Selective inactivation of rat lung and liver microsomal NADPH-cytochrome c reductase by acrolein

Addition of acrolein to rat lung or liver microsomal suspensions resulted in total inactivation of NADPH-cytochrome c reductase and partial conversion of cytochrome P-450 to P-420 in a concentration- and time-dependent fashion. Acrolein also caused total loss of nonprotein sulfhydryl content in both preparations, whereas protein sulfhydryl content was decreased by 40% and 28% in lung and liver preparations, respectively. Maxima of about 60% of the total lung cytochrome P-450 and 50% of the liver cytochrome P-450 in acrolein-treated microsomes did not support the N-demethylation of benzphetamine or ethylmorphine or hydroxylation of aniline because of the total loss of NADPH-cytochrome c reductase. Addition of purified NADPH-cytochrome c reductase to the acrolein-treated lung or liver microsomal suspension largely restored these monooxygenase activities. Addition of glutathione or dithiothreitol to the lung or liver microsomal suspension prior to the addition of acrolein significantly protected cytochrome P-450 from conversion to cytochrome P-420 as well as NADPH-cytochrome c reductase from inactivation. Thus, selective conjugation of acrolein with lung and liver NADPH-cytochrome c reductase but not cytochrome P-450 was responsible for total loss of these lung and liver monooxygenase activities.     

Induction of xenobiotic-metabolizing enzymes and peroxisome proliferation in rat liver caused by dietary exposure to di(2-ethylhexyl)phosphate

Exposure of rats to 1% or 3% (w/w) di(2-ethylhexyl)phosphate in the diet for five days results in two- to three-fold inductions of liver cytosolic epoxide hydrolase activity and microsomal cytochrome P-450 content. Cytochromes P-450b + e were induced 20- to 35-fold, but no increase was observed in cytochrome P-450c. Considerably smaller effects were obtained on NADPH-cytochrome c reductase, microsomal epoxide hydrolase and microsomal cytochrome b5 content, and there was no effect on cytosolic glutathione transferase activity, under the same conditions. A dramatic increase in cyanide-insensitive palmitoyl-CoA oxidation and total mitochondrial protein, together with smaller increases in total catalase and cytochrome oxidase activities, were observed after treatment with di(2-ethylhexyl)phosphate, indicating that this compound causes proliferation of both peroxisomes and mitochondria. It is suggested that the induction of cytosolic epoxide hydrolase and the proliferation of peroxisomes may be related processes.     

Effects of motorcycle exhaust inhalation exposure on cytochrome P-450 2B1, antioxidant enzymes, and lipid peroxidation in rat liver and lung

The effects of motorcycle exhaust (ME) on metabolic and antioxidant enzymes and lipid peroxidation were determined using male rats exposed to 1:10 diluted ME by inhalation 2 h daily for 4 wk. For microsomal cytochrome P-450 enzymes, ME resulted in threefold increases of 7-ethoxyresorufin and pentoxyresorufin O-deethylase activities in liver and a sixfold increase of 7-ethoxyresorufin O-deethylase activity and an 80% decrease of pentoxyresorufin O-dealkylase activity in lung. The results of immunoblot analysis of microsomal proteins revealed that ME increased liver and lung cytochrome P-450 1A1 with minimal effects on cytochrome P-450 2E1. ME increased cytochrome P-450 2B1/2 proteins in liver but decreased cytochrome P-450 2B1 in lung. ME did not change microsomal cytochrome P-450 enzyme activity or protein level in kidney. For phase II enzymes, ME resulted in 53% and twofold increases of cytosolic NAD(P)H:quinone oxidoreductase activities in liver and lung, respectively, and no effect on microsomal UDP-glucuronosyltransferase activities. For antioxidant enzymes, ME produced 23% and 35% decreases of superoxide dismutase, 9% and 27% decreases of catalase, and no changes of glutathione peroxidase activities in liver and lung cytosols, respectively. For lipid peroxidation, the results of thiobarbituric acid assay showed that ME resulted in a twofold increase of formation of malondialdehyde by liver microsomes incubated with FeCl(3) -ADP. ME produced a threefold increase of malondialdehyde formation by lung microsomes. The present study demonstrates that ME inhalation exposure differentially modulates cytochrome P-450 2B1 and antioxidant enzymes and increases susceptibility to lipid peroxidation in rat liver and lung.     

Sex difference in responsiveness to Aztreonam of monooxygenase system in liver microsomes from rats

Effect of successive administration of Aztreonam on microsomal monooxygenase system was investigated in male and female Sprague-Dawley rats. The activities of benzphetamine N-demethylase, aminopyrine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes from male rats were decreased dose-dependently by Aztreonam. On the contrary, the activities in liver microsomes from female rats were slightly increased rather than decreased by the administration of Aztreonam. In addition, Aztreonam was found to decrease the specific content of microsomal cytochrome P-450 in male rats but not in female rats. The decreases in the activities observed in male rats were accompanied by a parallel decrease in the specific content of cytochrome P-450. Furthermore, the results of quantitation of P-450 (M-1), one of the male specific forms of cytochrome P-450, indicated that the administration of Aztreonam resulted in a dose-dependent decrease in the content of P-450 (M-1) in liver microsomes from male rats.     

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 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 p-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.     

The effect of age on inducibility of various types of rat liver cytochrome P-450.

1. The content and specific activities of inducible cytochrome P-450 enzymes were determined in liver microsomes of rats of various ages after maximal induction with phenobarbital, isosafrole of 3-methylcholanthrene, and in untreated animals. 2. With age an increase in liver weight was observed both in untreated rats and in maximally induced ones; the microsomal protein content/g of liver decreased with age in untreated animals but not in induced ones. Total cytochrome P-450 content/mg microsomal protein remained unchanged with age in all experimental groups. 3. Immunologically detectable levels of cytochrome P4501A1/1A2 and 2B1/2B2 remain unchanged with age both in untreated animals and in maximally induced ones. 4. Several cytochrome P-450 activities showed an age-related decrease in untreated animals, but no change with age was observed in the activities of cytochrome P4501A1, 2A2 and 2B1/2B2 in rat liver microsomes. This indicates that ageing affects only the activity of some constitutive forms of cytochrome P-450 in male rats, but not the activity of inducible types of P-450. 5. Although previous results indicated decreased inducibility of the cytochrome P-450 mRNA levels with age, the present study clearly demonstrates that this is not reflected in decreased enzyme levels or activities after maximal induction. From this it is concluded that the decreased mRNA levels might rather be reflected in a decreased rate at which maximal induction can be achieved.     

Comparison of monooxygenase activities and cytochrome P-450 isozyme concentrations in human liver microsomes

Concentrations of three human liver microsomal cytochrome P-450 isozymes and 20 different monooxygenase activities were determined in human liver microsomal preparations. The results of correlation analysis suggest that: there are important variations in the amounts of the three cytochrome P-450 isozymes measured, particularly P-450(8) and P-450(9); aldrin epoxidase, d-benzphetamine N-demethylase, and S-warfarin 4-hydroxylase activities are linked to cytochrome P-450(5); aryl hydrocarbon (benzo(a)pyrene) hydroxylase and 4-nitroanisole-O-demethylase activities are linked to P-450(8); hydroxylations at the 4'-, 6-, 7-, and 8-positions of R-warfarin are closely linked to each other but are not correlated with other measured monooxygenase activities or P-450 isozyme levels; and P-450(9) is not related to any of the catalytic activities tested. Thus, certain monooxygenase activities can be attributed to specific cytochrome P-450 isozymes. This approach should be useful in suggesting the roles of different cytochromes P-450 in drug metabolism in man which can be further examined using in vitro and in vivo methods.     

Induction of rat liver microsomal and nuclear cytochrome P-450 by dietary 2-acetylaminofluorene and butylated hydroxytoluene

The influence of dietary 2-acetylaminofluorene (AAF) on the cytochrome P-450 content of rat liver microsomal and nuclear fractions was immunochemically probed with monoclonal and polyclonal antibodies to cytochromes P-450c and P-450d. Cytochrome P-450d but not P-450c was immunodetected in microsomes, nuclear envelopes, and nuclei from untreated rats. The levels of both cytochromes P-450c and P-450d were elevated after a diet of either 0.1% AAF for 1 week or 0.05% AAF for 3 weeks. However, the level of cytochrome P-450c relative to P-450d was lower after the more prolonged AAF feeding. Supplementation of AAF-containing diets with 0.3% butylated hydroxytoluene (BHT), which affords protection against AAF hepatocarcinogenesis in high-fat fed rats, protected and/or induced total (spectral) nuclear envelope cytochrome P-450 content. Immunochemical studies of liver fractions showed that BHT enhanced the AAF-dependent induction of cytochrome P-450c, but not of P-450d. This was a concerted effect of AAF + BHT since dietary BHT by itself did not affect the levels of cytochrome P-450c or P-450d as compared to control rats. Since 1- to 3-week dietary AAF had little effect on total (spectral analyses) microsomal cytochrome P-450 but markedly reduced total P-450 in nuclear envelopes, the coordinated induction of specific cytochrome P-450s in the different fractions suggests selective induction and depression of different forms of cytochrome P-450 and provides additional evidence for independent regulation of the drug-metabolizing system in nuclear envelope and microsomes. In addition, these results suggest that regulation of cytochrome P-450 may play a crucial role in the nutritional modulation of AAF hepatocarcinogenesis.     

Effect of linoleic acid hydroperoxide on liver microsomal enzymes in vitro.

Rat liver microsomes incubated with linoleic acid hydroperoxide (LAHPO) lost cytochrome P-450 specifically among the enzymes of microsomal electron transport systems. The loss of cytochrome P-450 content and glucose-6-phosphatase activity by LAHPO was accompanied by an increase in malondialdehyde (MDA) production. Turbidity of microsomal suspensions was decreased with increasing MDA production, but not proportionately. Diethyldithiocarbamate (DTC), N,N'-diphenyl-p-phenylenediamine and alpha-tocopherol inhibited almost completely the LAHPO-induced MDA production of microsomes, however no perfect protection against the loss of cytochrome P-450 content and glucose-6-phosphatase activity was observed. The decrease of microsomal turbidity by LAHPO was little affected in the presence of DTC. Purified cytochrome P-450 was destroyed by LAHPO, with minimal protection by the compounds described above. These results suggest the possibility that the loss of microsomal enzyme activities during lipid peroxidation may be attributed largely to a direct attack on enzyme proteins by lipid peroxides rather than indirectly to a structural damage of microsomal membranes resulting from peroxidative breakdown of membrane lipids.     

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.     

Comparison of xenobiotic biotransformation enzymes in kidney and liver of rainbow trout (Salmo gairdneri)

The microsomal cytochrome P-450 content in kidney of rainbow trout (Salmo gairdneri) was approximately 5-fold lower than the content in liver. The renal ethoxycoumarin- and ethoxyresorufin-O-deethylase activities calculated on a per-cytochrome P-450 basis were, however, found to be about 10-fold higher than the hepatic activities. The patterns of time-dependent increase and subsequent decrease of microsomal cytochrome P-450-dependent monooxygenase activities after a single injection of beta-naphthoflavone (BNF) were similar in the kidney and liver. The microsomal ethoxyresorufin- and ethoxycoumarin-O-deethylase activities were maximally induced in liver (120- and 10-fold, respectively) by a single BNF injection (50 mg/kg body wt), whereas in kidney the maximal levels of induction (135- and 21-fold, respectively) were reached after three injections with BNF. The induction of cytochrome P-450 systems was associated with synthesis of a new microsomal protein of 58,000 Da in both kidney and liver. UDP-glucuronosyl transferase activity toward p-nitrophenol was about 8-fold lower in kidney than in liver. A significant 2.5-fold elevation in microsomal UDP-glucuronosyltransferase activity was found in the kidney 14 days after a single injection with BNF (50 mg/kg). In the liver, a 2-fold increase of this activity was seen 3 days after the treatment. The results indicate that the rainbow trout kidney in addition to the liver is of great importance in biotransformation of lipophilic xenobiotics.     

Hypophysectomy differentially alters P-450 protein levels and enzyme activities in rat liver: pituitary control of hepatic NADPH cytochrome P-450 reductase

Pituitary-determined hormones regulate the expression of hepatic cytochromes P-450 through processes involving both negative and positive controls. Accordingly, protein levels of several P-450 forms are elevated in rat liver following hypophysectomy [P-450 forms designated 2a (gene IIIA2), RLM2 (gene IIA2), and PB-4 (gene IIB1)], whereas protein levels of others are suppressed [e.g., P-450 2c (gene IIC11)]. In the present study, microsomal steroid hydroxylase activities associated with these same P-450 forms were found to be decreased by hypophysectomy, despite elevations in protein levels for several of them. Studies were, therefore, undertaken to determine the biochemical basis for this decrease in microsomal P-450 enzyme specific activity. In vivo treatment of hypophysectomized rats with gonadotropin, under conditions that restore heme to testis P-450, and heme reconstitution experiments carried out with liver homogenates indicated that a deficiency in P-450-associated heme is unlikely to account for the observed decreases in liver P-450 enzyme specific activity. Analysis of the flavoprotein P-450 reductase, however, revealed that the reductase protein and its associated cytochrome c reductase activity are decreased by 50 to 75% in liver microsomes isolated from hypophysectomized rats. Moreover, supplementation of isolated liver microsomes with exogenous purified P-450 reductase stimulated microsomal steroid hydroxylase activity preferentially in the hypophysectomized rats, to levels consistent with the observed changes in P-450 protein levels. Thus, a deficiency in P-450 reductase, which is a rate-limiting component for many P-450-dependent hydroxylation reactions, appears to be responsible for the decrease in steroid hydroxylase specific activity in the hypophysectomized rats. Although growth hormone, adrenocorticotropic hormone, and chorionic gonadotropin were each ineffective at restoring hepatic P-450 reductase when administered to hypophysectomized rats, substantial restoration of P-450 reductase levels could be achieved by treatment of the hypophysectomized rats with thyroxine. Thyroxine treatment of these rats also elevated the microsomal steroid hydroxylase activities associated with the individual hepatic P-450 forms to levels commensurate with their respective P-450 protein levels. These results establish that hepatic P-450 reductase is subject to hormonal controls that are distinct from those governing cytochrome P-450 expression and further demonstrate the complexity of endocrine control of hepatic steroid hormone metabolism.     

Effect of the suicide substrate 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine on the metabolism of xenobiotics and on cytochrome P-450 apoproteins

Treatment of rats with the cytochrome P-450 suicide substrate, 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), produced a 95% inhibition of the in vivo demethylation of either aminopyrine or morphine within 2 hr. One-carbon metabolism of formaldehyde or formate to carbon dioxide was not altered. DDEP also produced a time-dependent decrease in total hepatic microsomal cytochrome P-450 but had no effect on either NADPH-cytochrome c reductase or p-nitrophenol glucuronyl-transferase activities up to 24 hr after administration. A rapid decrease in rat liver microsomal aniline hydroxylation and ethoxyresorufin deethylation was observed in vitro following DDEP administration. Although in vitro testosterone metabolism to 16 alpha-, 16 beta-, and 2 alpha-hydroxy metabolites was depressed profoundly by DDEP in microsomes from untreated and 3-methylcholanthrene-treated animals, 7 alpha-hydroxylation of testosterone was much less affected. Immunochemical quantification of various microsomal cytochrome P-450 protein moieties showed that cytochromes P-450 beta NF-B, P-450UT-A, P-450PCN-E, and P-450PB-C were decreased in hepatic microsomes from DDEP-treated rats. However, the protein moiety of cytochrome P-450UT-H was not diminished and the immunoreactive protein for cytochromes P-450UT-F, P-450PB-B, and P-450ISF-G was only slightly decreased. These results show that DDEP treatment leads to marked decreases in holoprotein and apoproteins of many but not all hepatic microsomal cytochrome P-450 isozymes.     

Selective damage to nonciliated bronchiolar epithelial cells in relation to impairment of pulmonary monooxygenase activities by 1,1-dichloroethylene in mice

A single ip dose of 1,1-dichloroethylene (DCE) to mice (125 mg/kg) caused a reduction within 24 hr in cytochrome P-450 and related monooxygenases in lung microsomes, with no corresponding changes in liver and kidney microsomes. Light microscopy revealed that at 24 hr, DCE caused a highly selective and complete loss of the bronchiolar nonciliated (Clara) cells at all levels of the tracheobronchial tree. Electron microscopy showed that at this time, the bronchiolar luminal surface was covered by flattened, elongated ciliated cells. Within 24 hr total microsomal cytochrome P-450 and NADPH cytochrome c reductase were maximally reduced to about 50% of control and cytochrome P-450-dependent enzyme activities decreased to about 60% of control. By contrast, coumarin 7-hydroxylase was reduced to approximately 10% of control within 4 days. Since pulmonary coumarin 7-hydroxylase has been shown to reside almost exclusively in the Clara cells, this finding is in agreement with the observed extensive necrosis of the Clara cells. The return of lung microsomal P-450-linked enzyme activities took between 3 and 6 weeks and was paralleled by a corresponding slow reappearance of the bronchiolar Clara cells.     

Microsomal cytochrome P-452 induction and peroxisome proliferation by hypolipidaemic agents in rat liver. A mechanistic inter-relationship

Eight structurally diverse hypolipidaemic agents have been examined for their ability to induce the microsomal cytochrome P-452-dependent fatty acid hydroxylase system and the enzymes of peroxisomal beta-oxidation in rat liver. Using a specific ELISA method, we have shown that the cytochrome P-452 isoenzyme is induced up to ten fold by hypolipidaemic challenge, concomitant with a pronounced elevation of the peroxisomal beta-oxidation enzymes, mirrored by an increase in peroxisomal volume as determined morphometrically. In addition, the induction of cytochrome P-452 is accompanied by a decrease in the activities of cytochromes P-450b and P-450c as measured by benzphetamine N-demethylase and ethoxyresorufin O-deethylase activities respectively, the latter being more extensively reduced by hypolipidaemic treatment. A hypothesis is presented whereby an early biological response is the hypolipidaemic induction of microsomal cytochrome P-452 resulting in omega-hydroxy fatty acids and their subsequent further oxidation to dicarboxylic acids, the latter providing the proximal stimulus for peroxisomal proliferation.     

Loss of rat liver microsomal cytochrome P-450 during methimazole metabolism. Role of flavin-containing monooxygenase

The role of flavin-containing monooxygenase (FMO) in the decrease in cytochrome P-450 content during the microsomal metabolism of methimazole (N-methyl-2-mercaptoimidazole) was investigated by heat inactivation of FMO. Incubation of liver microsomes from untreated Fischer 344 rats with NADPH and methimazole resulted in a 25% loss of cytochrome P-450 detectable as its ferrous-carbon monoxide complex. The same extent of cytochrome P-450 loss was observed with 1 and 20 mM methimazole, suggesting saturation of the process. There was no significant loss of cytochrome P-450 when microsomal FMO was heat-inactivated prior to incubation with NADPH and methimazole. Heat pretreatment of the microsomes did not affect cytochrome P-450 concentrations and cytochrome P-420 was not observed. These results indicate that FMO-catalyzed metabolism of methimazole is necessary for the loss of cytochrome P-450 in microsomes from untreated rats. Sulfite and N-methylimidazole, the ultimate products of methimazole metabolism, did not cause a significant loss of cytochrome P-450. There was no loss of cytochrome P-450 when glutathione was included in the incubation with methimazole, suggesting that cytochrome P-450 loss was due to an interaction with oxygenated metabolites of methimazole formed by FMO. Losses of cytochrome P-450 were also observed after incubation of microsomes from phenobarbital- (31%) of beta-naphthoflavone-pretreated rats (44%) with NADPH and methimazole. In contrast to microsomes from untreated rats, heat inactivation of FMO did not prevent the loss of cytochrome P-450 in microsomes from the pretreated rats. These results indicate that both phenobarbital and beta-naphthoflavone induce isozymes of cytochrome P-450 capable of directly activating methimazole.     

Induction of xenobiotic-metabolizing enzymes and peroxisome proliferation in rat liver caused by dietary exposure to di(2-ethylhexyl)phosphate

Abstract

1. Exposure of rats to 1% or 3% (w/w) di(2-ethylhexyl)phosphate in the diet for five days results in two- to three-fold inductions of liver cytosolic epoxide hydrolase activity and microsomal cytochrome P-450 content. Cytochromes P-450b + e were induced 20- to 35-fold, but no increase was observed in cytochrome P-450c.

2. Considerably smaller effects were obtained on NADPH-cytochrome c reductase, microsomal epoxide hydrolase and microsomal cytochrome b₅ content, and there was no effect on cytosolic glutathione transferase activity, under the same conditions.

3. A dramatic increase in cyanide-insensitive palmitoyl-CoA oxidation and total mitochondrial protein, together with smaller increases in total catalase and cytochrome oxidase activities, were observed after treatment with di(2-ethylhexyl)phosphate, indicating that this compound causes proliferation of both peroxisomes and mitochondria.

4. It is suggested that the induction of cytosolic epoxide hydrolase and the proliferation of peroxisomes may be related processes.     

Comparison of the form(s) of cytochrome P-450 induced by ethanol and glutethimide in cultured chick hepatocytes

In this study, using a combination of immunological and enzymatic characterizations, we compared the forms of cytochrome P-450 induced by ethanol and glutethimide in primary cultures of chicken embryo hepatocytes. Recently we purified a cytochrome P-450 of 50K molecular weight from chicken embryo liver using glutethimide as a prototypic inducer. Antibodies to both this chicken cytochrome P-450 and to rabbit cytochrome P-450 form 3a from the IIE subfamily detected microsomal proteins of 50K induced by either ethanol or glutethimide in cultured chick embryo hepatocytes, indicating the antigenic homology of these subfamilies of cytochromes P-450 among different animal species. However, the antibody to glutethimide-induced chick cytochrome P-450 of 50K inhibited p-nitrophenol hydroxylase and benzphetamine demethylase activities 85-90% in microsomes from both ethanol- and glutethimide-treated cells, indicating similar epitopes whose integrity is required for catalytic activity. In contrast, antibodies to rabbit cytochrome P-450 form 3a had little to no effect on these same microsomal activities. Both ethanol and glutethimide induced microsomal p-nitrophenol and aniline hydroxylase activities in cultured chick embryo hepatocytes. In microsomes from ethanol-treated cells, the turnover of p-nitrophenol per cytochrome P-450 was 2-fold greater than that induced by glutethimide treatment, suggesting that ethanol is inducing a form of cytochrome P-450 that has greater catalytic activity with this substrate than glutethimide-induced forms. Thus, in cultured chick embryo hepatocytes, ethanol may induce cytochromes P-450 from both the IIB and IIE subfamilies.     

Influence of chronic ethanol consumption on hamster liver microsomal O-dealkylase activities and cytochrome b5 content

The effects of two different methods of administering ethanol to hamsters on liver microsomal cytochrome levels and the activities of ethoxyresorufin O-deethylase and p-nitroanisole O-demethylase have been examined. Administration of ethanol in liquid diets resulted in enhanced levels of cytochrome P-450, NADPH-supported aniline hydroxylase (Form I), and both NADPH- and NADH-supported p-nitroanisole O-demethylase. NADH-ferricyanide reductase was also increased. No change in NADPH-cytochrome c reductase or in the NADPH-supported rate of ethoxyresorufin O-deethylase was observed. In contrast, both NADH-supported ethoxyresorufin O-deethylase and cytochrome b5 levels were decreased. Administration of ethanol in the drinking water to chow-fed animals had no effect on total cytochrome P-450 levels; however, the rates of NADPH-supported aniline hydroxylase (Form I) and p-nitroanisole O-demethylase activity were increased. No changes in NADPH-cytochrome c reductase, NADH-ferricyanide reductase, or NADH-supported p-nitroanisole O-demethylase activity were noted. Cytochrome b5 levels were decreased as were both the NADPH- and NADH-supported rates of ethoxyresorufin O-deethylase. These data suggest that chronic consumption of ethanol by hamsters either in liquid diet form or as ethanol-water solutions to chow-fed animals lowers cytochrome b5 levels. When cytochrome b5 levels are lowered and total chromosome P-450 levels remain unchanged, the NADPH-supported rate of microsomal O-dealkylation of ethoxyresorufin is decreased. These data suggest that cytochrome b5 participates in the NADPH-supported microsomal O-dealkylation of ethoxyresorufin.