Stimulation of microsomal dimethylnitrosamine-N-demethylase by pretreatment of mice with acetone

To further investigate the relationship between in vivo microsomal enzyme modifiers and in vitro dimethylnitrosamine (DMN) metabolism, male C57BL/6J mice were pretreated with acetone or Aroclor 1254, two compounds known to influence DMN-N-demethylase activity. Pretreatment with acetone enhanced the in vitro microsomal activity of DMN-N-demethylase, as measured by formaldehyde production from DMN. Accompanying this acetone-enhanced demethylase activity was an increase in the covalent binding of [14C]DMN to RNA, protein and DNA. Four distinct Km values dependent on the substrate concentration were observed for the N-demethylase present in control microsomes. Only one Km value was observed for the demethylase in microsomes from acetone-treated animals, but it was significantly lower than the lowest Km observed in the control microsomes. At DMN concentrations of 1 and 10 mM, acetone significantly increased N-demethylation of DMN as compared to control, but not at 100 mM DMN. Aroclor 1254 pretreatment repressed DMN-N-demethylase at 1 mM DMN but enhanced it at 100 mM. These results suggest that there may be multiple forms of DMN-N-demethylase which are dependent on DMN concentration and respond differently to modifiers of the microsomal drug-metabolizing enzymes.     

The effect of vitamin E deficiency on enzyme activity and the status of the membrane fraction of rat liver microsomes

The vitamin E deficiency was studied for its effect on the activity of enzymes participating in metabolism of xenobiotics. Experiments with 54 rats have demonstrated that the maintenance of animals on the vitamin-E-deficient diet within 13-14 weeks decreases the activity of microsomal monooxygenases (demethylase and hydroxylase), NADH- and NADPH-reductases, aryl- and aliesterases in the liver and lungs, which is a result of disturbance of hydrophobic and polar interactions in microsomal membranes. Vitamin E deficiency makes the extent of solubilization of these enzymes higher under the influence of deoxycholate and trypsin and intensifies inactivation of these enzymes under the effect of urea. In the lungs and in the liver of the vitamin E deficient rats the content of reduced glutathione decreases as well as the activity of glutathione reductase, glutathione-S-transferase, aldehyde dehydrogenase, while the activity of gamma-glutamyltransferase increases; glutathione disulphide is accumulated.     

The relationship of the intensity of liver microsomal oxidation to individual longevity

Positive correlation between the activity of enzymes of microsomal oxidation of the liver and individual lifespan was shown in experiments on Wistar female rats. The use of test of duration of pentobarbital hypnosis provides for the distinction between the animals with low and high life expectancy.     

Effect of denervation of the liver on the system of microsomal oxidation in adult and old rats

The role of adrenergic and cholinergic neural regulation in the functional activity of the liver microsomal oxidation enzymes has been studied. The experiments on adult and old rats using surgical denervation of the liver (vagotomy and sympathectomy++) have revealed changes in the monooxygenase activity (aminopyrine demethylase and aniline hydroxylase), in isoform composition and inductive synthesis of cytochrome P-450. The neural control over detoxication function of the liver is found to weaken in old age.     

Partial purification of trimethylamine-N-oxide (TMAO) demethylase from crude fish muscle microsomes by detergents

Detergent treatments were examined for their efficacy in purifying trimethylamine-N-oxide (TMAO) demethylase activity from fish muscle microsomes. Tritons X-100 and X-45, deoxycholate, Brijs, Tweens 20, 65, and 80, and SDS were generally ineffective in solubilizing demethylase activity from this membrane fraction, at concentrations up to 10 mg detergent per mg protein. In all of these cases, specific activity became enriched in the particulate fraction obtained post-treatment. Highest fold-purification was achieved by using 10 mg SDS per mg protein in 5 mM histidine, pH 7.0 at 10-14 degrees C. Activity was relatively stable to the presence of SDS at this level, and with this treatment, TMAO demethylase activity became purified in the resultant particulate fraction 28- and 58-fold for activity stimulated by ascorbate-iron-cysteine and FMN-NADH, respectively. The presence of urea or 2-mercaptoethanol, or sonication of the SDS-microsome suspension during purification resulted in significant losses of recovered activity. This partially purified fraction represented about 1% of the original microsomal protein and SDS-PAGE revealed the presence of several protein components. The partially purified demethylase could utilize the same two cofactor systems as the native microsomes. It displayed a curvilinear dependence on iron for activity and a sigmoidal response for cysteine. Utilization of NADH, FMN, and ascorbate differed for the purified fraction as compared to the microsomes. Substrate inhibition by TMAO was observed for the partially purified preparation, whereas saturation kinetics were previously noted for microsomal activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of precursors of endogenously synthesized dimethylnitrosamine on the activity of the demethylase of that carcinogen in rat liver

Oral administration of dimethylamine, sodium nitrite and a combination of these precursors of endogenously synthesized dimethylnitrosamine to rats increased the activity of demethylase of this carcinogen in the liver microsomes. Under conditions of chronic experiment inclusion of dimethylamine into the diet of rats intensified the demethylase activity even in the presence of casein, an inductor of this enzymatic system. An inhibitor of protein synthesis--actinomycin D prevented an increase of demethylase activity in the microsomal fraction caused by dimethylamine.     

Involvement of cytochrome b5 in the hepatic microsomal metabolism of benzo(a)pyrene

Ethanol consumption decreased the specific content of microsomal cytochrome b5 in both chow-and liquid diet-fed hamsters while cytochrome P450 levels were unchanged in chow-fed animals and increased in liquid diet-fed animals. Microsomes from animals receiving ethanol in their drinking water exhibited decreased rates of microsomal aryl hydrocarbon hydroxylase activity and postmitochondrial supernatant mediated mutagenicity of benzo(a)pyrene. In contrast, microsomes from hamsters receiving ethanol in liquid diets showed no changes in either of these two activities. When the observed rates of 7,8 and 9,10 diol formation per nmole P450 for chow-fed animals are plotted vs. the b5/P450 ratio a positive correlation was observed suggesting that cytochrome b5 participates directly in the microsomal metabolism of benzo(a)pyrene.     

In vivo effect of cholesterol feeding on the short term regulation of hepatic hydroxymethylglutaryl coenzyme A reductase during the diurnal cycle

Light-dark-cycled rats were fed a 3% cholesterol-supplemented diet at the beginning of the dark phase. Cholesterol-fed and control animals were taken at intervals throughout the following 12 h and the microsomal and solubilized hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase was isolated. Immunotitrations of this microsomal and solubilized enzyme were performed with a monospecific antibody to 3-hydroxy-3-methylglutaryl coenzyme A reductase. In contrast to the specific activity of the enzyme, which differs extremely during the diurnal cycle, the immunotitrations obtained from cholesterol-fed and control animals, yielded in identical antisera equivalence points. On the other hand, when the enzyme was phosphorylated in vitro, the antisera equivalence points corresponded to the alterations of the specific activity. In contrast to the results published by Higgins and Rudney ((1973) Nature New Biol. 246, 60-61), our data prove that even the in vivo short term changes in enzyme activity are due to changes in the quantity of enzyme rather than to a modulation of the catalytic activity.     

Changes in the longevity of rats under the influence of enzyme imprinting by phenobarbital

Experiments on the Wistar rats showed that neonatal daily injections of phenobarbital (35 mg/kg) during first 3 days of life resulted in the enzyme imprinting of liver microsomal monooxygenases. Rise in the activities of liver microsomal oxidation enzymes is constantly maintained during all the life leading to increase in average lifespan of rats. Analysis of the survival curves in Gompertz equation co-ordinates showed that enzyme imprinting by phenobarbital caused changes in mortality patterns at different stages of ontogenesis. The phenomenon of enzyme imprinting by phenobarbital and lifespan prolongation was registered only in females but not in males. An inverse correlation was found between the duration of phenobarbital sleeping time and lifespan of rats.     

Effect of spermine on the inhibition by fatty acid on AMP deaminase reaction as a control system of the adenylate energy charge in yeast

Treatment of rats with pyrazole elevated the hepatic microsomal dimethylnitrosamine demethylase activity (DMNd) by several fold. Methylethylnitrosamine demethylase activity was also increased by pyrazole, but some classical monooxygenase activities were not induced. The treatment induced a new protein species which has an apparent molecular weight of 52,000 dal and is believed to be a cytochrome P-450 isozyme. The involvement of a hemoprotein in the pyrazole-induced DMNd was demonstrated in an experiment with CoCl₂ which decreased both the microsomal cytochrome P-450 content and DMNd. The induced enzyme with a single K_m value of 0.061 mM and V_max of 12.1 nmol/min/mg is probably the most efficient enzyme known to metabolize nitrosamines. NADPH-cytochrome P-450 reductase was also demonstrated to be an essential component enzyme of the DMNd. These results further substantiate the idea that the P-450-containing monooxygenase is responsible for the metabolism of dimethylnitrosamine in both the control and pyrazole induced microsomes.     

Cold lability and dissociation of the F1-ATPase from Bacillus stearothermophilus

Effects of inadequate vitamin E (E) and/or selenium (Se) nutrition on the activities of cytochrome P-450 mixed function oxidase system (heme hydroperoxidase, p-nitroanisole O-demethylase), and epoxide hydrolase have been investigated. Heme hydroperoxidase activity of liver and lung microsomes was significantly decreased in E deficiency. In the liver, Se deficiency resulted in a significant increase in hydroperoxidase activity. In contrast to the peroxidase activity, liver demethylase activity was only marginally affected in $\text{E}\text{Se}$ deficiency states. However, kidney demethylase activity was increased two fold in Se deficient states. Liver microsomal epoxide hydrolase activity was significantly increased in both E and Se deficiency states.     

Mechanism of induction of cytochrome P-450ac (P-450j) in chemically induced and spontaneously diabetic rats

Previous studies demonstrated that a microsomal high-affinity N-nitrosodimethylamine demethylase activity and cytochrome P-450ac (an acetone/ethanol-inducible form) were induced by streptozotocin-induced diabetes in rats. In the present work, the induction was studied in detail in two chemically induced (by streptozotocin and alloxan) diabetic rat models and one spontaneously (BB/Wor) diabetic rat model. All the diabetic conditions caused increases in three parameters: (a) microsomal N-nitrosodimethylamine demethylase activity which is known to be a good indicator of the level of P-450ac; (b) the levels of P-450ac as determined by immunoblot analysis; and (c) the levels of mRNA of P-450ac as determined by hybridization assays with a cDNA probe for this enzyme. These increases were abolished by treatment of the diabetic rats with insulin. The results suggest that the pathophysiological condition of diabetes is responsible for the induction of P-450ac and elevation of mRNA is involved in all of the three diabetic models investigated.     

Expression of cytochrome P-450 isozymes in the liver of hypophysectomized rats. Evidence for different regulation mechanisms concerning P450IIB and P450IIIA subfamilies

1. Monooxygenase activities have been examined in rat liver to determine the effects of castration and hypophysectomy on cytochrome P-450 species. In adult males, hypophysectomy caused a decrease of total P-450 concentration, aniline hydroxylase, benzopyrene hydroxylase, benzphetamine demethylase, testosterone hydroxylase and imipramine hydroxylase and demethylase activities. The treatment of hypophysectomized animals with human growth hormone or testosterone did not restore the full activity. 2. When probed with antibodies, microsomes from hypophysectomized males and females exhibited an intense reaction with a polyclonal anti-(phenobarbital-induced P-450) which was not observed with a monoclonal antibody of anti-(phenobarbital-induced P-450). 3. These microsomal preparations also reacted with an antibody raised against a developmentally regulated P-450. No sex difference could be detected with this antibody. Furthermore, administration of human growth hormone to hypophysectomized males prevented this immunoreaction. 4. Total RNA has been prepared from the same liver; when probed with cDNAs, no changes occurred in the content in P-450 b/e, PB 24 (a constitutive member of the phenobarbital subfamily) and phenobarbital-inducible mRNA for UDP-glucuronosyltransferase. 5. In contrast, P-450 mRNA induced by pregnenolone 16 alpha-carbonitrile was modulated by hormonal manipulations: lower in females and castrated males than in intact males, increased in both sexes after hypophysectomy. Treatment of hypophysectomized males with human growth hormone abolished this rise in pregnenolone-16 alpha-carbonitrile-induced P-450 mRNA accumulation. Data collected in this study support the assumption that hypophysectomy acts differently on the regulation of various P-450 isozymes and that this regulation clearly does not involve the phenobarbital subfamily of P-450s.     

The dependence on vitamin E and selenium of drug demethylation in rat liver microsomal fractions.

1. The effects of vitamin E deficiency, and of vitamin E and selenium deficiency, on rat liver microsomal aminopyrine demethylase activity were investigated. It was found that, over a wide range of substrate concentrations, the enzyme activity in preparations from deficient animals was significantly lower than that in controls. 2. Addition of antioxidants in vitro, either to the homogenization or to the assay media, was without significant effect on the depressed enzyme activity. Castration and alteration in dietary protein concentration were also without effect. The rate of oxidation of NADPH was however, lower in preparations from deficient animals. 3. Lineweaver-Burk plots of the reciprocal of enzyme activity and substrate concentration showed a higher Km value in preparations from vitamin E-deficient animals, irrespective of whether selenium was present; the Vmax. was unaffected. These parameters were unchanged when antioxidants were added in vitro. Induction with phenobarbitone and 3-methylcholanthrene showed large changes in Km value which, for preparations from vitamin E-deficient animals, was higher than that for corresponding controls. 4. Examination of the synergism between NADH and NADPH as donors of reducing equivalents for aminopyrine demethylation showed that vitamin E and selenium were only minimally involved in the phenomenon. However, both the initial rate and the extent of demethylation were significantly lower in vitamin E- and selenium-deficient preparations and both nutrients were required for the restoration of full activity. 5. The significance of these results is discussed in the light of our working hypothesis.     

Partial purification of pisatin demethylase,a cytochrome P-450 from the pathogenic fungus Nectria haematococca

The plant pathogen Nectria haematococca can demethylate pisatin, a phytoalexin from pea. Demethylation is apparently necessary for virulence on pea and is catalyzed by a microsomal cytochrome P-450 monooxygenase system. The cytochrome P-450 and NADPH-cytochrome P-450 reductase of this system were solubilized with sodium cholate and partially purified by chromatography on blue A-agarose and -aminohexyl-agarose. The reductase was further purified by chromatography on 2,5-ADP-agarose to a specific activity of about 16 moles cytochrome c reduced per min per mg protein. Upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the reductase fraction contained one major band of molecular weight 84,000. The partially purified cytochrome P-450 fraction contained a number of minor bands and three major bands of molecular weights 52,000, 56,000 and 58,000. This fraction lost all demethylase activity during concentration after -aminohexyl-agarose chromatography, so it could not be purified further. The purified reductase could reconstitute demethylase activity of cytochrome P-450 fractions and appeared to be rate-limiting for demethylase activity in microsomal extracts.     

A study of the glycerol phosphate acyltransferase and dihydroxyacetone phosphate acyltransferase activities in rat liver mitochondrial and microsomal fractions. Relative distribution in parenchymal and non-parenchymal cells, effects of N-ethylmaleimide, palmitoyl-coenzyme A concentration, starvation, adrenalectomy and anti-insulin serum treatment.

1. GPAT (glycerol phosphate acyltransferase) and DHAPAT (dihydroxyacetone phosphate acyltransferase) activities were measured both in subcellular fractions prepared from fed rat liver and in whole homogenates prepared from freeze-stopped pieces of liver. 2. GPAT activity in mitochondria differed from the microsomal activity in that it was insensitive to N-ethylmaleimide, had a higher affinity towards the palmitoyl-CoA substrate and showed a different response to changes in hormonal and dietary status. 3. Starvation (48 h) significantly decreased mitochondrial GPAT activity. The ratio of mitochondrial to microsomal activities was also significantly decreased. The microsomal activity was unaffected by starvation, except after adrenalectomy, when it was significantly decreased. Mitochondrial GPAT activity was decreased by adrenalectomy in both fed and starved animals. 4. Acute administration of anti-insulin serum significantly decreased mitochondrial GPAT activity after 60 min without affecting the microsomal activity. 5. A new assay is described for DHAPAT. The subcellular distribution of this enzyme differed from that of GPAT. The highest specific activity of DHAPAT was found in a 23 000 gav. pellet obtained by centrifugation of a post-mitochondrial supernatant. This fraction also contained the highest specific activity of the peroxisomal marker uricase. DHAPAT activity in mitochondrial fractions or in the 23 000 gav. pellet was stimulated by N-ethylmaleimide, whereas that in microsomal fractions was slightly inhibited by this reagent. The GPAT and DHAPAT activities in mitochondrial fractions had a considerably higher affinity for the palmitoyl-CoA substrate. 6. Total liver DHAPAT activity was significantly decreased by starvation (48 h), but was unaffected by administration of anti-insulin serum. 7. The specific activities of GPAT and DHAPAT were lower in non-parenchymal cells compared with parenchymal cells, but the GPAT/DHAPAT ratio was 5--6-fold higher in the parenchymal cells.     

Mutation of C. elegans demethylase spr-5 extends transgenerational longevity

Complex organismal properties such as longevity can be transmitted across generations by non-genetic factors. Here we demonstrate that deletion of the C. elegans histone H3 lysine 4 dimethyl (H3K4me2) demethylase, spr-5, causes a trans-generational increase in lifespan. We identify a chromatin-modifying network, which regulates this lifespan extension. We further show that this trans-generational lifespan extension is dependent on a hormonal signaling pathway involving the steroid dafachronic acid, an activator of the nuclear receptor DAF-12. These findings suggest that loss of the demethylase SPR-5 causes H3K4me2 mis-regulation and activation of a known lifespan-regulating signaling pathway, leading to trans-generational lifespan extension.     

Effects of phenobarbital upon triacylglycerol metabolism in the rabbit.

1. The association between hepatic microsomal enzyme induction and triacylglycerol metabolism was examined in fasting male rabbits (2kg body wt.) injected intra-peritoneally with 50 mg of phenobarbital per kg for 10 days. 2. Occurrence of enzyme induction was established by a significant increase in hepatic aminopyrine N-demethylase activity and cytochrome P-450 content, as well as a doubling of microsomal protein per g of liver and a 54% increase in liver weight. Parallel increments in hepatic gamma-glutamyltransferase (EC 2.3.2.2) activity occurred; these were more pronounced in the whole homogenate than in the microsomes, which only accounted for 12.5% of the total enzyme activity in the controls and 17.0% in the animals given phenobarbital. Increased activity of gamma-glutamyltransferase activity was also observed in the blood serum of the test animals. 3. The rabbits given phenobarbital manifested increased hepatic triacylglycerol content and the triacylglycerol concentration of blood serum was also elevated. These changes were accompanied by a significantly enhanced ability of cell-free fractions of liver from the test animals (postmitochondrial supernatant and microsomal fractions) to synthesize glycerolipids in vitro from sn-[14C] glycerol 3-phosphate and fatty acids, when expressed per whole liver. Relative to the protein content of the fraction, glycerolipid synthesis in vitro was significantly decreased in the microsomes, presumably consequent upon the dramatic increase in their total protein content, whereas no change occurred in the postmitochondrial supernatant, possibly due to the protective effect of cytosolic factors present in this fraction and known to enhance glycerolipid synthesis. 4. Microsomal phosphatidate phosphohydrolase accounted for 85% of the total liver activity of this enzyme and its specific activity was 20-fold higher than that of the cytosolic phosphatidate phosphohydrolase (EC 3.1.3.4), when each was measured under optimal conditions. A significant increase in the activity of both enzymes per whole liver occurred in the rabbits given phenobarbital. A closer correlation between hepatic triacylglycerol content and and microsomal phosphatidate phosphohydrolase, as well as the above observation, suggest that this, rather than the cytosolic enzyme, may be rate-limiting for triacylglycerol synthesis in rabbit liver. 5. Significant correlations were observed between the various factors of hepatic microsomal-enzyme induction (aminopyrine N-demethylase and gamma-glutamyltransferase activity as well as cytochrome P-450 content) and hepatic triacylglycerol content, suggesting that that microsomal enzyme induction may promote hepatic triacylglycerol synthesis and consequently hypertriglyceridaemia in the rabbit.     

UDP-glucuronosyltransferase, epoxide hydrolase and glutathione S-transferase activities in the liver of diabetic mice

The activities of UDPglucuronosyltransferase, microsomal epoxide hydrolase and cytosolic glutathione S-transferase were measured in the liver of spontaneously (db/db and ob/ob) or streptozotocin-induced diabetic mice. An important (2-3-fold) increase of most phase II activities was observed in streptozotocin-treated animals, whereas slighter changes were detected in spontaneously diabetic animals. The latter also exhibited physico-chemical modifications of the liver microsomal membranes, as shown by the temperature-induced variations of epoxide hydrolase activity.