Components Of Mixed Function Oxidase Research Articles

Sex and age-related differences in aminopyrine N-demethylase activity in DA- and Wistar-strain rat liver microsomes. Effect of ovariectomy

1. Liver microsomal mixed-function oxidase components were studied in Wistar and Dark Agouti (DA) rats (4-45 weeks) with regard to sex- and age-related differences. Total cytochrome P-450 ranged from 0.29 to 1 nmol/mg in Wistar rats and from 0.21 to 1.27 nmol/mg in DA rats, males had higher levels than females (P less than 0.0025). Cytochrome b5 ranged between 0.42-1.37 nmol/mg and 0.42-1.56 nmol/mg in Wistar and DA strains, respectively, and NADPH-reductase activity ranged between 14-43 and 11-46 nmol/min per mg (Wistar and DA respectively). 2. Significant age-related differences were found in DA rats with four- to six-fold increase in N-demethylase activity from young to adult rats. Sex-related differences were found in both Wistar- and DA-strain rats, with males having higher (about twice) metabolic activity than females. In contrast, no significant sex- or age-related differences in cytochrome b5 content, or NADPH-reductase activity, were found. 3. Ovariectomy of 10-13-week-old females did not affect N-demethylase activity, cytochrome P-450, cytochrome b5 or NADPH-reductase activity in Wistar or DA rats. 4. Cytochrome P-450 content did not correlate (r = 0.35) with aminopyrine N-demethylase activity. 5. Results indicate that sex- and age-related differences are due to changes in the isozymic composition of cytochrome P-450, and that these changes are not subject to oestrogen regulation.

Comparison of hepatic drug metabolizing enzymes in three-month-old lambs and kids

1. The comparative activity of hepatic cytochrome P-450 monooxygenase system, glucuronyltransferase, glutathione S-transferase and N-acetyltransferase was studied in three-month-old male and female Lacaune lambs and male Saanen kids. 2. The study of mixed-function oxidase components showed that total cytoehromc P-450 ranged from 0.54 in kids to 0.85–0.88 nmol/mg −1 in lambs. Male lambs had higher levels than kids (122–165%) for aminopyrine, benzphetamine, ethylmorphine and erythromycin demethylases or benzo(a)pyrene hydroxylase whereas NADPH-cytochrome c reductase was 1.19-fold lower in lambs. 3. Sex-related changes were observed in lambs in case of microsomal benzo(a)pyrene hydroxylase activity which appeared 1.31-fold more potent in male liver. Cytosolic N-acetyltransferase accepting sulfamethazine as substrate was about 8-fold higher in female than in male lambs. 4. The analysis of samples from various liver lobes, indicated the heterogenous distribution of micro- somal proteins which is related to higher concentrations of both cytochrome b5, NADPH-cytochrome c reductase and p-nitrophenol glucuronyltransferase in left lobes.

Cytochrome P-450 and oxidative metabolism in molluscs.

1. Cytochrome P-450 and the associated components and oxidative activities of a mixed-function oxidase (MFO) system are localized primarily in the microsomes of the digestive gland of molluscs. 2. Cytochrome P-450 and putative cytochrome P-450-catalysed oxidative activities, measured in vitro and/or in vivo, have variously been detected in 23 species of mollusc. 3. Cytochrome P-450 and other MFO components and activities may be increased by exposure to xenobiotics, but the results are variable and no correlation is obvious between changes in cytochrome P-450 content and measured MFO activities (benzo[a]pyrene hydroxylase (BPH) and 7-ethoxycoumarin O-deethylase (ECOD)). 4. Type II binding compounds (clotrimazole, miconazole, ketoconazole, metyrapone and pyridine) give type II difference spectra with mussel digestive gland microsomal P-450, whereas type I binding compounds (testosterone, 7-ethoxycoumarin, alpha-naphthoflavone, SKF525-A) give apparent reverse type I difference spectra. 5. The existence of multiple or particular forms (P450 IVA or LAw) of cytochrome P-450 is indicated from enzyme kinetics and inhibition studies, seasonality, purification studies and cDNA probes. 6. Microsomal MFO activities are observed even in the absence of added or generated NADPH, and the NADPH-independent BPH, ECOD and N,N-dimethylaniline N-demethylase activities are inhibited by reducing agents, including NADPH. 7. The major metabolites of microsomal benzo[a]pyrene metabolism are quinones. 8. One-electron oxidation is considered to be one possible mechanism of molluscan cytochrome P-450 catalytic action.

Effect of diisopropyl 1,3-dithiolan-2-ylidenemalonate(NKK-100) on the components of hepatic microsomal mixed function oxidase in male broiler chicks.

Effect of oral administration of diisopropyl 1, 3-dithiolan-2-ylidenemalonate (NKK-100) on the components of mixed function oxidase system in hepatic microsomes was investigated in male broiler chicks. Feeding NKK-100 increased relative liver weight and microsomal protein content of the liver. Contents of cytochrome P 450 and b 5 per mg microsomal protein and g liver increased with NKK-100 levels. The activities of NADPH-cytochrome c reductase and NADH-cytochrome b 5 reductase per g liver were enhanced by feeding NKK-100, while those activities per mg microsomal protein remained unchanged. Pretreatment with clopidol (an anticoccidial agent) and colistin (an antibiotic) did not affect the components of MFO. These results suggests that NKK-100 has a property to activate the hepatic microsomal MFO and the effect of NKK-100 are more prominent in the increases in specific activities of cytochrome P 450 and b 5 rather than in those of NADPH-cytochrome c reductase or NADH-cytochrome b 5 reductase.

Age- and sex-related changes in the components of the hepatic microsomal mixed function oxidase system in Sprague-Dawley rats.

1. A study of the sex- and age- (4 to 103 weeks) related changes in liver microsomal mixed-function oxidase components of Sprague-Dawley rats, showed that total cytochrome P-450 ranged from 12.0 to 34.0 nmol/g, peaking between weeks 26 and 39; males had higher levels than females at weeks 4 to 51. Cytochrome b5 ranged from 5.0 to 15.0 nmol/g with inconsistent age- and sex-related differences. 2. NADPH:cytochrome C reductase ranged from 2.6 to 4.8 mumol/min per g, with maximal activity at week 39; males had greater activity at weeks 12 to 78. NADH:cytochrome C reductase ranged from 5.2 to 26.9 mumol/min per g, peaking between weeks 51 and 78; females had greater activity at weeks 12 to 51. 3. This age- and sex-related pattern of changes in these components is specific to the Sprague-Dawley strain. There are slight but significant differences between sexes only in rats less than 1 year old. There was no significant correlation between rates of p-nitroanisole or aniline metabolism and cytochrome P-450 concentrations.

Characterization of the liver cytochrome P450 in the marine fish Mugil cephalus and effects of some hydrocarbons on the mixed-function oxidase components

The marine fish Mugil cephalus was caught in the Bay of Marseilles (France) in 1985 and the liver microsomal cytochrome P450 was identified at a concentration of about 0.230 nmol mg-1 protein. Optimal assay conditions were established to measure two mixed-function cytochrome P450-dependent oxidases: aniline hydroxylase and p-nitroanisole O-demethylase. Induction of cytochrome P450 occurred after exposure of mullet to an aromatic hydrocarbon and a water-soluble petroleum fraction. The liver cytochrome P450 was solubilized and purified. The monomer molecular weight of partially purified cytochrome P450, determined by sodium dodecyl-sulfate-gel electrophoresis, was 51 000.

Enlarged livers and hepatic microsomal mixed‐function oxidase components in tumor‐bearing brown bullheads from a chemically contaminated river

AbstractHepatic mixed‐function oxidase (MFO) components in tumor‐bearing brown bullheads (Ictalurus nebulosus) from a chemically contaminated river and in bullheads from two relatively uncontaminated sites were compared to determine if the components in the river bullheads were induced. Induction of MFO components could be indicative of chemical contamination of the river water and of contaminant stress in the fish. In general, biochemical hepatic microsomal MFO component levels in bullheads from the river were the same as, or lower than, those in bullheads from uncontaminated sites. However, the liver weight as a percentage of body weight was significantly greater in bullheads from the river than in those from the uncontaminated sites. Thus, a comparison of bullheads from the river and the uncontaminated sites appeared to show that relative liver weight was indicative of chemical contamination of the river water and of contaminant stress in its fish. However, biochemical hepatic microsomal components measured in this study were apparently not adequate as indicators of chemical contamination of the river water or of contaminant stress in bullheads from the river.

A modified protocol for measuring alterations in human placental arylhydrocarbon hydroxylase (AHH) activity associated with environmental chemical exposure

Levels of mixed-function oxidase (MFO) enzymes were measured in adriamycin(ADR)-sensitive murine leukemia P-388 and its ADR-resistant subline P-388/ADR. The subcellular fractions of the resistant cells showed decreased contents of MFO components, cytochrome P-450 and cytochrome b5, in comparison with the identically prepared fractions of the parental tumor. Similarly, the levels of 7-ethoxycoumarin O-deethylase and the rate of ascorbate induced lipid peroxidation in vitro showed lower values in resistant tumor cells than those of P-388 tumor cells. The observed differences in the two tumor cell types were found to be considerably enhanced if the tumor cells were exposed in vitro to ADR before fractionation. The magnitude of induction of the MFO enzymes was significantly greater in the ADR exposed P-388 cells. The corresponding inducibility was suppressed in the drug exposed resistant tumor cells.

Mixed function oxidase in the mammary gland and liver microsomes of lactating rats: Effects of 3-methylcholanthrene and β-naphthofla vone

Mammary gland and liver microsomes of lactating rats were examined for the components of mixed function oxidase and related enzyme activities. Cytochrome b 5, NADH- and NADPH-dependent cytochrome c reductase activities were 15-, 6- and 10-fold lower, respectively, in the mammary gland than in the liver microsomes. The determination of cytochrome P-450 (P-448) in the mammary gland microsomes required elimination of the spectral interferences by hemoglobin and cytochrome aa 3. The presence of the latter in this fraction was also shown by cytochrome c oxidase activity. Cytochrome aa 3 was reduced by anaerobic incubation of mammary gland microsomes, in the presence of antimycin A, with sodium succinate, phenazine ethosulfate, and sodium ascorbate for 30 min at room temperature. Spectral resolution of the dithionite-reduced cytochrome P-450 (P-448) carbon monoxide complex occurred 30 min after gassing. The basal level of cytochrome P-450 was about 500-fold greater in the liver than in the mammary gland microsomes. Pretreatment of lactating rats with the inducers of hepatic cytochrome P-448, 3-methylcholanthrene and β-naphthoflavone, increased the cytochrome content 3- to 10-fold and 2-fold, in the mammary gland and liver microsomes, respectively. The induction of cytochrome P-448 in microsomes of both tissues was also shown by type I binding spectra obtained with N-2-fluorenylacetamide. Using hydroxylation of benzo[ a]pyrene and N-2-fluorenylacetamide as a measure of mixed function oxidase activity, we found that the basal activities, which were 4- to 8-fold greater in the liver microsomes, were increased in both tissues after treatment of rats with the inducers. The induced activities were inhibited by 0.1 mM α-napthoflavone in vitro, indicating a dependence on cytochrome P-448. The data suggest that the mammary gland, an extrahepatic target for carcinogens, is capable of their metabolism.

Mixed-function oxidase enzymes in adriamycin-sensitive and resistant sublines of P-388 leukemia

Levels of mixed-function oxidase (MFO) enzymes were measured in adriamycin(ADR)-sensitive murine leukemia P-388 and its ADR-resistant subline P-388/ADR. The subcellular fractions of the resistant cells showed decreased contents of MFO components, cytochrome P-450 and cytochrome b 5, in comparison with the identically prepared fractions of the parental tumor. Similarly, the levels of 7-ethoxycoumarin O-deethylase and the rate of ascorbate induced lipid peroxidation in vitro showed lower values in resistant tumor cells than those of P-388 tumor cells. The observed differences in the two tumor cell types were found to be considerably enhanced if the tumor cells were exposed in vitro to ADR before fractionation. The magnitude of induction of the MFO enzymes was significantly greater in the ADR exposed P-388 cells. The corresponding inducibility was suppressed in the drug exposed resistant tumor cells.

Enhanced aryl hydrocarbon hydroxylase activity after interaction between solubilized cytochrome P-448 and microsomes low in endogenous cytochrome P-450

The effects of cumene hydroperoxide on microsomal mixed-function oxidase components and enzyme activities were determined. In vitro cumene hydroperoxide treatment decreased cytochrome P-450 content, benzphetamine N-demethylase activity and aryl hydrocarbon hydroxylase activity of hepatic and renal microsomes from adult male and female rats, and of hepatic microsomes from fetal rats. Cumene hydroperoxide-treated microsomes, as well as fetal liver and adult renal microsomes, which are naturally low in cytochrome P-450 and mixed-function oxidase activity, were used to incorporate partially purified hepatic cytochrome P-448 isolated from 2,3,7,8-tetrachlorodibenzo- p-dioxin-pretreated immature male rats. This resulted in an enhanced rate of benzo[ a]pyrene hydroxylation. Aryl hydrocarbon hydroxylase activity was increased 12-, 26-. 31- and 53-fold when 1.0 nmole of partially purified cytochrome P-448 was incubated with fetal liver microsomes, microsomes from kidney cortex of female rats, and cumene hydroperoxide-pretreated hepatic microsomes from female and male rats, respectively. The increased rate of benzo[ a]pyrene hydroxylation was linear with cytochrome P-448 over the range 0.25 to 1.0 nmole. Because cumene hydroperoxide-pretreated microsomes from male rat liver and the hepatic and renal microsomes from female rats have a combination of high NADPH-cytochrome c reductase activity and low mixed-function oxidase activity, they are an attractive choice for catalytic studies of the interaction between cytochrome P-448 and microsomes.

A seasonal study of microsomal mixed-function oxidase components in insecticide-resistant and susceptible mosquitofish, Gambusia affinis

The mixed-function oxidase components which are involved in xenobiotic hydroxylations were studied in liver microsomes from two populations of mosquitofish ( Gambusia affinis) for 26 months. One population is highly resistant to organochlorine insecticides and is exposed periodically to agricultural chemical contamination; the other population is susceptible to these chemicals and is not exposed to such pollutants. The following parameters were measured: cytochromes P-450 and b 5; NADPH-cytochrome c, NADPH-dichloroindophenol, NADH-cytochrome c, and NADH-cytochrome b 5 reductases; liver weight to body weight ratios; and microsomal protein. Most parameters demonstrated seasonal cycles with the highest values occurring during the late fall and winter. The cyclic nature of the parameters investigated may be the result of the relative magnitude of microsomal hydroxylations during the year in relationship to other microsomal processes such as biosynthesis. The range of concentrations or specific activities of most of the components was about the same for both fish populations, although the resistant fish possessed relatively larger livers and, therefore, greater amounts of these microsomal components than the susceptible fish. The results suggest the induction of these microsomal oxidase components in the resistant population during their annual exposure to environmental pollutants in the agricultural runoff.

The specificity of the rat-liver cholesterol 7alpha-hydroxylase.

Experiments have been performed which investigate the relationship of the cholesterol 7α‐hydroxylase enzyme to the cytochrome‐P ‐450‐dependent mixed‐function oxidase system of rat‐liver microsomes. There appears to be no simple relationship between the activity of the cholesterol 7α‐hydroxylase enzyme and the cytochrome P ‐450 content of rat liver microsomes. Rat liver microsomal cholesterol 7α‐hydroxylase was sensitive to cycloheximide treatment. Its activity decayed in vivo following this treatment more rapidly than the aminopyrine demethylase enzyme or the mixed‐function oxidase components cytochrome P ‐450 and NADPH‐cytochrome c reductase. Freezing and thawing of rat liver microsomes resulted in a marked decrease in the activity of the cholesterol 7α‐hydroxylase.The specificity of the cholesterol 7α‐hydroxylase enzyme has been investigated by comparing the liver microsomal oxidation of cholesterol with that of other steroids having a similar structure to cholesterol.The substrates whose oxidation was studied were androst‐5‐en‐3β‐ol‐17‐one, pregn‐5‐en‐3β ol‐20‐one, androst‐5‐en‐3β‐ol and pregn‐5‐en‐3β‐ol. All these steroids were metabolised to one or more products when incubated with rat liver microsomes, NADPH and oxygen. Treatment of rats with cholestyramine which increased the activity of cholesterol 7α hydroxylase, had no effect on the rate of oxidation of any of the four steroids studied.The conversion of [4‐14C]cholesterol to 7α‐hydroxy[4‐14]cholesterol was significantly increased by preincubating the added tracer cholesterol with liver microsomes prior to starting the hydroxylation reaction with NADPH.It is suggested that the observed differences between the mechanism of control of the cholesterol 7α‐hydroxylase enzyme and other mixed‐function oxidase enzymes in rat liver microsomes might be due to presence of different rate‐limiting steps in the overall enzyme complex.

The distribution of the components of mixed-function oxidase between the rough and the smooth endoplasmic reticulum of liver cells.

Mixed-function oxidase activity, when measured by the N-demethylation of ethylmorphine or the hydroxylation of aniline, is significantly higher in the smooth hepatic endoplasmic reticulum than in the rough. In the rabbit the smooth membrane/rough membrane activity ratios are significantly greater than 1 whether the activities are expressed per g. of liver (ratio 5), per mg. of protein (ratio 3-5), per mug. of phospholipid phosphorus (ratio 2), per unit of cytochrome P-450 (ratio 1.7) or per unit of NADPH-cytochrome c reductase activity (ratio 2). On the other hand, if the activities are normalized to the NADPH-cytochrome P-450 reductase, there is no significant difference between the rough and smooth membranes. These results suggest that, in the rabbit, the rate-limiting step is the reduction of cytochrome P-450. In contrast, in the rat the difference in activities can be explained by differences in the concentration of cytochrome P-450.