, compared to metabolism under anaerobic conditions.     

The development of sex differences in the demethylation of ethylmorphine and in its interaction with components of the hepatic microsomal cytochrome P450 system in mice.

The development of sex differences in ethylmorphine N-demethylation and several components of the reaction chain were studied in hepatic microsomes from mice of the CPB-SE strain between 3–11 weeks of age. Sex-specific changes were observed in demethylation rate, type 1 spectral interaction, cytochrome P450 content, and ethylmorphine-induced stimulation of NADPH-cytochrome P450 reductase activity. These changes occurred mainly between weeks 3–7 and were confined to females. It is concluded that the development of the cytochrome P450 system is repressed by androgen during sexual maturation. The kinetic constants of demethylation developed differently from ethylmorphine binding constants. Changes in demethylase were mainly restricted to K_m, whereas the changes in type 1 binding only involved the maximum spectral change. In combination with differences observed between the developmental patterns of demethylation rate and cytochrome P450 reductase activities, this demonstrated that the reduction of cytochrome P450-substrate complex is not rate-limiting in ethylmorphine demethylation. The type 1 spectral change was correlated with the amount of cytochrome P450 only when a large portion of the cytochrome was considered inactive in ethylmorphine binding. It is suggested that immature animals possess a low basal level of ethylmorphine binding type 1 sites, which is elevated selectively in females during sexual maturation.     

Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism--III. The influence of the 1,4,5,6-tetrahydronicotinamide analogue of NADP on the NADPH kinetics of aminopyrine-N-demethylation.

1,4,5,6-Tetrahydronicotinamide adenine dinucleotide (NADH₃), a structural analogue of NADH, was unable to support the demethylation of aminopyrine or the reduction of the cytochrome P450-aminopyrine complex. However, the combination of NADH₃ with NADPH stimulated the NADPH dependent reduction of the cytochrome P450-aminopyrine complex. There was no significant alteration in the apparent K_m (NADPH) value, but there was an 80 per cent increase in apparent V of NADPH for NADPH-cytochrome P450-reductase (plus aminopyrine) when the kinetic constants were determined in the presence of 100 μM NADH₃. The inclusion of NADH₃ in the medium for aminopyrine demethylation also resulted in a significant increase in apparent V compared to the value obtained in the absence of NADH₃. The results suggest that the structure of NADH, as well as its capacity to donate the electron, is responsible for the NADH mediated increase in aminopyrine metabolism.     

Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism--I. Factors that influence NADPH kinetic estimations during mixed function oxidase reactions.

The localisation of significant amounts of nucleotide pyrophosphatase activity in the rat hepatic microsomal fraction results in erroneous values of apparent K_m (NADPH) for aminopyrine-Ndemethylase. This was demonstrated by the inclusion of 20 mM pyrophosphate, which inhibits nucleotide pyrophosphatase activity and reduces the apparent K_m (NADPH) value from 27.9 μM to 7.92 μM. The apparent K_m (NADPH) values determined in the presence of three Type I substrates were not statistically different from each other, but the value in the presence of aniline was lower. The kinetic constants of NADPH for NADPH cytochrome P450-reductase in the presence of either aminopyrine, ethylmorphine or aniline, are also reported.     

Partial purification and characterization of a reduced nicotinamide adenine dinucleotide phosphate-linked aldehyde reductase from heart.

The presence of a nonspecific NADPH-linked aldehyde reductase (alcohol-NADP oxidoreductase, EC 1.1.1.2) from heart was observed in the soluble portion of tissue homogcnates. The reductase activity was present in at least two forms. The enzyme which accounted for the major portion of activity was purified some 800-fold over the crude homogenate. The enzyme was capable of reducing a number of aromatic and medium chain length aldehydes in the presence of NADPH. No ketones were utilized as substrates and the enzyme was inactive with NADH. The enzyme was shown to have a pH optimum at 6.4 for the reduction of aldehydes. Oxidation of alcohols occurred optimally at pH 9.6 with NADP as cofactor. although the reaction proceeded at less than 10 per cent of the rate observed in the forward direction. A molecular weight of 29,000 was estimated by gel filtration on Sephadex G-75. Biogenic aldehydes prepared from β-hydroxylated phenylethylamines were actively reduced. The K_m values for 3,4-dihydroxyphenylglycolaldehyde, 4-hydroxy-3-methoxyphenylglycolaldehyde and 4-hydroxyphenylglycolaldehyde were: 0.53, 0.50 and 0.03 mM respectively. The aldehydes of non-β-hydroxylated phenylethylamines were not efficiently utilized as substrates. The K_m for NADPH was determined to be 0.013 mM in the presence of ρ-nitrobenzaldehyde. Inhibitor studies show the enzyme to be different from “classical” alcohol dehydrogenase. Various anticonvulsants and diuretics were inhibitory at concentrations of 0.01 mM. The enzyme is postulated to be responsible for the reduction of biogenic aldehydes in heart in vivo.     

Characterization of the enzymes involved in the in vitro metabolism of amrubicin hydrochloride

The in vitro metabolism of amrubicin by rat and human liver microsomes and cytosol was examined. The main metabolic routes in both species were reductive deglycosylation and carbonyl group reduction in the side-chain. In vitro metabolism of amrubicinol by rat and human liver microsomes and cytosol was also examined and the main metabolic route of this active metabolite was reductive deglycosylation. Metabolism of amrubicin in human liver microsomes was inhibited by TlCl₃ and that in human liver cytosol was inhibited by dicumarol and quercetin. Generation of amrubicinol was inhibited only by quercetin. The results indicate that metabolism of amrubicin is mediated by NADPH-cytochrome P450 reductase, NADPH:quinone oxidoreductase and carbonyl reductase. In addition, generation of amrubicinol is mediated by carbonyl reductase. Metabolism of amrubicinol in human liver microsomes was inhibited by TlCl₃ and that in human liver cytosol was inhibited by dicumarol. The results indicate that metabolism of amrubicinol is mediated by NADPH-cytochrome P450 reductase and NADPH:quinone oxidoreductase. To investigate the influence of cisplatin on the metabolism of amrubicin and amrubicinol, human liver microsomes and cytosol were pre-incubated with cisplatin. This did not change the rates of amrubicin and amrubicinol metabolism in either human liver microsomes or cytosol.     

Tetrachloroethylene metabolism by the hepatic microsomal cytochrome P-450 system

The interaction of tetrachloroethylene with hepatic microsomal cytochromes P-450 has been investigated using male Long-Evans rats. The spectral binding of tetrachloroethylene to cytochromes P-450 in hepatic microsomes from uninduced rats was characterized by a K_s of 0.4 mM. The K_s was not affected by phenobarbital induction, but was increased following pregnenolone-16α-carbonitrile induction. The K_M of 1.1 mM, calculated for the conversion of tetrachloroethylene to total chlorinated metabolites by the hepatic microsomal cytochrome P-450 system, was decreased by phenobarbital induction and increased by pregnenolone-16α-carbonitrile induction. The maximum extents of binding (ΔA_max) and metabolism (V_max) of tetrachloroethylene were increased by both phenobarbital and pregnenolone-16α-carbonitrile induction. Induction with β-naphthoflavone was without effect on any of the above parameters. The effects of the inducing agents on tetrachloroethylene-stimulated CO-inhibitable hepatic microsomal NADPH oxidation followed the same trend as their effects on V_max for the metabolism of tetrachloroethylene, although in all cases the extent of NADPH oxidation was 5- to 25-fold greater than the extent of metabolite production. The inhibitors of cytochromes P-450, viz. metyrapone, SKF 525-A, and CO, inhibited the hepatic microsomal binding and metabolism of tetrachloroethylene. Free trichloroacetic acid was found to be the major metabolite of tetrachloroethylene from the hepatic microsomal cytochrome P-450 system. Neither 2.2,2-trichloroethanol nor chloral hydrate was produced in measurable amounts from tetrachloroethylene. A minor but significant metabolite of tetrachloroethylene by cytochrome P-450 was the trichloroacetyl moiety covalently bound to components of the hepatic microsomes. Incubation of tetrachloroethylene. an NADPH-generating system. EDTA and hepatic microsomes was without effect on the levels of microsomal cytochromes P-450, cytochrome b₅, beme, and NADPH-cytochrome c reductase. It is concluded that hepatic microsomal cytochromes P-450 bind and metabolize tetrachloroethylene. The major product of this interaction is trichloroacetic acid, which is also the major urinary metabolite of tetrachloroethylene in vivo. The forms of cytochrome P-450 that bind and metabolize tetrachloroethylene include those induced by pregnenolone-16α-carbonitrile and by phenobarbital. Cytochrome P-448. which was induced in rat liver by β-naphthoflavone, does not appear to spectrally bind or metabolize tetrachloroethylene. The metabolism and toxicity of tetrachloroethylene are considered in relation to other chlorinated ethylenes.     

Oxygen radical formation and DNA damage due to enzymatic reduction of bleomycin-Fe(III)

Aerobic incubations of bleomycin, FeCl₃, DNA, NADPH, and isolated liver microsomal NADPH-cytochrome P-450 reductase resulted in NADPH and oxygen consumption and malondialdehyde formation, indicating that the deoxyribose moiety of DNA was split. All parameters measured depended on the active enzyme, bleomycin and FeCl₃. In the absence of oxygen malondialdehyde formation was very low.When bleomycin, FeCl₃ and the reductase were incubated with methional ethene (ethylene) was formed, suggesting that during the enzyme-catalyzed redox cycle of bleomycin-Fe(III/II) hydroxyl radicals were formed. Ethene formation also depended on oxygen, NADPH, the enzyme, bleomycin, and FeCl₃.During aerobic incubations of bleomycin, FeCl₃, NADPH, and isolated liver nuclei oxygen and NADPH were consumed and malondialdehyde was formed. Oxygen and NADPH consumption and malondialdehyde formation depended on bleomycin and FeCl₃. In the absence of oxygen malondialdehyde was not formed. These results indicate that nuclear NADPH-cytochrome P-450 reductase redox cycles the bleomycin-Fe(III/II) complex and that the reduced complex activates oxygen, whereby hydroxyl radicals are formed which damage the deoxyribose of nuclear DNA.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Mechanisms responsible for the thermal sensitivity of adrenal microsomal monooxygenases.

Studies were done to determine the mechanism(s) responsible for the thermal lability of adrenal microsomal monooxygenases. Preincubation of guinea pig adrenal microsomal suspensions at 37 degrees C caused large time-dependent declines in benzo(a)pyrene (BP) hydroxylase and benzphetamine (BZ) demethylase activities. Similar preincubations with hepatic microsomes had little effect on enzyme activities. The decreases in adrenal enzyme activities were completely prevented by co-incubation of microsomes with cytosol, but were not diminished by reduced glutathione, ascorbic acid, or bovine serum albumin. Partial protection was afforded by EDTA, suggesting that lipid peroxidation might be involved, but malonaldehyde production was not demonstrable and MnCl2, a potent inhibitor of lipid peroxidation, did not affect the decline in enzyme activities. The decreases in the rates of BP and BZ metabolism were prevented by including NADPH or NADP+ in the preincubation medium. The preincubation conditions causing losses of adrenal enzyme activities did not affect cytochrome P-450 concentrations or substrate binding to cytochromes P-450, as indicated by type I difference spectra. NADH-cytochrome c reductase activity also was not affected, but there were decreases in NADPH-cytochrome c reductase activity that were proportionately similar to the declines in drug-metabolizing activities. Direct assessment of NADPH-cytochrome P-450 reductase revealed similarly large decreases in enzyme activity resulting from preincubation of adrenal microsomes. The results demonstrate a need for extra caution when doing preincubation experiments with adrenal microsomal preparations, and suggest that the thermal lability of adrenal monooxygenases is attributable to effects at the active site of NADPH-cytochrome P-450 reductase.     

Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism--II. Evidence for a co-operative interaction between NADPH and NADH.

There was a significant reduction in apparent K_m (NADPH) values for both aminopyrine and ethylmorphine demethylases when the kinetic constants for NADPH were determined in the presence of constant NADH concentrations. NADH was also shown to significantly stimulate apparent V for NADPH cytochrome P450-reductase (in the presence of aminopyrine) without changing the apparent K_m (NADPH) value. Further, NADH stimulated the initial rapid phase of the biphasic reduction kinetics of NADPH cytochrome P450-reductase in the presence of aminopyrine. These findings suggest that in the presence of both pyridine nucleotides, there has been a change in the rate limiting step which, with NADPH alone, is generally accepted to be the reduction of the cytochrome P450-substrate complex. It has been necessary to make certain modifications to a previously proposed mechanism to explain the results obtained in the present study.     

Modification of carbon tetrachloride hepatotoxicity by chemicals.

Cobaltous chloride (60 mg/kg, sc, daily for 2 days), which was found to effectively decrease the microsomal cytochrome P-450 content of mouse liver to approximately half of its normal value and which impaired the oxidative metabolism or hydroxylation of aminopyrine, ethylmorphine, and hexobarbital, offered no protection against CCl₄-induced liver damage. However, this hemoprotein inhibitor had no effect on the rate of reduction of cytochrome P-450 by NADPH and exerted a slight effect on aniline hydroxylation. SKF-525A (50 mg/kg, ip) also failed to protect against CCl₄ hepatotoxicity though it has been shown to inhibit the hydroxylation of a number of substrates. This inhibitor, a type I compound, was found to enhance cytochrome P-450 reduction by NADPH. Further studies revealed that CCl₄-induced hepatic injury could be prevented by phenazine methosulfate (2 mg/kg, ip, 5 doses at 0.5-hr intervals), which in vitro was found to inhibit NADPH-cytochrome c reductase noncompetitively. All of these findings are not satisfactorily explainable by electron transfer from NADPH-cytochrome c reductase to CCl₄ as the activation reaction for CCl₄ but are compatible with the hypothesis previously proposed by others that cytochrome P-450 is the critical site for CCl₄ activation.     

Pyridine nucleotide cofactor requirements of indicine N-oxide reduction by hepatic microsomal cytochrome P-450

Indicine N-oxide is reduced to indicine under anaerobic conditions by rat hepatic microsomal fraction in the presence of either NADH or NADPH. CO completely inhibits reduction, indicating the involvement of cytochrome P-450. In contrast to cytochrome P-450-catalyzed oxidations, for which NADH is about 15 per cent as effective as NADPH, NADH is 80 per cent as effective as NADPH in supporting indicine N-oxide reduction. In the presence of 3 mM NADH, the K_m for indicine N-oxide is 0.37 mM, and the V_max is 2.65 nmoles indicine formed/min/mg; with 3 mM NADPH the K_m is 0.51 mM, and the V_max is 3.00 nmoles/min/mg. NADH- and NADPH-dependent indicine N-oxide reduction appear to involve different pathways. NADH-supported reduction is inhibited 48 per cent by 0.5 mM KCN and 45 per cent by 0.8 M acetone, while NADPH-supported reduction is inhibited 3 per cent by 0.5 mM KCN and stimulated 28 per cent by 0.8 M acetone. The ability of NADH and NADPH at saturating concentrations to support indicine N-oxide reduction is additive, although this effect is not seen with phenobarbital- or 3-methylcholanthrene-pretreated animals. Phenobarbital pretreatment produces a selective increase in the V_max for NADPH-dependent reduction, to 5.75 nmoles/min/mg, but has no significant effect upon K_tm with NADPH or upon either the K_m for the f_max for NADH-supported indicine N-oxide reduction. Pretreatment with 3-methylcholanthrene has no significant effect upon the K_m or V_max for NADPH- or NADH-supported reduction. A possible explanation for the observations is a form of cytochrome P-450 which can accept electrons from NADH and catalyze indicine N-oxide reduction but which does not contribute to oxidative microsomal drug metabolism     

Metabolism of tetraorganolead compounds by rat-liver microsomal mono-oxygenase: II. Enzymic dealkylation of tetraethyl lead

1. The biological degradation of tetraethyl lead to the triethyl lead cation by rat-liver microsomes from untreated, phenobarbital-pretreated and methylcholanthrene-pretreated rats has been studied; NADPH and oxygen are essential.

2. The reaction is inhibited by CO and can be reactivated in the presence of O₂ by irradiation with u.v. light with a max. at 450nm.

3. Substrate binding to cytochrome P-450 is of type 1.

4. Apparent K_m values for triethyl lead formation in microsomes were determined. The highest activities (i.e. about 2 nmol triethyl lead per nmol cytochrome P-450 per min) and the lowest apparent K_m values (i.e. 7 × 10^-6 M) are found in microsomes from methylcholanthrene-pretreated rats.

5. In microsomes from control and phenobarbital-pretreated rats K_s values from substrate-binding studies (about 2 × 10^-6M) are one order of magnitude lower than the apparent K_m values (3 × 10^-5 M).