Induction Of Microsomal Monooxygenases Research Articles

Role of lipoproteins transporting lipophilic xenobiotics in the induction of microsomal monooxygenases.

We studied the effects of lipopolysaccharide on activity of liver microsomal enzymes against the background of xenobiotics treatment. Against the background of lipopolysaccharide stimulation of macrophages we observed in vivo activation of cytochromes P-450 1A subfamily in liver microsomes with Arochlor 1254, but not induction of cytochrome P-450 2B subfamily with phenobarbital.

Changes in the plasmatic membrane characteristics during microsomal monooxygenase induction in the liver of adult and old rats

The experiments on adult (6–8 months) and old (24–26 months) male Wistar rats have shown that treatment of animals with phenobarbital results in a significant increase in hepatic microsomal enzyme content, plasmatic membrane Na +,K +-ATPase activities and the elevation of hepatocyte membrane potential value. It is presumed that the changes in plasmatic membrane characteristics during microsomal monooxygenase induction are related to the synthesis of specific intracellular factors (invertors). This assumption was verified by the experiments with ‘cellular hybrid’ system (cytosol — plasmatic membranes). Using this cross-systems, it was shown that the hepatocyte cytosol of rats treated with phenobarbital produced Na +,K +-ATPase activity. The extent of Na +,K +-ATPase activation was essentially lower when cytosol derived from old rat hepatocytes was used. The presence of specific factors that activated Na +,K +-ATPase in hepatocyte plasmatic membrane was also discovered in blood serum of induced adult and old rats.

Interactive effects between EBI fungicides (prochloraz, propiconazole and penconazole) and OP insecticides (dimethoate, chlorpyrifos, diazinon and malathion) in the hybrid red-legged partridge

The toxicokinetic interactions between the ergosterol-biosynthesis-inhibiting (EBI) fungicides prochloraz, propicon-azole, and penconazole and the organophosphorus (OP) insecticides dimethoate, chlorpyrifos, and diazinon have been studied in the hybrid red-legged partridge. The inhibition of serum butyrylcholinesterase (BuChE) activity provided a useful biochemical indicator of the generation of the toxic oxon metabolites of these OP insecticides. Birds pretreated with 180 mg/kg prochloraz tended to show a greater inhibition of serum BuChE activity at 1, 4, and 24 h following oral exposure to either of the OPs dimethoate (3 mg/kg) or chlorpyrifos (9 mg/kg) compared to birds pretreated with corn oil. Prochloraz-pretreated birds also showed a tendency toward an increased inhibition at 24 h following dosing with the OP diazinon (4.3 mg/kg) compared to corn oil controls. In the case of dimethoate, the inhibition of serum BuChE activity was significantly greater in treated birds than controls at 24 h. Birds pretreated with the EBI fungicide propiconazole (200 mg/kg) showed a similar inhibition of serum BuChE activity to those pretreated with corn oil following administration of 167 mg/kg malathion. Pretreatment with the EBI fungicide penconazole (200 mg/kg) produced significantly greater depression of serum BuChE activity at 1, 4, and 24 h after dosing with malathion, when compared to corn oil controls. The tendency toward increased inhibition of serum BuChE activity by each of the OPs in prochloraz-pretreated birds was attributed to an increased activation of the compound to its active oxon form as a consequence of induction of microsomal monooxygenases by prochloraz.

The invertor mechanism of changes in the plasma membranes of hepatocytes during induction of microsomal monooxygenases in adult and old rats

Age-specific changes of some parameters of the state of the plasma membrane are studied for genetic induction of enzymes of microsomal oxidation. Changes of the state of plasma membrane of hepatocytes are shown to be associated with the synthesis of a specific intracellular regulator (invertor).

Liver microsomal mono-oxygenase induction in winter flounder ( Pseudopleuronectes americanus) from a gradient of sediment PAH concentrations at Sydney Harbour, Nova Scotia

Winter flounder (Pseudopleuronectes americanus) were sampled in summer 1989 and 1990 in Sydney Harbour, Nova Scotia, along a gradient of sediment polynuclear aromatic hydrocarbon (PAH) concentrations arising from industrial contamination; an uncontaminated bay nearby was used as a reference site. Hepatic mono-oxygenase activity, estimated by measurements of catalytic activity and by immunochemical analysis of cytochrome P-450 1A, increased with sediment PAH concentrations; the extent of induction between the reference and the most contaminated sites ranged up to 50-fold. Cyanoethoxycoumarin O-de-ethylase activity was at least as sensitive an indicator of mono-oxygenase induction as ethoxyresorufin O-de-ethylase or benzo(a)pyrene hydroxylase, and all three enzyme activities were well correlated with cytochrome P-450 1A concentrations and with sediment PAH concentrations.

Alteration of liver microsomal monooxygenases and substrate competition with aniline hydroxylase from rats chronically fed low-fat and high-fat-containing alcohol diets.

Male Sprague-Dawley rats fed ethanol (EtOH) 36% of total calories for four weeks in a liquid diet containing either 34% (HF) or 12% (LF) of calories as fat were studied with respect to induction of microsomal monooxygenases (MFO) and substrate competition with EtOH-inducible aniline hydroxylase. The specific activity and turnover of aniline hydroxylase were induced to similar extents by HF-EtOH and LF-EtOH diets. Whereas, both LF-EtOH and HF-EtOH caused a decrease in the turnover of arylhydrocarbon (benzo[a]pyrene) hydroxylase (AHH) and aldrin epoxidase compared to pair-fed (PF) controls, LF-EtOH but not HF-EtOH increased the turnover of ethoxycoumarin and ethoxyresorufin O-deethylase (ECOD and EROD). The increase in ECOD and EROD and the decrease in AHH by EtOH is contrary to the parallel induction of these activities by 3-methylcholanthrene (3-MC) and Aroclor 1254 (Aroclor). Benzo(a)pyrene (BaP) stimulated aniline hydroxylase in the HF-EtOH and PF systems, whereas with LF diet, stimulation was seen only in the EtOH group. Ethoxycoumarin (EC) inhibited aniline hydroxylase by microsomes from EtOH- and pyrazole-treated rats, whereas it stimulated aniline hydroxylase by control microsomes, suggesting that the EC effects were associated with EtOH-inducible cytochrome P-450. Ethoxyresorufin (ER) inhibited aniline hydroxylase in EtOH and PF groups, thus the differential effects of EC were not nonspecific O-deethylase effects. The effects of EtOH feeding on ECOD, EROD, and AHH (ie, substrates for 3-MC-inducible cytochrome P-450) displayed a greater differential between the experimental and control group with the LF- than with the HF-containing diet. The findings suggest that the alteration of certain MFO activities by chronic EtOH ingestion can be modified by the content of dietary fat. Moreover, the competition dynamics of MFO substrates toward EtOH-inducible aniline hydroxylase are altered by EtOH feeding and, in turn, modified by dietary fat.

Comparison of hexachlorobenzene-induced alterations of microsomal membrane composition and monooxygenase activity in male and female rats

The effect of hexachlorobenzene (HCB) on microsomal cytochromes P-450 and b 5, monooxygenase activity and membrane composition was examined in male and female Fischer rats. Cytochrome P-450 was induced more in male than in female animals while cytochrome b 5 was induced only in males. Analysis of patterns of induction of microsomal monooxygenases showed that aminopyrine- N-demethylase activity doubled in both sexes after treatment while aryl hydrocarbon hydroxylase activity was 16 times the control value in the females and 1.5 times in the males. After HCB treatment the phospholipid content of microsomal membranes per gram of liver was increased in both sexes while cholesterol was unchanged. Analysis of the phospholipids (PL) pattern showed that the percentage of sphingomyelin (SPH) decreased significantly (50% of the control value) while phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) did not change. These changes resulted in a reduction of membrane microviscosity and indicate that HCB interferes with the biosynthesis of phospholipids containing choline. Free fatty acid (FFA) content also dropped in both sexes but females were more affected; free arachidonic acid rose in females. HCB induction of microsomal cytochromes and monooxygenases is thus accompanied by marked modifications of membrane composition. Comparing the 2 sexes, HCB showed more pronounced features of ‘PB type’ inducers in males.

Nonadditive interactive effects of polychlorinated biphenyl congeners in rats: role of the 2,3,7,8-tetrachlorodibenzo-p-dioxin receptor.

Administration of 3,3',4,4',5,5'-hexa-,3,3',4,4',5-penta-, and 2,3,3'4,4'5-hexa-chlorobiphenyl to immature male Wistar rats caused a thymic atrophy at high dose levels (1.25, 1.0, and 100 mumol/kg, respectively) and induced the hepatic cytochrome P-448 dependent monooxygenases (benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase) at both high and low (0.25, 0.01, and 5 mumol/kg, respectively) doses. In contrast, 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP) (300 mumol/kg) did not elicit any of these effects but elevated hepatic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cytosolic receptor protein levels (threefold) as previously reported. The effects of hepatic receptor modulation by 2,2',4,4',5,5'-HCBP (300 mumol/kg) on the enzyme induction activities of 3,3'4,4',5-penta-, 3,3'4,4',5,5'-hexa-, and 2,3,3',4,4',5-hexa-chlorobiphenyl were dose-dependent; no interactive effects were observed at high (toxic) doses of these compounds, whereas apparent synergistically increased hepatic microsomal monooxygenase induction activities were noted at the lower submaximal induction doses. It was concluded that the increased responsiveness of the rats was due to elevated hepatic 2,3,7,8-TCDD receptor levels.

Host Plant Induction of Microsomal Monooxygenases in Relation to Organophosphate Activation in Fall Armyworm Larvae

Various host plants were found to induce microsomal monooxygenase activity that activates organophosphorus insecticides in larvae of the fall armyworm, Spodoptera frugiperda (J. E. Smith). Of these plants, cotton and parsley were best inducers of microsomal organophosphate desulfuration and sulfoxidation, respectively. The induction was associated with increased toxicity of numerous phosphorothionate and thioether-containing insecticides. Analyses of internal insecticide revealed that, at various intervals, induced larvae retained less phorate and its oxidative metabolites than did the controls, indicating that induction caused an overall increase in the rate of insecticide metabolism in this insect.

Induction of cytochrome p-450 in congenic c57bl/6j mice by isosafrole: lack of correlation with the ah locus

Isosafrole induction of cytochrome P-450 was compared in congenic strains of C57BL/6J mice, one of which expresses normal levels of the Ah receptor [B6(Ahb)], and another that does not contain a measurable receptor concentration [B6(Ahd)]. Using sucrose gradient analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding, an Ah receptor concentration of 69.1 +/- 3.8 fmol/mg protein was measured in the hepatic cytosol from B6(Ahb) mice, while no receptor could be detected in the cytosol from B6(Ahd) mice. Isosafrole treatment (75 mg/kg X 3 days) increased the total hepatic microsomal cytochrome P-450 content to the same extent in the two congenic strains. The level of microsomal monooxygenase induction in the isosafrole-treated B6(Ahd) mice was greater than that of B6(Ahb) mice for ethylmorphine N-demethylase and isosafrole metabolite-complex formation, the latter a measure of cytochrome P2-450. In the case of 7-ethoxycoumarin O-deethylase only the isosafrole-treated B6(Ahd) mice had elevated microsomal activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) also revealed a similar induction pattern for the two congenic strains, following isosafrole treatment. Thus, the isosafrole treated B6(Ahd) mice produced an equivalent or slightly larger induction of cytochrome P-450 than the B6(Ahb) mice, suggesting that there is no direct role for the Ah receptor in the regulation of these cytochrome P-450 monooxygenase activities by isosafrole.

Differential effects of aging on hepatic microsomal monooxygenase induction by phenobarbital and β-naphthoflavone

The influence of aging on hepatic microsomal monooxygenase induction by phenobarbital (PB) or β-naphthoflavone (BNF) was investigated in male Fischer 344 rats maintained in a constant environment. PB-induced increases in microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity were similar in rats aged 3–5 months (young-adult) and 24–25 months (old), but increases in benzephetamine N-demethylase activity were markedly diminished in the old rats. Separation of hepatic microsomal proteins by sodium dodecylsulfate gel electrophoresis demonstrated that aging decreased the induction by PB of a polypeptide with a molecular weight of 52,500. BNF-induced increases in microsomal cytochrome P-450 and nitroanisole O-demethylase activity were greater in old than in young-adult rats, and BNF induction of 55,000 and 57,000 molecular weight microsomal polypeptides was increased slightly in livers from old rats. The results indicate that age-related effects on monooxygenase induction vary with different inducers of the hepatic microsomal enzyme system.

Substrate specificity of age-related changes in the inducibility of hepatic microsomal monooxygenases in middle-aged rats

Hepatic microsomal monooxygenase induction was investigated in young-adult and middle-aged male Fischer 344 rats. Monooxygenase components and drug metabolism activities were determined in liver microsomes prepared from rats treated with phenobarbital (PB), β-naphthoflavone (BNF) or methyltestosterone (MT) and compared with values from untreated rats. PB and BNF effects on cytochrome P-450 concentration and cytochrome c reductase activity were similar in young-adult and middle-aged animals. However, the extent of cytochrome P-450 induction by MT was less in the older animals. The age-related changes in induction of drug metabolism activities differed with different substrates for the monooxygenase system. In contrast to the inducibility of benzphetamine N-demethylation and aniline hydroxylation, which was dimished in the older rats, the inducibility of nitroanisole O-demethylation was enhanced. The results imply that qualitative changes in the microsomal enzyme system occurred as the animals progresses from young to middle adulthood.

The effect of hepatic microsomal monooxygenase induction on the metabolism and toxicity of the organophosphorus insecticide chlorfenvinphos

The induction of rat liver microsomal monooxygenase by pretreatment of rats with dieldrin affords a 10-fold protection against the acute toxic effects of the organophosphorus insecticide, chlorfenvinphos. Metabolism studies were carried out to confirm that the protection was due to an enhanced rate of detoxification (via oxidative deethylation). At low doses of chlorfenvinphos (2.5 mg · kg −1), dieldrin pretreatment caused minimal changes in the metabolic profiles. However, at a higher dose (13.2 mg · kg −1), giving clinical signs of intoxication in the control animals, the dieldrin pretreated rats produced 5 times more deethylchlorofenvinphos than did the control animals. The results support the conclusion that the effect of enzyme induction on the metabolism of substrates of that enzyme are dosedependent. Alterations in metabolism, therefore, are not an automatic consequence of enzyme induction.

Induction of drug-metabolizing enzymes by phenobarbitone: structural and biochemical aspects.

Two aspects of the induction of microsomal monooxygenases by phenobarbitone have been investigated. First, structural associations between mitochondria and single cisternae of rough endoplasmic reticulum (mitochondria--RER complexes) may operate as functional units in the biosynthesis of cytochrome P-450. This was deduced from (i) studies on the subcellular distribution of the phenobarbitone-induced incorporation of leucine into microsomal proteins including apocytochromes P-450 and (ii) the incorporation of labelled delta-aminolaevulinic acid into the haem prosthetic group of cytochrome P-450. Secondly, in hepatocytes from chick embryo in primary monolayer culture, induction of cytochrome P-450-haemoproteins was markedly influenced by changes in the proliferative activity of hepatocytes. Inducibility of cytochrome P-450 by phenobarbitone and by beta-naphthoflavone was decreased in cultures with 'spontaneous' or experimentally increased proliferative activity of hepatocytes. Treatment with inhibitors of DNA synthesis increased the induction response.

The inhibition of induction of microsomal monooxygenase activity by 1,3-diamino-2-propanol, an inhibitor of ornithine decarboxylase

The p.o. administration of 1,3-diamino-2-propanol, an indirect inhibitor of ornithine decarboxylase, to rats and mice inhibited in a dose-dependent manner the induction of cytochrome P-450, benzo(a)pyrene hydroxylase, and 7-ethoxycoumarin O-deethylase by phenobarbital, β-naphtoflavone or Clophen C, a mixture of polychlorinated biphenyls. The results are consistent with the hypothesis that the induction of ornithine decarboxylase is a necessary step in the induction of microsomal monooxygenases.

Respiration of mitochondria and state of phosphorylation of rat liver adenine nucleotides after repeated administration of 3-methylcholanthrene and phenobarbital

After four injections of phenobarbital (PB) and 3-methylcholanthrene (MC) in olive oil and a single injection of olive oil into rats the acyl-CoA content in the liver (in % of the control) was 73, 167, and 230 respectively. The liver mitochondria of rats receiving injections of oil and MC were characterized by a decrease in the respiration rate in Chance's 3rd metabolic state, but this was abolished by preincubation with carnitine. The blood ketone body level after injection of PB, MC, and oil was 31, 136, and 342% respectively. The phosphate potential was lowered only after injection of oil, when the ATP concentration in the liver was considerably reduced. The AMP concentration in the liver was doubled after injections of PB and oil. Comparison of the data for induction of microsomal monooxygenases of PB and MC leads to the conclusion that acyl-CoA metabolism proceeds in different directions in the two cases.

Induction of liver microsomal monooxygenases in experimental cholestasis

The dependence of expressiveness of microsomal mono-oxygenase induction by phenobarbital upon the amount of binding sites at cytochrome P-450 active center(s) has been studied. The experimental cholestasis is accompanied by accumulation of hydroxylated derivatives of cholesterol, which possess the detergent characteristics and destruct the substrate binding sites in P-450 molecule. The possibility has been demonstrated of phenobarbital induction under conditions when the inducer-monooxygenase primary binding and metabolic steps are not involved. It is assumed that the activation of de novo microsomal protein synthesis is effected by the molecule of phenobarbital itself and not by the products of its primary hydroxylation in the microsomes.

Possible mechanism of induction of liver microsomal monooxygenases by phenobarbital

A study has been made of a model of “consecutive” induction of microsomal monooxygenases following treatment with phenobarbital which was administered after maximal induction by 3-methylcholanthrene. It has been found that in this “consecutive” system, notwithstanding the discontinuation of 3-methylcholanthrene, phenobarbital stimulates the further increase of the CO-binding hemoprotein content in the form of P-448. At the same time there is a considerable gain in the content of low-spin species in hemoprotein without an accompanying rise in the content of its high-spin species. An increase has been observed in the velocity of NADPH-dependent reduction of the CO-binding hemoprotein as well as a pronounced elevation in the activity of aminopyrine N-demethylase. The data presented indicate that the location of the CO-peak of Na 2S 2O 4-reduced hemoprotein is not the criterion of monooxygenase functional specificity; the latter may, possibly, be defined by the relation between the contents of the high-spin and low-spin species of the cytochrome. The possibility has been demonstrated of phenobarbital induction under conditions when the inducer-microsomal hemoprotein primary binding step is not involved, inasmuch as cytochrome P-448 lacks binding sites phenobarbital. In this connection it is assumed that in the mechanism of phenobarbital induction of microsomal monooxygenases the activation of a portion of the genome and subsequent protein synthesis are effected by the substrate itself and not by the products of its primary metabolism in the microsomes.