Effect of endotoxin to differentially affect cytochrome P-450 monooxygenase activities of untreated rats and animals induced with phenobarbital or 3-methylcholanthrene

The effect of endotoxin in decreasing the cytochrome P-450-dependent metabolism of aniline, aminopyrine and ethoxycoumarin was examined in untreated rats, and in rats pretreated with either phenobarbital or 3-methylcholanthrene. Ethoxycoumarin metabolism was determined at two substrate concentrations (5 microM and 500 microM) to determine the effect of endotoxin on the high and low affinity enzyme activities. In untreated animals, endotoxin depressed both aniline and ethoxycoumarin metabolism by the high and low affinity enzymes by approximately 70%, but aminopyrine was decreased by only 47%. In phenobarbital pretreated rats, endotoxin decreased enzyme activities less than in untreated animals. Aniline metabolism and low affinity ethoxycoumarin metabolism were decreased by only 24%, and aminopyrine metabolism was decreased by 35%. The high affinity ethoxycoumarin metabolism was least affected, being decreased by only 12%. In 3-methycholanthrene pretreated rats, aniline and ethoxycoumarin (500 microM) metabolism were decreased by approximately 45%, but aminopyrine metabolism was only decreased by 20%. In these animals, endotoxin did not significantly affect the activity of ethoxycoumarin metabolism assayed with the low substrate concentration. Endotoxin decreased total cytochrome P-450 level of untreated rats by 32%, of phenobarbital pretreated rats by 39%, and in 3-methylcholanthrene pretreated animals the decrease was only 21%. Heme oxygenase activity of untreated animals was induced most by endotoxin administration and least in phenobarbital treated rats. The data suggest that endotoxin may differentially affect the various isozymes of cytochrome P-450 associated with the metabolism of aniline, aminopyrine and ethoxycoumarin. The results also suggest that the isozymes associated with these activities in untreated, phenobarbital or 3-methylcholanthrene pretreated rats may differ in their sensitivity to the effect of endotoxin.     

Effect of PSK, a protein-bound polysaccharide from Coriolus versicolor, on drug-metabolizing enzymes in sarcoma-180 bearing and normal mice

The effects of PSK and Propionibacterium acnes (anaerobic Corynebacterium) on hepatic drug-metabolizing enzymes were studied using sarcoma-180 bearing and non-tumor bearing mice. PSK had no influence on aminopyrine N-demethylase and aniline hydroxylase activities, cytochrome P-450 concentration in hepatic microsomes, and the reductase activity of cytochrome c in normal mice. The content of cytochrome P-450 was not significantly reduced in S-180 bearing mice. On the other hand, P. acnes administration significantly decreased the amount of cytochromes P-450 and b5 and aminopyrine N-demethylase activity. When FT-207 (Tegafur) was administered orally to S-180 bearing mice combined with the immunoadjuvants, only P. acnes significantly reduced the 5-FU levels in the serum and some organs.     

Changes in hepatic xenobiotic-metabolising enzymes in mouse liver following infection with Leishmania donovani

Infection of mice with Leishmania donovani resulted in decreased activities of several liver enzymes involved in the metabolism of xenobiotics. Microsomal membranes from infected livers contained reduced amounts of cytochromes P450 and b5 and NADPH-cytochrome P450 reductase. Several cytochrome P450 isoenzymes (P450-PB1, P450-PB3, P450-PCN and P450-UT1) and P450-mediated reactions (aminopyrine demethylase, aniline hydroxylase, benzphentamine demethylase and ethoxycoumarin deethylase) were affected similarly. The metabolism of two carcinogens (nitrosodimethylamine and 7,12-dimethylbenz[a]anthracene) by liver microsomal membrane preparations was also reduced. Leishmania infection caused an increase of cytosolic epoxide hydrolase and microsomal epoxide hydrolase and NADH-cytochrome b5 reductase were unaffected. The results suggest that Leishmania-infected animals are likely to have altered responses to exogenous toxins compared to uninfected animals.     

Hepatic drug metabolism during development in food-restricted female Sprague-Dawley rats.

Hepatic drug metabolism was investigated in female Sprague-Dawley rats fed ad libitum (A) or a restricted diet (R) (implemented from age 1 month), at 1.5, 4.5 and 12 months to determine the short- and long-term effects of caloric restriction. Microsomal cytochrome P-450 content and NADPH cytochrome c reductase activity were not modified by age. While dietary restriction did not affect cytochrome P-450, it significantly increased NADPH cytochrome c reductase activity at all time periods when compared to corresponding A-fed groups. Aniline hydroxylase and aminopyrine N-demethylase activity tended to decrease with age in the A-fed groups but the differences did not prove to be statistically significant. A significant decrease of aminopyrine N-demethylase was observed with age in R rats. A significant reduction of aniline hydroxylase activity was noted in the R groups compared to age-matched A-fed controls. In contrast, aminopyrine N-demethylase activity increased significantly, but only at 1.5 months of age. Glutathione S-transferase activity was augmented between 1.5 and 4.5 months of age, and this was followed by a significant decrease at age 12 months in both A and R groups. Dietary restriction had no effect on this enzymatic activity. The microsomal cholesterol and phospholipid content as well as the cholesterol/phospholipid molar ratio changed significantly between 1.5 and 4.5 months of age but not between 4.5 and 12 months of age. These parameters were unaltered by dietary restriction. In conclusion, in the female Sprague-Dawley rat there are no statistically significant changes in hepatic microsomal components and drug metabolizing capacity between 1.5 and 12 months of age. Dietary restriction resulted in significant changes in enzymes related to drug metabolism which varied with the enzyme examined. In general, these changes were similar after short- or long-term dietary intervention.     

Effect of Plasmodium yoelii nigeriensis infection and chloroquine on the hepatic mixed function oxidase system of mice

Plasmodium yoelii nigeriensis infection in albino mice significantly altered the hepatic microsomal mixed function oxidase system. Cytochrome P-450 (the terminal monooxygenase) and other monooxygenases, viz. aniline hydroxylase, aminopyrine-N-demethylase and benzo(a)pyrene hydroxylase were significantly lowered while microsomal heme showed 4-fold increase at 80% parasitaemia. Noticeable impairment in the other components like NADH:cytochrome b5 reductase, NADPH:cytochrome c reductase, cytochrome b5 and glucose-6-phosphatase was also observed. Oral treatment of normal and P. y. nigeriensis infected mice with chloroquine (64 mg per kg body weight for 4 days) caused lowering of mixed function oxidase activities which however showed a recovering trend, a week after cessation of treatment.     

Effect of acute and repeated chlordimeform treatment on rat hepatic microsomal drug metabolizing enzymes

The effect of chlordimeform treatment on the hepatic microsomal drug metabolizing enzymes was examined in male and female rats following either acute or repeated treatment. After acute administration of chlordimeform (100 mg/kg, ip one hr prior to sacrifice) differential effects were observed in various parameters of the hepatic microsomal mixed function oxidase system with significant decreases in ethylmorphine metabolism, cytochrome P-450 content, NADPH cytochrome c reductase, and in the spectral binding of hexobarbital and aniline while no changes were found in the metabolism of aniline or p-nitroanisole. Durations of zoxazolamine-induced paralysis and pentobarbital-induced hypnosis were increased significantly after acute chlordimeform administration. Following repeated administration of chlordimeform (75 mg/kg ip for four days) to adult male rats, a decrease was observed in zoxazolamine-induced paralysis time while pentobarbital-induced hypnosis was not altered. Metabolism studies using isolated hepatic microsomal fractions showed a decrease rate of biotransformation of ethylmorphine and aniline while the activity of p-nitroanisole O-demethylase was not changed. No differences were found in cytochrome P-450 levels whereas microsomal spectral binding of hexobarbital was reduced while that of aniline was not affected. Following acute or repeated administration of chlordimeform to adult female rats, decreases in the hepatic microsomal metabolism of aniline, but not ethylmorphine or p-nitroanisole, were observed. Addition of chlordimeform to microsomal suspensions yielded a Type I spectral binding curve.     

Effects of ethanol on microsomal drug metabolism in aging female rats. I. Induction

The purpose of this study was to determine how aging affects the induction by ethanol or acetone of the hepatic microsomal monooxygenase system of female Fischer 344 rats. Young-adult, middle-aged and old rats (4, 14 and 25 months) were fed an ethanol-containing or control liquid diet for 15 days. Cytochrome P-450, cytochrome c reductase, aniline hydroxylase, nitrophenol hydroxylase, nitroanisole O-demethylase and benzphetamine N-demethylase activities were measured in hepatic microsomes. All of the drug metabolism activities except benzphetamine N-demethylase were 20-35% lower in old than in young-adult rats fed the control diet. In addition, the increase in drug metabolism produced by feeding the regular ethanol diet (36% of calories as ethanol) was 50-60% lower in the old rats. However, there was no difference in the magnitude of ethanol induction when ethanol intakes were matched. The effects of chronic acetone consumption (1.2g/day per kg body weight for 15 days) paralleled those of ethanol consumption, except that the extent of induction was greater with acetone. Acetone-induced levels of hepatic microsomal cytochrome P-450, nitrophenol hydroxylase, nitroanisole O-demethylase and aniline hydroxylase were similar in all three age groups. The results of this study indicate that induction of hepatic microsomal drug metabolism by ethanol or acetone is unaffected by the aging process.     

Induction of tolerance in mice and rats to the effect of endotoxin to decrease the hepatic microsomal mixed-function oxidase system. Evidence for a possible macrophage-derived factor in the endotoxin effect

Daily administration of low, non-lethal doses of bacterial endotoxin to mice and rats has been shown to induce tolerance to the effect of an acute challenge dose of endotoxin to decrease the hepatic microsomal drug metabolizing activity, the level of cytochrome P-450, and to increase heme oxygenase activity. The serum collected at various times after injection of endotoxin into control animals when injected into untreated animals markedly depressed aniline hydroxylase activity, ethylmorphine N-demethylase activity, and the level of cytochrome P-450. Tolerant animals were not affected by the post-endotoxin serum injection, suggesting the decreased activity caused by the serum in untreated animals was probably due to endotoxin contained in the serum. Injection of tolerant mice and rats with supernatant medium obtained from cultures of peritoneal macrophages incubated with 100 micrograms/ml of endotoxin caused a loss of hepatic microsomal drug-metabolizing activity, and a decrease in the level of cytochrome P-450. These results suggest that peritoneal macrophages release a factor in response to endotoxin that mediates the decreased hepatic mixed-function oxidase activity.     

Hepatic microsomal alterations during chronic trypanosomiasis in the field vole, Microtus montanus

The field vole, Microtus montanus, was used as a model system to evaluate the chronic effects of infection by Trypanosoma brucei gambiense on hepatic mixed-function oxidase activity. At day 28 post inoculation there was a 97% increase in liver wet weight per g body weight. A portion of the increase (21%) was accounted for by tissue edema which occurred after day 14 of infection. Total hepatic cytochrome P-450 content and related total tissue mixed-function oxidase activities were decreased to about 60% of control levels at day 28 post inoculation. The decrease in total tissue mixed-function oxidase activity was partly due to a small decrease in microsomal protein per cell, and partly to a large decrease in cytochrome P-450 concentration in the endoplasmic reticulum. Although the decrease in total liver monooxygenase activity in several substrates roughly paralleled the loss in cytochrome P-450 content, several other microsomal enzyme markers not related to cytochrome P-450 monooxygenation were elevated in proportion to total liver microsomal protein content. The results suggest that in M. montanus during trypanosomiasis, there is proliferation of hepatic cells with normal content of endoplasmic reticulum. Furthermore, there appears to be selective toxicity for hepatic cytochrome P-450 and related monooxygenase activities. This may compromise the animals' ability to metabolize and dispose of other drugs to which the animal may be exposed in the course of infection.     

Effects of ischemia on drug-metabolizing microsomal enzymes in rat liver.

The activities of some enzymes and the content of some coenzymes associated with membranes of the endoplasmic reticulum are affected by ischemia in a way which is progressive with the duration of ischemia and selective for the various enzymes. Aminopyrine demethylation and aniline hydroxylation are strongly inhibited; cytochrome P-450 content decreases, though with a different pattern; NADPH- and NADH-cytochrome c reductases and cytochrome b₅ are unchanged; NAD(P)⁺ glycohydrolase is only slightly reduced. In the microsomal enzyme systems which catalyze drug metabolism the step affected by ischemia seems to be at the level of the hydroxylating enzymes proper or at the site of coupling between these enzymes and the rest of the pathway. Phenobarbital pretreatment exerts a marked protective effect on the decay of induced aminopyrine demethylase and aniline hydroxylase and on the content of newly formed cytochrome P-450. Differences in turnover and/or in location and stability within the membranes of these enzymes and coenzymes are discussed as possible causes of the differential damage caused by ischemia. Increased oxidation of NADH and NADPH added to microsomal preparations from ischemic livers is another sign of disturbed function which can be tentatively ascribed to some as yet undefined biophysical change of the microsomal membranes.     

Effect of a catatoxic steroid pregnenolone-16alpha-carbonitrile, on rat liver microsomal subfractions

Pregnenolone-16α-carbonitrile (PCN), a potent catatoic steroid without known classical hormonal effects, was administered per os to female rats. Its effects were studied on mixed function oxygenases and on various phosphatases in liver microsomal subfractions: rough microsomes, smooth I, and smooth II microsomes. For comparison, phenobarbital (PB) and 3-methylcholanthrene (MC) were also administered. The inducers increased the protein content in total, rough and smooth I microsomes in the following order: PB, PCN, and MC, whereas the protein amount in smooth II microsomal fraction remained unchanged. The content of cytochrome P-450 was about doubled in all subfractions except the smooth II membranes, following treatment with any of the inducers.PCN differed in inducing specificity from PB in increasing benzo(α)pyrene hydroxylase and from MC in stimulating aminopyrine demethylase in total microsomes. PCN also differed from PB in enhancing the capacity not only for rough and smooth I microsomes, but also for smooth II microsomes to demethylase aminopyrine. No major difference in magnitude of effects among the inducers was noted between rough and smooth I microsomes.In contrast to the altered substrate specificities produced in the monooxygenase system by different inducers, a more uniform pattern of specificities was seen in microsomal phosphatases. PCN, MC and PB all increased the activities of nucleoside diphosphatase (IDPase), whereas G6Pase and ATPase activities were little affected. Cycloheximide was partially effective in depressing the increase of both the monooxygenase system (cytochrome P-450 and benzo(α)pyrene hydroxylase) and nucleoside diphosphatase activity. We conclude that (1) treatment with PCN results in different substrate specificity when compared to PB and MC; that (2) PCN is the most potent inducer of the three in stimulating drug hydroxylation in smooth II microsomes, that (3) various smooth microsomal membranes of liver cells are affected differently by inducers of drug metabolism, and finally (4) that drugs also alter some microsomal phosphatase activities but not all.     

Biotransformation enzymes in Cunninghamella blakesleeana (NCIM-687)

Presence of higher enzyme levels of aminopyrine N-demethylase, aniline hydroxylase and 11-beta hydroxylase activities were observed in Cunninghamella blakesleeana grown in potato-dextrose medium for 96 h. The enzyme activity preferred NADPH as a cofactor and showed inhibition with CO, indicating cytochrome P450 mediated reactions. A significant increase in aniline hydroxylase enzyme activity was observed when mycelia incubated in incubation medium containing different inducers (viz. camphor, cholesterol, naphthalene, veratrole, phenobarbital, n -hexadecane and ethyl alcohol) when compared with mycelia incubated in same way but in absence of inducers. Cunninghamella blakesleeana (NCIM 687) have shown the ability to degrade cholesterol, camphor and naphthalene when 96 h grown mycelia incubated in incubation medium containing these organic compounds.     

Effects of aging and caloric restriction on hepatic drug metabolizing enzymes in the Fischer 344 rat. I: The cytochrome P-450 dependent monooxygenase system

The effects of long-term caloric restriction on the hepatic cytochrome P-450 dependent monooxygenase system were investigated in the 22-month-old Fischer 344 rat. Caloric restriction decreased the age-related changes in hepatic testosterone metabolism, which are associated with demasculinization of the liver. Caloric restriction also increased hepatic microsomal testosterone 6 beta-hydroxylase, lauric acid 12-hydroxylase and 4-nitrophenol hydroxylase activities over corresponding values in both ad libitum fed 22-month and 60-day-old control male rats. This suggests that cytochrome P-450 isozymes, P-450 pcn1&2, P-452 and P450j may be induced by caloric restriction. Such changes in cytochrome P-450 isozyme profiles could result in altered carcinogen activation, radical formation or drug detoxication in the calorically restricted rat.     

Vanadium: genetical and biochemical investigations

Ammonium metavanadate was studied for its ability to induce mitotic gene conversion and reverse point mutation in the D7 strain of Saccharomyces cerevisiae. Metavanadate increased the convertant and revertant frequencies; the highest activity was observed without metabolic activation. This indicated that the S9 hepatic fraction and yeast cells in logarithmic phase (and containing a high level of cytochrome P450) biotransform vanadate, probably reducing it to vanadyl. In addition, the effect of ammonium metavanadate on the hepatic monooxygenase system was studied in mice by measuring the level of cytochrome P450 and determining the activities of aminopyrine N-demethylase, p-nitroanisole O-demethylase and 7-ethoxycoumarin O-deethylase in mouse liver microsomal fraction. The results indicated that this compound reduced mono-oxygenase activity and also the level of cytochrome P450.     

Biochemical and histopathological alterations in golden hamster during infection with Ancylostoma ceylanicum

Ancylostoma ceylanicum infection in golden hamsters (Mesocricetus auratus) caused marked biochemical and histopathological derangements. Jejunum, the primary site of infection, showed pronounced alterations compared with liver. Though the biochemical composition of jejunum was not significantly altered, activities of a few lysosomal enzymes were enhanced during hookworm infection. Marked damage to mitochondrial and microsomal membranes was reflected in changes in the activities of the marker enzymes from jejunal tissue. Lipid content, especially phospholipids and neutral lipids of hepatic tissue, exhibited marked elevation. Levels of hexokinase, phosphofructokinase, and lactate dehydrogenase were enhanced in jejunal as well as hepatic tissues, indicating activation of the glycolytic machinery during hookworm infection. A decrease in the levels of mucosal disaccharidases indicated damage to intestinal brush border membranes. However, alkaline phosphatase activity was increased in intestinal mucosa during the infection. Light microscopic examination of jejunal tissue revealed peeling off of the upper epithelial layer, activation of the goblet cells, and thickening of muscularis mucosa. However, hepatic tissue did not show gross alterations, except for slight necrosis in the centrilobular region.     

Morphological and biochemical effects of carbon disulfide on rat liver

The early biochemical and ultrastructural effects on the liver of CS₂ administration in vivo to fasted rats were compared in animals treated and not treated with phenobarbital, to ascertain whether these effects differed quantitatively and/or qualitatively. Ultrastructural examination showed that in phenobarbital-treated rats, the main lesion induced by CS₂ was an increase in the number and size of the lysosomes containing cell debris from other cell organelles such as mitochondria. The endoplasmic reticulum was unchanged compared to that of rats treated only with phenobarbital. In particular, there was no decrease in the rough endoplasmic reticulum. By contrast, in rats not treated with phenobarbital, the main effect of CS₂ was a decrease in the number of ribosomes bound to the rough endoplasmic reticulum, with little or no change in other cell organelles. Cytochrome P-450 decreased more in phenobarbital-treated than in untreated rats, and cytochrome b5 decreased in phenobarbital-treated rats only. CS₂ lowered aniline hydroxylase activity, expressed per nanomole of cytochrome P-450 in untreated rats, but increased this activity in phenobarbital-treated rats. The opposite was observed for microsomal peroxidase and cytosolic glutathione reductase activities. Liver catalase diminished more in phenobarbital-treated than in untreated rats. The effects of CS₂ on liver endoplasmic reticulum as well as on the respective activities of aniline hydroxylase, microsomal peroxidase, and glutathione peroxidase show that untreated and phenobarbital-treated rats respond to CS₂ administration in qualitatively different ways. It is suggested that in untreated rats, the effects observed are mainly due to CS₂ itself, whereas in phenobarbital-treated animals, the effects are mainly caused by reactive CS₂ metabolites formed by the mixed function oxidase microsomal system.     

Aberration of heme and hemoprotein in aged female rats

The magnitude and duration of drug action is determined partially by the activity of the drug metabolizing enzyme systems in the liver. The pharmacological effectiveness of many drugs is altered during the aging process. In this study, the regulation of heme metabolism and hemoprotein content was examined in livers of aged female rats. The activities of hexobarbital hydroxylase and aniline hydroxylase, indicators of mono-oxygenase function, were decreased in aged rats by 31% and 24%, respectively, as compared to values in young rats. This was accompanied by a proportional decrease in the level of cytochrome P-450 (26%). Additionally, the activity of delta-aminolevulinic acid synthetase (ALA-S), the rate-limiting enzyme in heme synthesis, and the microsomal concentration of heme were also decreased by 33% and 26%, respectively, in these animals. In contrast, the basal activity of microsomal heme oxygenase (MHO), the rate-limiting enzyme in heme degradation, and the percent heme saturation of tryptophan pyrrolase (TPO), a sensitive indicator of changes in the availability of heme in the "regulatory" heme pool, were increased by (87%) and (31%), respectively, in the aged rats. The serum concentration of bilirubin, an indicator of erythrocyte breakdown and/or liver function was likewise increased in these animals. In view of these findings, we suggest that the high activity of MHO and the low level of ALA-S may be a significant causative factor for the decreased microsomal concentration of heme, cytochrome-P-450 and its dependent monooxygenase activities in senescent female rats.     

A comparison of the effect of two bovine serum albumin preparations on benzo(alpha)pyrene hydroxylase in rat liver and lung microsomes

One of two commercial bovine serum albumin preparations caused decreases in rat liver and lung microsomal benzo(alpha)pyrene hydroxylase activities when measured by the fluorescence assay. The decreased activities were not due to a decreased recovery of a reaction product, 3-hydroxybenzo (alpha)pyrene, the presence of unmasked fatty acid binding sites or decreased content of cytochrome P450. The decreased enzyme activity may be due to a component present in the preparation. The results indicate that bovine serum albumin preparations should be carefully checked before use in the benzo(alpha)pyrene hydroxylase assay.     

Cytochrome P450 modification by a new oxazine derivative with hypolipidemic activity

Plasma lipoprotein levels, related to atheromatosis, are influenced by liver function. Microsomal enzyme inducers are reported to modify serum lipoproteins and triglycerides. In this study, the effects of subchronic and acute treatment of rats with 3-(4-biphenyl)-3-n-propoxy-octahydro-1,4-pyrido[2,1-c]oxazine, a novel compound with hypolipidemic and antioxidant activities, on rat hepatic microsomal protein and total cytochrome P450, as well as on p-nitrophenol hydroxylase (CYP2E) and erythromycin N-demethylase (CYP3A) activities are examined. The subchronic treatment had no significant effect on liver weight, microsomal protein and total cytochrome P450. The acute administration lowered considerably cytochrome P450 content. The metabolic activities of CYP2E1 and CYP3A1/2 were not altered by the subchronic treatment, but were notably decreased after the single administration of 3-(4-biphenyl)-3-n-propoxy-octahydro-1,4-pyrido[2,1-c]oxazine. The inhibition of drug metabolism by 3-(4-biphenyl)-3-n-propoxy-octahydro-1,4-pyrido[2,1-c]oxazine cannot be completely correlated with the modification of plasma cholesterol, triglycerides and LDL cholesterol, although published data connect microsomal enzyme induction with a decrease of these parameters. This discrepancy could be attributed to the different biochemical events involved in enzyme induction and inhibition.     

Enzyme-inductive effect of a hypolipidemic compound N-bis-(p-chlorophenoxy)-acetyl-urea in man and rat

Besides its antilipidaemic effect, the new clofibric acid derivative (N-bis-(p-chlorohenoxy)-acetyl-urea) has an enzyme-inductive effect. The drug was administered (100 mg/kg orally) to male, Wistar rats for three days. The treatment raised the weight of the liver, the content of liver microsomal protein and cytochrome p-450 and shortened the hexobarbital sleeping time. The increase of cytochrome p-450 dependent biotransformation was found by in vitro methods in 9000-g supernatant of liver homogenate. There was a growth in biotransformation of substrates of type I (ethylmorphine, aminopyrine) and an extreme increase in reduction of nitrobenzene. We did not find any change in biotransformation of the type-II substrate aniline. In 16 patients suffering from Gilbert's syndrome, there was a decrease in the level of serum bilirubin, and increase of D-glucuric-acid output in urine and bromsulphophthalein transport maximum following the treatment of this drug given in 150 mg/day orally for three weeks. After this treatment, the level of gamma-glutamyl-transpeptides did not change. The authors highly recommend the serious consideration of metabolic interaction during the clinical application.