Effect of diabetes on rat liver cytochrome P-450: Evidence for a unique diabetes-dependent rat liver cytochrome P-450

Detergent-solubilized hepatic microsomal fractions from alloxan diabetic rats exhibited a 52,000 molecular weight hemeprotein band that was not present in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles of identically solubilized hepatic microsomal fractions from normal, 3-methylcholanthrene- or phenobarbital-treated rats. This 52,000 mol. wt hemeprotein band disappeared from the protein profile of insulin-treated diabetic rat liver to yield the SDS-PAGE profile of normal rat liver. When P-450 hemeproteins were purified by lauric acid affinity and hydroxylapatite chromatography from solubilized microsomes, only the diabetic rat had a 52,000 mol. wt P-450. This distinct 52,000 mol. wt diabetes-induced P-450 interacted with type II compounds to yield a 2-fold greater absorbance change than was observed with the purified P-450s from either the normal or the chemically induced rats. The properties of this unique 52,000 mol. wt P-450 suggest that it may be the catalytic component responsible for the increased rate of type II substrate (aniline) metabolism observed in the diabetic rat.     

Polyamines as modulators of drug oxidation reactions catalyzed by cytochrome P-450 from liver microsomes

The effect of polyamines on the activity of the mixed-function oxidase (MFO) system from human, rat and rabbit liver microsomes was investigated in detail. It was shown that polyamine (spermine) stimulates NADPH-dependent activity of the MFO system several-fold whatever the substrate (foreign drug or natural), not only with microsomes but also with the reconstituted system consisting of highly purified cytochrome P-450 (LM₂ isozyme), cytochrome P-450 NADPH reductase and dilauroylphosphorylcholine. Stimulation (extent and concentration dependence) appeared to be dependent on a number of parameters such as ionic strength, pH, animal species and treatment, nature of the substrate, and was stereospecific (different effect on 6β-and 16α-testosterone hydroxylation). Further, the spermine effect was evaluated on some elementary steps of the cytochrome P-450 reaction cycle, like substrate binding, P-450 reduction and second electron transfer. Finally, it was shown that the organic peroxide dependent activity was not stimulated by spermine with microsomes nor with the purified P-450 LM₂ isozyme.On the basis of this study, it was concluded that the locus of polyamine action is cytochrome P-450 and that stimulation could result either from increased stability of the oxyferrous intermediate of P-450 or from an increased rate of second electron transfer from reductase to P-450.     

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 in inhibited in the presence of SKF-525A, 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.     

Cellular distribution of cytochrome P-450 loss in rats of different ages treated with alkyl halides

Challenge of male rats with a single dose of the alkyl halides, 1,2-dibromo-3-chloropropane (DBCP), ethylene dibromide (EDB), carbon tetrachloride (CCl₄), and epichlorohydrin (EPI), resulted in significant decreases in cytochrome P-450 in microsomes isolated from liver, kidney, testis, lung, and small intestinal mucosa 48 hr after treatment. Treatment with CCl₄, but not DBCP, EDB, or EPI, was characterized by rapid loss of cytochrome P-450, detectable within 4 hr. Evidence of lipid peroxidation was found only in hepatic microsomes from rats treated with CCl₄, but not in hepatic or extrahepatic microsomes from rats treated with other compounds. In liver tissue, treatment with DBCP and CCl₄ resulted in a decrease in cytochrome P-450 in both rough and smooth microsomal fractions and nuclei, but not in mitochondrial fractions. Mixed-function oxidase (MFO) activities in hepatic microsomes decreased in parallel with cytochrome P-450 content after treatment with DBCP and EDB. In microsomes and nuclei after treatment with CCl₄ and in nuclei after treatment with DBCP, however, the response of the MFO depended on the substrate tested. Microsomal cytochrome P-450, which is susceptible to proteolytic cleavage, and microsomal and nuclear cytochrome P-450, which increased with maturation and decreased with aging of the rat, appeared to be the most responsive of the forms of cytochrome P-450 to alkyl halide treatment. These results suggest that treatment with alkyl halides may preferentially affect specific isozymes of cytochrome P-450.     

The substituted pyridines metyrapone and nicotinamide are inducers of 5-aminolevulinate synthase and cytochrome P-450 in hepatocyte culture

The effects of metyrapone and nicotinamide, two substituted pyridines, were studied in cultured chick embryo hepatocytes, a system characterized by preserved inducibility of cytochrome P-450 hemoproteins. Both metyrapone and nicotinamide caused a dose-dependent increase in cytochrome P-450 concentration. Their inducing potencies differed by one to two orders of magnitude and correlated with the known difference in the binding affinity of these two pyridines to cytochrome P-450. The increase of cytochrome P-450 concentration after metyrapone and nicotinamide was additive to the induction of cytochrome P-450 by phenobarbital and beta-naphthoflavone and was abolished by cycloheximide. Treatment of hepatocyte cultures with metyrapone resulted in an increase in a microsomal protein with an apparent mol. wt of 52,000. In addition, induction of cytochrome P-450 by the substituted pyridines was associated with enhanced 5-aminolevulinate synthase, the rate-limiting enzyme of heme biosynthesis. These data suggest that in cultured chick embryo hepatocytes the substituted pyridines metyrapone and nicotinamide induce cytochrome P-450.     

The role of cytochrome P-450-inducing agents in potentiaing the toxicity of fluroxene (2,2,2-trifluoroethyl vinyl ether)

The effects of the hepatic microsomal cytochrome P-450-inducing agents 3-methylcholanthrene (MC), benzo[α]pyrene (BP), β-naphthoflavone (BNF), mirex, 8-hydromirex, Kepone, spironolactone, pregnenolone-16α-carbonitrile (PCN), barbital, phenobarbital (PB), mephobarbital, hexobarbital, and pentobarbital in potentiating the toxicity of the anesthetic fluroxene (2,2,2-trifluoroethyl vinyl ether) in male rats have been investigated. The toxicity was expressed as death, hepatic necrosis, or destruction of hepatic cytochrome P-450. Only PCN, mephobarbital, and phenobarbital induction caused death within 48 hr of the administration of fluroxene (2.5 g/kg, ip), and this was prevented by administration of cytochrome P-450 inhibitors. With PB induction the percentage of mortality correlated with the induced cytochrome P-450 concentrations. Fluroxene destroyed microsomal cytochrome P-450 in vivo, within 90 min of administration, and this destruction was enhanced over twofold by pretreatment of the rats with MC, BP, and all the barbiturates, and less significantly with BNF, Kepone, and PCN. Only the livers of mirex-, 8-hydromirex-, or MC-induced rats showed necrosis within 48 hr of administration of fluroxene, with 8-hydromirex-induced livers showing the most widespread necrosis. We conclude that a number of different forms of cytochrome P-450 can metabolize fluroxene to toxic products. Some forms of cytochrome P-450, particularly cytochrome P-448, are highly susceptible to destruction by fluroxene metabolites which probably arise from metabolism of the fluroxene vinyl group. Fluroxene-induced liver necrosis is not related to the destruction of cytochrome P-450, nor to the death of the experimental animals. Thus, the three aspects of fluroxene toxicity—mortality, cytochrome P-450 destruction and hepatic necrosis—are differentially affected.     

Metabolic oxidation of acetaminophen (Paracetamol) mediated by cytochrome P-450 mixed-function oxidase and prostaglandin endoperoxide synthetase in rabbit kidney

Long-term abuse of acetaminophen, in combination with other antipyretic analgesics, is thought to be responsible for papillary necrosis in analgesic nephropathy. Cytochrome P-450-mediated oxidative metabolism requiring NADPH and O₂ has been believed, to date, to be the sole pathway for the toxic metabolic activation of acetaminophen. Using microsomes from different regions of rabbit kidney, protein covalent binding of acetaminophen by NADPH-dependent metabolism was highest in cortex, less in outer medulla, and minimal in inner medulla. In vivo studies have shown that the highest binding of acetaminophen occurred in renal inner medulla compared to liver and renal cortex. However, cytochrome P-450 could not be detected in rabbit renal inner medulla. Hence, another metabolic pathway for the activation of acetaminophen was presumed to be operative in renal inner medulla. This alternate pathway was recognized as cooxidation of acetaminophen mediated by prostaglandin endoperoxide synthetase, requiring arachidonic acid as well as O₂, and was found to be active predominantly in renal inner medulla. Glutathione, ascorbic acid, and ethoxyquin inhibited protein covalent binding of acetaminophen arising from both pathways. Indomethacin and aspirin inhibited only the arachidonic acid-dependent cooxidation of acetaminophen. Butylated hydroxyanisole inhibited both NADPH- and arachidonic acid-dependent metabolism, the latter more effectively. Arachidonic acid-dependent metabolism of acetaminophen is probably mediated by the hydroperoxidase activity of prostaglandin endoperoxide synthetase. This alternate pathway could be a significant contributing factor for the genesis of papillary necrosis, as manifested in analgesic nephropathy.     

Identification and partial purification of hamster microsomal cytochrome P-450 isoenzymes

We have identified and partially purified three forms of cytochrome P-450 from hamster liver microsomes. Phenobarbital (PB) treatment induced three major polypeptides with relative mobilities (M_r) of 47,000, 50,000 and 51,500. The 47,000 polypeptide was assigned as epoxide hydrolase, since it was also enhanced by trans-stilbene oxide (TSO) treatment. Two polypeptides (M_r = 48,500 and 53,500) were induced by both 3-methylcholanthrene (3-MC) and β-naphthoflavone (BNF) treatments. Treatment with Aroclor 1254 induced three polypeptides (M_r = 48,500, 50,000 and 53,500), indicating the induction of both drug- and carcinogen-inducible cytochrome P-450s. Liver microsomal benzo[a]pyrene hydroxylase activity was not affected significantly by any of these inducers. In contrast, it was induced 2- to 3-fold in lung microsomes by 3-MC, BNF or Aroclor 1254 treatment. Benzphetamine N-demethylase and 7-ethoxycoumarin O-deethylase activities, expressed as nmoles of product formed per min per mg of liver microsomal protein, were increased 3- to 4-fold by either PB or Aroclor treatment. The activity of 7-ethoxycoumarin O-deethylase was the only one enhanced significantly by 3-methylcholanthrene or β-naphthoflavone treatment in liver microsomes. Pregnenolone-16-α-carbonitrile (PCN) and TSO did not alter any of these activities. The major polypeptides induced by PB (M_r = 50,000) and 3-MC (M_r = 48,500 and 53,500 respectively) were partially purified, to a specific content of 6–10 nmoles P-450/mg of protein and were active in catalyzing N-demethylation of benzphetamine, hydroxylation of benzo[a]pyrene, and O-deethylation of 7-ethoxycoumarin with different substrate specificity. None of these isoenzymes immuno-cross-reacted with antibodies prepared against rabbit cytochrome P-450_LM2 or P-450_LM4.     

Formaldehyde production promoted by rat nasal cytochrome P-450-dependent monooxygenases with nasal decongestants, essences, solvents, air pollutants, nicotine, and cocaine as substrates

To identify compounds which might be metabolized to formaldehyde in the nasal cavity, 32 potential substrates for cytochrome P-450-dependent monooxygenases were screened with rat nasal and, for comparison, liver microsomes. Tested substrates included 6 nasal decongestants, cocaine, nicotine, 9 essences, 3 potential air pollutants, and 12 solvents. Each test substrate, with the possible exception of the air pollutants, contained one or more N-methyl, O-methyl, or S-methyl groups. Eighteen of the tested materials were metabolized to produce formaldehyde by nasal microsomes. Five substrates, namely, the solvents HMPA and dimethylaniline, cocaine, and the essences dimethyl anthranilate and p-methoxyacetophenone, were metabolized to produce formaldehyde at rates exceeding 1000 pmol/mg microsomal protein/min by nasal microsomes. Eight substrates, including four nasal decongestants, nicotine, and an extract of diesel exhaust particles, were metabolized to produce formaldehyde at rates of 200 to 1000 pmol/mg microsomal protein/min. Five other substrates were metabolized to formaldehyde at detectable rates. The results indicate that a variety of materials which often come in contact with the nasal mucosa can be metabolized to formaldehyde by nasal enzymes. The released formaldehyde may influence the irritancy of inhaled compounds and has been suggested to play a role in the tumorigenicity of some compounds.     

Sex differences in cytochrome P-450 and mixed-function oxygenase activity in gonadally mature trout

Levels of microsomal cytochrome P-450 and aminopyrine demethylase activity in liver and of cytochrome P-450 in kidney of gonadally mature rainbow and brook trout were markedly greater in males than in females. Similar differences appeared in hepatic microsomal NADH- but not in NADPH-cytochrome c reductase activity or cytochrome b₅ content. When normalized to cytochrome P-450 content, benzo[a]pyrene hydroxylase activity in both liver and kidney was greater in females. In liver, there was a pronounced sex difference in the response of this activity to 7,8-benzoflavone, suggesting cytochromes P-450 of different catalytic function. Electron paramagnetic resonance spectra of hepatic microsomal cytochromes P-450 in mature brook trout were not demonstrably different between males and females, and crystal field parameters indicate that axial ligands to the neme are the same in these as in other cytochromes P-450. Mixed-function oxygenase activities in liver of gonadally immature brook trout differed from those in mature fish, and there was no sex difference. The appearance of seasonally dependent sex differences suggests that fish may provide interesting models for studying regulation of sex-specific forms of cytochromes P-450.     

Postnatal development of constitutive forms of cytochrome P-450 in liver microsomes of male and female rats

In a previous paper (1), we reported on the purification of constitutive forms of cytochrome P-450, namely P-450-male and P-450-female, from liver microsomes of male and female rats, respectively. Immunochemical examinations of these hemoproteins showed that P-450-male and P-450-female were detectable specifically in respective liver microsomes of adult male and female rats. The synthesis of P-450-male was apparently dependent on testosterone, and that of P-450-female was dependent on estradiol.Thus, in this study we examined the postnatal development of P-450-male and P-450-female. We show herein that P-450-female is primarily synthesized before the occurrence of P-450-male in male rats. This and other results support the view that the synthesis of unknown pre-existing forms of cytochrome P-450 is depressed in association with the appearance of P-450-male and P-450-female during postnatal periods before sexual maturation.     

Isopropanol enhancement of cytochrome P-450-dependent monooxygenase activities and its effects on carbon tetrachloride intoxication

Acute or chronic treatment of rats with isopropanol caused a significant increase in hepatic cytochrome P-450 content and a two- to threefold increase in aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities, but no significant change in ethylmorphine N-demethylase or benzo(a)pyrene hydroxylase activity. In rats treated with isopropanol and challenged with CCl4, liver toxicity of CCl4 was characteristically potentiated, as assessed by elevation of serum glutamic-pyruvic transaminase (SGPT) levels. Isopropanol pretreatment also potentiated CCl4-induced damage to the hepatic monooxygenase system. In addition to a decrease in cytochrome P-450, rats treated with isopropanol and challenged with CCl4 showed a nonspecific decrease not only in aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities, but also in ethylmorphine N-demethylase, benzo(a)pyrene hydroxylase, and NADPH-cytochrome c reductase activities. These results were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized microsomes. The electrophoretic results showed that isopropanol pretreatment markedly potentiated the CCl4-caused destruction of cytochrome P-450 hemeproteins. The data strongly suggest that isopropanol increases one or more forms of cytochrome P-450 which selectively enhance the metabolism of CCl4 to an active metabolite. This active metabolite then causes a nonselective damage to the microsomal mixed-function oxidase system.     

Induction of cytochrome P-450 by methylenedioxyphenyl compounds: Importance of the methylene carbon

Induction of hepatic microsomal cytochrome P-450 in Dub:ICR male mice treated with phenobarbital, 3-methylcholanthrene, safrole, isosafrole, 5-tert.-butyl-1,3-benzodioxole (BBD), 2-methyl-5-tert.-butyl-1,3-benzodioxole (MBBD), and 2,2-dimethyl-5-tert.-butyl-1,3-benzodiozole (DBBD) was evaluated by measuring the cytochrome P-450 content, Type II:Type 1 binding ratio, ethylisocyanide pH equilibrium point, biphenyl 2- and 4-hydroxylase, ethylmorphine N-demethylase, ethoxyresorufin O-deethylase, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Safrole and isosafrole treatment of mice produced a phenobarbitaltype induction. BBD, but not MBBD and DBBD, induced cytochrome P-450 and formed a Type III metabolite-cytochrome P-450 complex, in vitro and in vivo. SDS-PAGE revealed that DBBD does induce proteins other than cytochrome P-450. These data suggest that the methylene carbon plays an important role in cytochrome P-450 induction.     

The 1,2-dichloroethylenes: Their metabolism by hepatic cytochrome P-450 in vitro

Cis- and trans-1,1-dichloroethylene bound to the active site of hepatic microsomal cytochrome P-450 with the production of a Type I difference spectrum and stimulated CO-inhibitable hepatic microsomal NADPH oxidation. Incubation of cis- and trans-1,2-dichloroethylene plus hepatic microsomes, NADPH-generating system-EDTA resulted in the production of measurable levels of 2,2-dichloroethanol and dichloroacetaldehyde but not of 2-chloroethanol, chloroacetaldehyde or chloroacetic acid and, also, resulted in decreased levels of hepatic microsomal cytochrome P-450 and heme. In addition, dichloroacetic acid was produced from trans-dichloroethylene under these experimental conditions. The omission of any component of the incubation mixture eliminated the above effects, while the inclusion of SKF-525A, metyrapone or CO: O₂ (80, v/v) diminished these effects. The effects of β-naphthoflavone and phenobarbital pretreatment on the values of K_s, ΔA_max, K_m and V_mam for the binding and metabolism of the 1,2-dichloroethylenes are reported. The binding and metabolism of the 1,2-dichloroethylenes and the 1,2-dichloroethylene-mediated inactivation of cytochrome P-450 were enhanced per mg of microsomal protein, but generally not per nmole of cytochrome P-450 by prior induction with β-naphthoflavone or phenobarbital. It is concluded that multiple forms of hepatic microsomal cytochrome P-450 bind and metabolize the 1,2-dichloroethylenes. The role of cytochrome P-450 in the metabolic activation of the dichloroethylenes is considered.     

Cumene hydroperoxide-supported microsomal hydroxylations of warfarin—A probe of cytochrome P-450 multiplicity and specificity

The hepatic microsomal metabolism of R and S warfarin, supported by NADPH or cumene hydroperoxide, has been investigated to probe the multiplicity and specificity of cytochromes P-450. Microsomes were uninduccd, and phenobarbital (PB)-, 3-methylcholanthrene (MC)- or 3β-hydroxy-20-oxopregn-5-ene-16-α-carbonitrile (PCN)-induced from rat liver. Cumene hydroperoxide supported the formation of all the NADPH-supported warfarin metabolites (4′-, 6-, 7- and benzylic hydroxywarfarin and dehydrowarfarin). except 8-hvdroxywarfarin. Comparisons of the rates of formation of the metabolites supported by NADPH or cumene hydroperoxide (with uninduced and induced microsomes) revealed that cumene hydroperoxide had the following effects: (1) rates of hydroxylation of the phenyl substituent of warfarin (4′-hydroxywarfarin) were increased; (2) rates of metabolism of the aliphatic portion of warfarin (benzylic hydroxywarfarin and dehydrowarfarin) were increased, except with S warfarin and uninduced microsomes; and (3) rates of hydroxylation of the phenyl ring of the coumarin group of warfarin were (a) decreased (7-. 8-hydroxywarfarin) or (b) decreased (6-hydroxywarfarin) with MC-induced microsomes and increased or unchanged with uninduced and PB- or PC'N-induced microsomes. We concluded from these studies that multiple cytochromes P-450 are implicated in the metabolism of warfarin: that the cytochromes P-450 catalyzing the formation of 7- and 8-hydroxywarfarin differ from those catalyzing the other metabolites. except foro-hydroxylation by MC-induced microsomes: that the cytochromes catalyzing 7- and 8- hydroxywarfarin formation differ from one another; that for each metabolite of warfarin, the cytochrome P-450 type predominantly responsible for its formation is the same. irrespective of the mode of induction of the microsomes: and that 6-hydroxylase activity is the exception to the previous point, and is predominantly associated with different cytochromes P-450 in differently induced microsomes. The effects of cumene hydroperoxide have been ascribed to differences in cumene hydroperoxide affinities, differences in cumene hydroperoxide-induced destruction, and differences in cumene hydroperoxidc inhibitions of warfarin binding to different cytochromes P-450. together with differences in the situation of cytochromes P-450 in the microsomal membrane.