Loss Of Cytochrome P-450 Research Articles

Suicidal inactivation of microsomal cytochrome P-450 by halothane under hypoxic conditions

Under anaerobic conditions the addition of halothane to NADPH-reduced liver microsomes from phenobarbital-pretreated male rats resulted in a pronounced inactivation of microsomal cytochrome P-450, presumably produced by covalent binding of reactive halothane metabolites such as the CF 3CHCl-radical. Compared with microsomes from phenobarbital-pretreated rats, the loss of active cytochrome P-450 was markedly decreased in microsomes from both 3-methylcholanthrene-pretreated and untreated rats. Increasing the O 2-partial pressure decreased the amount of cytochrome P-450 inactivated by halothane metabolites. At an O 2-partial pressure of approximately 40 mm Hg the inactivation was virtually eliminated.

Suicidal inactivation of hepatic cytochrome P-450 in vitro by some aliphatic olefins

Vinylalicyclic olefins destroyed hepatic cytochrome P-450 from phenobarbital-pretreated male mice, in the presence of NADPH; the loss of the enzyme was always accompanied by a quantitatively correlated loss of microsomal heme. The same compounds caused much smaller losses of cytochrome P-450 and heme in 3-methylcholanthrene-microsomes than with PB-ones. The effect has been shown to be specific for cytochrome P-450, it is not stimulated by NADH, it is inhibited by CO, Methyrapone, and it shows pseudo first-order kinetics. The epoxides of the parent active olefins did not intervene in the destruction of the enzyme. No cytochrome P-450 loss was observed after incubation with cyclohexene, 1,3- and 1,4-cyclohexadiene, and isopren.

The reaction of phenylhydrazine with microsomal cytochrome P-450. Catalysis of heme modification.

Phenylhydrazine interacted with oxidized and reduced cytochrome P-450 of rat liver microsomes to produce binding difference spectra typical of many nitrogenous compounds. The phenylhydrazine-induced difference spectrum observed with oxidized microsomal cytochrome P-450 was converted, in a time-dependent process, to yield a new spectral intermediate with an absorbance maximum around 480 nm. The time required to form this new phenylhydrazine-induced spectral intermediate was decreased from hours to minutes when either NADPH or NADH was added to the reaction mixture. Phenyldiazene generated by addition of the decarboxylation product of methyl phenyldiazenecarboxylate or by addition of potassium ferricyanide and phenylhydrazine (2:1 molar equivalents) instantly formed the new spectral intermediate. This suggests that phenyldiazene is formed during the NADPH-dependent reaction. The appearance of the new spectral intermediate occurred concomitant with the loss of CO-reactive cytochrome P-450 (less than 90%) and loss of absorbance at 418 nm. The interpretation of the optical spectral changes was supported by a loss of the low spin signals characteristic of oxidized cytochrome P-450 as determined by electron paramagnetic resonance spectroscopy. The loss of CO-reactive cytochrome P-450 apparently resulted from the formation of a binary complex of phenyldiazene and the heme of oxidized cytochrome P-450 giving rise to the 480 nm spectral intermediate. In addition, the diazene-bound heme of cytochrome P-450 apparently was modified irreversibly in the presence of oxygen. The effects observed with phenylhydrazine could be produced to a lesser degree by other hydrazine derivatives. The possible role of phenylhydrazine as a new type of suicide substrate is discussed.

The destruction of cytochrome P-450 by alclofenac: Possible involvement of an epoxide metabolite

The metabolism of alclofenac (4-allyloxy-3-chlorophenyl acetic acid) and its ability to induce destruction of cytochrome P-450 was studied in mouse hepatic microsomes obtained from control animals or animals pretreated with phenobarbitone (PB) or 3-methyl-cholanthrene (3-MC). No evidence was obtained for metabolism of alclofenac in control microsomes although in induced microsomes alclofenac was metabolised to both the dihydroxy (DHA) and phenolic (4-hydroxy-3-chlorophenyl acetic acid: HCPA) metabolites. Significant destruction of cytochrome P-450 was observed when alclofenac was incubated with microsomes from mice pretreated with PB but not from untreated or 3-MC-treated mice. This destruction is dependent on the presence of a NADPH-regenerating system and is inhibited in the presence of SKF525A, metyrapone, glutathione and cysteine. The stable metabolites DHA and HCPA caused no loss of cytochrome P-450 whereas the reactive intermediate, alclofenac epoxide, was a potent inducer of destruction in the absence of NADPH. These results suggest that destruction of cytochrome P-450 by alclofenac in vitro is mediated, at least in part, through the formation of a reactive epoxide metabolite.

Destruction of microsomal calcium pump activity: a possible secondary mechanism in BrCCl3 and CCl4 liver cell injury.

In vitro rat liver microsomes free of Fe2+ ions peroxidize minimally at 37° when NADPH and either CC14 or BrCC13 are added. Although the lipid peroxidation dependent on toxigenic haloalkanes in these Fe2+-free microsome systems is very low, it is considerably more efficient in causing loss of cytochrome P-450 and glucose6-phosphatase than is the far more vigorous lipid peroxidation dependent on presence of Fe2+ ions. In particular, the Ca2+-pump activity of isolated microsomes was almost completely destroyed when malonic dialdehyde (MDA) production was as little as 8 pg per gram equivalent microsomes. Moore et al. (1976) had shown previously that the capacity of liver microsomes to sequester Ca2+ was severely depressed 30 min after CC14 administration to rats. We have shown that this effect is already manifested within 3 min after CC14 administration, by which time peroxidative decomposition of microsomal lipids can be detected. The time course of the destruction of the liver microsomal Ca2+ pump after CC14 administration to rats was essentially identical to the time course of microsomal lipid peroxidation, as revealed by the appearance of conjugated diene configurations in microsomal lipids.KeywordsLipid PeroxidationCarbon TetrachlorideMalonic DialdehydeLipid Peroxidation ProcessMicrosomal Lipid PeroxidationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

N-Alkylprotoporphyrin IX formation in 3,5-dicarbethoxy-1,4-dihydrocollidine-treated rats. Transfer of the alkyl group from the substrate to the porphyrin.

Administration of 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) to rats causes the accumulation of N-methylprotoporphyrin IX, a potent inhibitor of ferrochelatase. To clarify the origin of the porphyrin N-methyl group, we have synthesized and administered to rats N-ethyl-3,5-dicarbethoxy-1,4-dihydrocollidine (N-ethyl DDC) and 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), the DDC analogue with a 4-ethyl rather than 4-methyl group. Only N-methylprotoporphyrin IX is isolated from rats treated with the former agent, and only N-ethylprotoporphyrin IX from those treated with the latter. All four isomers of N-ethylprotoporphyrin IX are formed biologically. The structure of the isolated porphyrins has been confirmed by complete spectroscopic comparison with the four synthetic isomers of N-ethylprotoporphyrin IX. DDEP has been shown to cause NADPH- and time-dependent in vitro loss of hepatic microsomal cytochrome P-450. These results unequivocally establish that the 4-alkyl groups in DDC and dDEP are the source of the N-alkyl group in N-methyl- and N-ethylprotoporphyrin IX, respectively, and strongly suggest that the alkyl group is transferred to the prosthetic heme of cytochrome P-450 during catalytic processing of the substrate by the enzyme. The mechanism of the group transfer is discussed.

Depression of cytochrome P-450-dependent drug biotransformation by adriamycin

The cytochrome P-450-dependent mixed function oxidase system was depressed following the administration of adriamycin. Cytochrome P-450, aminopyrine N-demethylase, and benzo( a)pyrene hydroxylase were significantly decreased in hepatic microsomes prepared from rats treated with a single dose of adriamycin (10 mg/kg subcutaneously) 4 days previously. Total microsomal protein levels and cytochrome b 5 levels remained unchanged. The administration of cysteamine 1 hr prior to adriamycin prevented the loss of the cytochrome P-450 and the decrease in drug biotransformation. The administration of diethylmaleate had no effect on the ability of adriamycin to decrease this enzyme system. The loss of drug biotransformation and the protection offered by cysteamine is correlated with the lipid peroxidation activity in hepatic microsomes. This suggests that the loss of cytochrome P-450 and related drug biotransformation is related to the generation of free radicals and subsequent lipid peroxidation in the liver.

CARBON TETRACHLORIDE-INDUCED LOSS OF MICROSOMAL GLUCOSE 6-PHOSPHATASE AND CYTOCHROME P-450 IN VITRO

AbstractRat liver microsomes were incubated in the presence of NADPH and CCU under various conditions, and losses of glucose 6-phosphatase (G-6-Pase) and cytochrome P-450 were examined in terms of lipid peroxidation and CCl4 metabolism. Loss of G-6-Pase activity correlated well with the enhancement of lipid peroxidation. Loss of cytochrome P-450 was also dependent on the lipid peroxidation, under aerobic conditions. However, the cytochrome was destroyed under anaerobic conditions in which lipid peroxidation and loss of G-6-Pase were greatly suppressed. This anaerobic loss of cytochrome P-450 may be linked with the metabolism of CCl4 by this hemoprotein, as evidenced by the observation that CCU metabolism occurred only under anaerobic conditions and was inhibited by carbon monoxide accompanied by the suppression of the loss of cytochrome P-450.

Carbon tetrachloride-induced loss of microsomal glucose 6-phosphatase and cytochrome P-450 in vitro.

Rat liver microsomes were incubated in the presence of NADPH and CCl4 under various conditions, and losses of glucose 6-phosphatase (G-6-Pase) and cytochrome P-450 were examined in terms of lipid peroxidation and CCl4 metabolism. Loss of G-6-Pase activity correlated well with the enhancement of lipid peroxidation. Loss of cytochrome P-450 was also dependent on the lipid peroxidation, under aerobic conditions. However, the cytochrome was destroyed under anaerobic conditions in which lipid peroxidation and loss of G-6-Pase were greatly suppressed. This anaerobic loss of cytochrome P-450 may be linked with the metabolism of CCl4 by this hemoprotein, as evidenced by the observation that CCl4 metabolism occurred only under anaerobic conditions and was inhibited by carbon monoxide accompanied by the suppression of the loss of cytochrome P-450.

Nutrient imbalance causes the loss of cytochrome P-450 in liver cell culture: formulation of culture media which maintain cytochrome P-450 at in vivo concentrations.

Nutrient imbalance causes the loss of cytochrome P-450 in liver cell culture: formulation of culture media which maintain cytochrome P-450 at in vivo concentrations.

Age-related changes in liver drug metabolism: structure vs function.

AbstractSeveral definitive statements concerning the effect(s) of animal age on liver drug metabolism are made, and at the same time certain crucial questions that remain to be resolved are posed. Between 16 and 30 months of age, or maturity and senescence, the male Fischer rat exhibits a significant loss of hepatic smooth-surfaced endoplasmic reticulum (SER) which constitutes a real quantitative change. However, this does not exclude the possibility of qualitative age-related changes in this membrane, such as compositional alterations which may be reflected in its functional integrity. Second, there is a significant decline in the noninduced activities or amounts of enzymes and hemoproteins, respectively, of the microsomal mixed function oxidase system. With the exception of the loss of cytochrome P-450, which represents a quantitative change, the age-dependent alterations in the two enzymes may reflect quantitative and/or qualitative changes. Last, the reduced hepatic responsiveness to phenobarbital is ...

Cytochrome P-450 lowering effect of alkyl halides, correlation with decrease in arachidonic acid

Treatment of male rats with carbon tetrachloride, bromotrichloromethane, chloroform, 1,2-dibromoethane, 1-bromo-2-chloroethane, and 1,2-dibromo-3-chloropropane results in a decrease in cytochrome P-450 content and alterations in the relative content of fatty acids in hepatic microsomes. A high correlation was found between the loss of cytochrome P-450, the decrease in arachidonic acid (r=0.93), and the increases in linoleic (r=−0.91) and oleic acids (r=−0.89).

Ligands maintain cytochrome P-450 in liver cell culture by affecting its synthesis and degradation

Rat hepatocytes cultured for 24 h lose 68% of their cytochrome P-450. It is shown that this loss is due to the failure of cultured hepatocytes to synthesize cytochrome P-450 as well as enhanced degradation. Compounds that form ligands with cytochrome P-450, eg metyrapone, prevent the loss of cytochrome P-450. Ligands are generally considered to protect proteins from degradation but the present work suggests that the effect of metyrapone on cytochrome P-450 synthesis is of equal importance to its effect on degradation in preventing the loss of cytochrome P-450 in hepatocyte culture.

Evidence that ligand formation is a mechanism underlying the maintenance of cytochrome P-450 in rat liver cell culture. Potent maintenance by metyrapone.

The loss of cytochrome P-450 in cultured rat hepatocytes can be prevented by substituted pyridines, especially isonicotinamide, 3-hydroxypyridine and metyrapone. The effect of these compounds is independent of protein synthesis, suggesting that they maintain pre-existing cytochrome P-450. The efficiency of pyridines at maintaining cytochrome P-450 in hepatocyte culture is highly correlated with their ability to bind to this cytochrome, suggesting that ligand formation with cytochrome P-450 prevents its accelerated turnover in liver cell culture.

The effects of carbon disulphide on rat liver microsomal mixed-function oxidases, in vivo and in vitro.

An intraperitoneal dose of CS(2) (500mg/kg) to male rats resulted in loss of liver microsomal mixed-function-oxidase activity (85% loss of biphenyl 4-hydroxylase), followed by denaturation of liver cytochrome P-450 to cytochrome P-420, and degradative loss of both cytochromes (50% loss). Losses of NADPH-cytochrome c reductase (20%) and cytochrome b(5) were considerably less. Intraperitoneal administration of CS(2) (100mg/kg) to rats pretreated wtih phenobarbitone or 3-methylcholanthrene resulted in similar losses, but the rate of destruction was greater with cytochrome P-450 than with cytochrome P-448. At 12h after intraperitoneal injection of CS(2) to non-pretreated rats, a new cytochrome (P-448) appeared. Rat liver microsomal preparations incubated with CS(2) in the presence of NADPH and O(2) resulted in loss of cytochrome P-450 and mixed-function-oxidase activity directly related to the concentration of CS(2) (10-100mum) and to the period of incubation. Addition of EDTA (1mm) completely inhibited this destruction of cytochrome P-450 by CS(2)in vitro. Addition of CS(2) to liver microsomal preparations resulted in moderate increases in the K(s) values for type-I or type-II substrates, but these were insufficient to account for the inhibition of the mixed-function oxidases. We therefore suggest that desulphuration of CS(2) leads to binding of the S to cytochrome P-450, denaturation of cytochrome P-450 to cytochrome P-420, and ultimately to destruction of these cytochromes by autoxidation.

The effect of an equimolar mixture of carbon tetrachloride and carbon disulphide on the liver of the rat

The inclusion of an equimolar mixture of CS 2 in an oral dose of about 4 ld50's of CCl 4 (5 mmoles kg ) to phenobarbitone pretreated, fasted 100 g male rats reduced the amount of liver injury due to the CCl 4 and prevented deaths occurring. At 24 hr after dosing with the CS 2 + CCl 4 mixture, the morphological changes and chemical composition of the liver more closely resembled those due to CS 2 alone rather than those due to CCl 4. Subsequently the liver lesions due to the mixture became those of a typical but non fatal CCl 4 toxicity. Elevation of conjugated dienes in the hepatic microsomal lipids occurred in the CS 2 + CCl 4 group but not in the CS 2 group, and were less than in the rats given CCl 4 alone. The inclusion of CS 2 with 14CCl 4 dosed orally to rats caused an initial increase followed by a reduction in the uptake of 14CCl 4 into the liver within 15 min of dosing, reduction in the amount of bound radioactivity in the liver from 50 per cent in the 14CCl 4 controls to 30 per cent and a decrease in the respiratory excretion over 24 hr of 14CO 2 derived from the 14CCl 4 of about 65 per cent, findings which indicate reduced metabolism in the animal of the CCl 4. In addition, the CS 2 + CCl 4 mixture caused a 50 per cent loss of cytochrome P450 from liver microsomes within 5–10 min of dosing, while CS 2 and CCl 4 alone, respectively, caused no change during this period. Since the toxic effect of CCl 4 on the liver is directly related to its metabolism in the organ, the present findings suggest that the protection caused by the inclusion of CS 2 in the mixture is due to reduction of hepatic microsomal oxidative metabolism of the CCl 4 through an enhanced loss of cytochrome P450. A theoretical basis is thus proposed for the beneficial effects on the liver of sheep of using a CS 2 + CCl 4 mixture rather than CCl 4 alone as a drug treatment in the field.

Metabolism of α-naphthylthiourea by rat liver and rat lung microsomes

α-Naphthylthiourea (ANTU) is metabolized by rat liver and lung microsomes to α-naphthylurea (ANU) and atomic sulfur. A portion of the atomic sulfur formed in this reaction covalently binds to macromolecules of the liver and lung microsomes. Approximately half the atomic sulfur bound to the liver and lung microsomes appears to have reacted with cysteine side chains of the microsomal proteins to form a hydrodisulfide. The loss of cytochrome P-450 and monooxygenase activity seen on incubation of liver microsomes with ANTU is likely the result of the covalent binding of atomic sulfur to cytochrome P-450. The available evidence suggests that the pulmonary toxicity of ANTU results, at least in part, from the covalent binding of a cytochrome P-450 monooxygenase catalyzed metabolite of ANTU to pulmonary macromolecules. This metabolite is most likely atomic sulfur or alternatively, one containing the carbonyl carbon of ANTU. However, it is possible that the binding of both metabolites may be responsible for the lung toxicity.

Inactivation of purified rat liver cytochrome P-450 during the metabolism of parathion (diethyl p-nitrophenyl phosphorothionate).

The mechanism of the inactivation of purified rat liver cytochrome P-450 during the metabolism of parathion has been investigated with special reference to the covalent binding to the enzyme of the atomic sulfur (S) released upon oxidation of the compound. In addition to the previously reported loss of cytochrome P-450 detectable as its carbon monoxide complex, a loss of heme detectable as pyridine-hemochromagen, and a loss of enzymatic activity toward benzphetamine and ethoxycoumarin are shown. The loss of heme is 80% of the loss of cytochrome P-450, which in turn is less than the loss of benzphetamine demethylase activity, the turnover number of the modified enzyme being two-thirds that of the native based on the amount of cytochrome P-450 detectable as its carbon monoxide complex. Treatment with performic acid or dithiothreitol removes 75% of the 35S covalently bound to the cytochrome P-450 during parathion metabolism. However, the dithiothreitol treatment does not restore cytochrome P-450 detectable as its carbon monoxide complex or enzymatic activity. Likewise, no protection against the loss of heme, cytochrome P-450, or enzymatic activity is afforded by inclusion of 1 mM dithiothreitol during the incubation with parathion, although the amount of irreversibly bound sulfur, i.e. sulfur not dissociable by dithiothreitol, is significantly reduced. In the absence of dithiothreitol, 4 nmol of 35S become bound to the cytochrome P-450 for each nanomole of P-450 no longer detectable as its carbon monoxide complex. Evidence is presented for the covalent binding of sulfur to at least three other amino acids than cysteine. It is suggested that structural changes resulting from the covalent binding of atomic sulfur to cysteine residues are responsible for at least some of the loss of enzymatic activity observed.

Chemical reactivity and metabolism of norethindrone-4β,5β-epoxide by rat liver microsomes in vitro

A method has been developed to separate norethindrone and norethindrone-4β,5β-epoxide by high performance liquid chromatography using isocratic solvent systems with either ODS-reverse phase or conventional silica gel columns. Using these techniques it was found that norethindrone epoxide, prepared chemically, was stable in aqueous buffer (pH 7.4) at 37°C for at least 1 h. Under similar conditions, in the presence of a 10-fold molar excess of cysteine or glutathione, the half life for norethindrone epoxide was 15 and 32 min respectively. In 0.01 M perchloric acid at 37°C the t 1 2 of norethindrone epoxide was 17 min. Norethindrone epoxide was rapidly degraded by rat liver microsomal epoxide hydratase to give a metabolite having properties consistant with it being norethindrone-4,5-dihydrodiol. Epoxide hydratase activities were stimulated about three fold by pretreating rats with phenobarbitone. The pH optimum for this reaction was pH 7.4. Conversion of norethindrone epoxide to norethindrone-dihydrodiol was inhibited by the epoxide hydratase inhibitor 1,2-epoxytrichloropropane. Although norethindrone was extensively metabolised in the presence of NADPH and rat liver microsomes, no conversion to norethindrone-4β,5β-epoxide could be demonstrated, either in the presence or absence of epoxytrichloropropane in the reaction mixture. If norethindrone epoxide was produced under these conditions it was suggested that it either reacted with microsomal proteins at or close to the site or production or was further metabolised. Norethindrone-4β,5β-epoxide did not cause any loss of cytochrome P-450 when incubated with rat liver microsomes in the absence of NADPH. Only in the presence of NADPH did further metabolism of norethindrone epoxide occur leading to the formation of active metabolites capable of breaking down cytochrome P-450. The initial rate of loss of cytochrome P-450 under these conditions was greater with norethindrone than with norethindrone epoxide as the substrate.

Degradation of cytochrome P-450 haem by carbon tetrachloride and 2-allyl-2-isopropylacetamide in rat liver in vivo and in vitro. Involvement of non-carbon monoxide-forming mechanisms.

Degradation of intrinsic hepatic [(14)C]haem was analysed as (14)CO formation in living rats and in hepatic microsomal fractions prepared from these animals 16h after pulse-labelling with 5-amino[5-(14)C]laevulinic acid, a precursor that labels bridge carbons of haem in non-erythroid tissues. NADPH-catalysed peroxidation of microsomal lipids in vitro (measured as malondialdehyde) was accompanied by loss of cytochrome P-450 and microsome-associated [(14)C]haem (largely cytochrome P-450 haem), but little (14)CO formation. No additional (14)CO was formed when carbon tetrachloride and 2-allyl-2-isopropylacetamide were added to stimulate lipid peroxidation and increase loss of cytochrome P-450 [(14)C]haem. Because the latter effect persisted despite inhibition of lipid peroxidation with MnCl(2) or phenyl-t-butylnitrone(a spin-trapping agent for free radicals), it was concluded that carbon tetrachloride, as reported for 2-allyl-2-isopropylacetamide, may promote loss of cytochrome P-450 haem through a non-CO-forming mechanism independent of lipid peroxidation. By comparison with breakdown of intrinsic haem, catabolism of [(14)C]methaemalbumin by microsomal haem oxygenase in vitro produced equimolar quantities of (14)CO and bilirubin, although these catabolites reflected only 18% of the degraded [(14)C]haem. This value was increased to 100% by addition of MnCl(2), which suggests that lipid peroxidation may be involved in degradation of exogenous haem to products other than CO. Phenyl-t-butylnitrone completely blocked haem oxygenase activity, which suggests that hydroxy free radicals may represent a species of active oxygen used by this enzyme system. After administration of carbon tetrachloride or 2-allyl-2-isopropylacetamide to labelled rats, hepatic [(14)C]haem was decreased and haem oxygenase activity was unchanged; however, (14)CO excretion was either unchanged (carbon tetrachloride) or decreased (2-allyl-2-isopropylacetamide). These changes were unaffected by cycloheximide pretreatment. From the lack of parallel losses of cytochrome P-450 [(14)C]haem and (14)CO excretion, one may infer that an important fraction of hepatic [(14)C]haem in normal rats is degraded by endogenous pathways not involving CO. We conclude that carbon tetrachloride and 2-allyl-2-isopropylacetamide accelerate catabolism of cytochrome P-450 haem through mechanisms that do not yield CO as an end product, and that are insensitive to cycloheximide and independent of haem oxygenase activity.