Rat Liver Enzyme System Research Articles

Impairment of peroxisomal beta-oxidation system by endotoxin treatment.

It is now clear that peroxisomes play a crucial role in many cellular processes, including the beta-oxidation of very long chain fatty acids. Recently, mammalian peroxisomes have been shown to contain the antioxidant enzymes, superoxide dismutase and glutathione peroxidase, in addition to catalase. The presence of these enzymes in peroxisomes suggests that peroxisomes undergo oxidative stress in normal and disease states. As an indicator of the potential impact of an oxidative stress on peroxisomal functions, we evaluated the effect of endotoxin exposure on the beta-oxidation enzyme system in rat liver. Peroxisomes were isolated from liver homogenates by differential and density gradient centrifugations. Endotoxin treatment decreased the beta-oxidation of lignoceric acid to 56% of control values (p < 0.01). The specific activity of the rate limiting enzyme in the system, acyl-CoA oxidase, was decreased to 73% of control values (p < 0.05). Immunoblot analysis revealed a 25% decrease in the 21KD subunit of the acyl-CoA oxidase protein. In contrast, the protein levels of the other enzymes in the pathway, trifunctional protein and 3-ketoacyl-CoA thiolase, were increased by 10 and 15%, respectively. These findings suggest that impairment of beta-oxidation of lignoceric acid by endotoxin treatment is due primarily to a reduction in the activity and protein level of the key enzyme, acyl-CoA oxidase. Oxidative stresses such as endotoxin exposure may have deleterious effects on important peroxisomal functions, such as beta-oxidation of very long chain fatty acids.

In vitro red blood cell assay for oxidant toxicity of petroleum oil

AbstractPetroleum oil has caused hemolytic anemia in birds and mammals. In birds, an oxidant damage on circulating red cells has been identified as the primary toxic effect of ingested petroleum oils. An in vitro red blood cell assay was developed to discriminate among the oxidant activities of different petroleum oils. The assay used rabbit red blood cells with a rat liver enzyme system and formation of methemoglobin was measured as an indicator of oxidant damage to the red cells. The assay was applied to five different petroleum oils and to naphthalene, a petroleum hydrocarbon known to cause hemolytic anemia. Different petroleum oils differed in their capacity to induce methemoglobin formation. Methemoglobin levels varied from 2.9% with Arabian light crude oil to 6.2% with South Louisiana crude oil. Naphthalene induced formation of up to 37% methemoglobin. Naphthalene and the five petroleum oils generated methemoglobin only in the presence of liver enzymes.

Effect of Miso on the Drug-Metabolizing Enzyme System in Rat Liver

Various kinds of miso (paste and freeze-dried, low salt-dried and desalt-dried powders; 20% in basal diet) with or without benzo (a)pyrene (B(a)P; 1, 000ppm in basal diet) in the diet were given to male rats for a period of 14 days, and changes in hepatic drug-metabolizing enzymes (cytochrome P-450, aniline hydroxylase, aminopyrine N-demethylase and glutathione S-transferase) were investigated. Each miso diet without B(a)P tended to increase all of the parameters studied. Furthermore, the miso diets with B(a)P generally elevated even the B(a)P-induced activities of drug-metabolizing enzymes. These data do not preclude the possibility that miso may have defensive action against carcinogenic chemicals by enhancing the activity of detoxification systems.

Studies on the cytochrome P-450 of avocado (Persa americana) mesocarp microsomal fraction

1. Because of the low concentration of cytochrome P-450 in avocado fruit, microsomal fractions were prepared using polyethylene glycol aggregation and low-speed centrifugation, thus avoiding the need for high-speed centrifugation of large volumes of post-mitochondrial supernatant. Recoveries of cytochrome P-450 by this means (0.29 nmol/g tissue) were similar to those after the usual high-speed centrifugation preparation (0.26 nmol/g). The cytochrome P-450 content of tulip bulb (0.30 nmol/g) was similar to that of avocado, but both plant tissues had much lower P-450 contents than did rat liver (13.0 nmol/g). 2. Spectral studies indicate that cytochrome P-450 of avocado mesocarp microsomal fraction binds fewer substrates than does the rat liver enzyme system. Type I binding spectra are given by fatty acids (C7-C14), aryl hydrocarbons (C7-C12), p-chloro-N-methylaniline and N,N-dimethylaniline. Type II binding is seen with inhibitors of mammalian cytochrome P-450 such as metyrapone, and with the imidazole antifungal agents such as clotrimazole. 3. These binding spectra provide a rapid method for identifying possible substrates and inhibitors of avocado cytochrome P-450, and also provide information concerning the nature of the active site of avocado cytochrome P-450. 4. Avocado cytochrome P-450 catalysed the N-demethylation of N,N-dimethylaniline (17.1 nmol/min per nmol P-450) and p-chloro-N-methylaniline (13.1 nmol/min per nmol P-450), and the hydroxylation of lauric (dodecanoic) acid (1.1 nmol/min per nmol P-450).

Effects of chronic exposure of ethylene oxide, especially on heme metabolism

The changes in the cytochrome P-450 enzyme system of rat liver, lung and brain after inhalation of ethylene oxide were studied. When Wistar male rats were exposed to 500 ppm ethylene oxide three times a week for three months, they showed a suppressed gain of body weight. Therefore, the present study was done using pair-fed rats. Haematological examination revealed normocytic and normochromic anemia. Liver and renal functions were normal. The cytochrome P-450 enzyme system in the lung and brain were not affected. However, hepatic cytochrome P-450 and protoheme decreased by 28% and 19%, respectively. Hepatic total microsomal protein, cytochrome b5, NADPH-cytochrome c reductase and NADH-ferricyanide reductase were not affected. The activity of hepatic heme oxygenase showed a 2-fold increase. These results suggest that the heme moiety of hepatic cytochrome P-450 was primarily attacked by exposure of ethylene oxide and the cellular heme balance in liver was altered.

F-244 specifically inhibits 3-hydroxy-3-methylglutaryl coenzyme A synthase

A β-lactone isolated from Scopulariopsis sp. shows a potent inhibition of cholesterogenesis. The structure of this β-lactone, termed F-244, is 3,5,7-trimethyl-12-hydroxy-13-hydroxymethyl-2,4-tetradecadiendioic acid 12,14-lactone. The inhibition site of F-244 in cholesterol synthesis was studied. The growth of Vero cells was inhibited at 6.25–12.5 μg/ml of F-244. The inhibition of growth was overcome by the addition of mevalonate to the culture medium, but not by the addition of acetate. In a rat liver enzyme system, the incorporations of [ 14C]acetate and [ 14C]acetyl-CoA into digitonin-precipitable sterol were 50% inhibited by 0.58 βg/ml of F-244. The incorporation of [ 14C]mevalonate was not affected. Studies on the effects of F-244 on the three enzymes involved in mevalonate biosynthesis demonstrated that the drug specifically inhibits HMG-CoA synthase with IC 50 value of 0.065 μg /ml. The effect of analogs of F-244 on HMG-CoA synthase was also investigated.

Effects of sodium metallochlorophyllins on the activity and components of the microsomal drug-metabolizing enzyme system in rat liver.

Several sodium metallochlorophyllins (Me-Chl-Na), i.e., sodium copper chlorophyllin (Cu-Chl-Na), sodium cobalt chlorophyllin and sodium iron chlorophyllin, decreased the activities of aminopyrine N-demethylase and aniline hydroxylase and the content of cytochrome P-450 in liver microsomes when given intraperitoneally to rats. The lowest levels of these variables were observed 24h after dosing. Me-Chl-Na administration did not modify the substrate-induced spectral changes of cytochrome P-450. Of the kinetic parameters studied in aminopyrine N-demethylation and aniline hydroxylation, only the Vmax values were markedly decreased by Cu-Chl-Na, indicating that the enzyme inhibition was non-competitive. Me-Chl-Na also reduced cytochrome b5 and total heme contents and NADPH-cytochrome c reductase activity in microsomes. Preincubation of liver microsomes from control rats with Cu-Chl-Na did not cause any reduction in the aminopyrine-and aniline-metabolizing enzyme activities and in the cytochrome P-450 content.These findings strongly suggest that the decreases in the activities of the hepatic microsomal drug-metabolizing enzyme system caused by Me-Chl-Na are correlated with the reduction in the contents of microsomal cytochromes P-450 and b5, and are brought about by primary action(s) of Me-Chl-Na on the control mechanism of microsomal hemoprotein levels, not by direct action of Me-Chl-Na on the system.

Effect of subchronic cholestasis on microsomal mixed-function oxidases and the glutathione-conjugating enzyme system in rat liver

Bile duct ligation in male rats for two weeks led to a marked increase in both serum sorbitol dehydrogenase activity and serum bile acid concentration indicating cholestatic liver injury. Furthermore, a rise in the hepatic hydroxyproline level indicating collagen accumulation was observed. As a consequence of these alterations, the hepatic microsomal mixed-function oxidase system was impaired as evidenced by a decrease in cytochrome P-450 content and in the activities of NADPH-cytochrome c reductase and aminopyrine-demethylase. While the hepatic glutathione content remained unaffected, the cytosolic GSH S-transferase activity was clearly suppressed due to subchronic cholestasis.

Evidence for a negative modulating effect of erucic acid on the peroxisomal β-oxidation enzyme system and biogenesis in rat liver

In male rats fed a 30 cal% high-erucic acid (22:1 (13) ( cis)) rapeseed oil diet for 4 weeks a transient, small increase in peroxisomal β-oxidation was observed in liver homogenates and isolated peroxisomes after approximately 1 week. Morphometric analysis revealed a progressive decrease in the average size of the liver peroxisomes (by approx. 20%), as well as their volume fraction (by more than 40%). A negative dose-response was observed when peroxisomal β-oxidation was determined in animals fed rapeseed oil diets with a variable content of erucic acid. When erucic acid was given as subcutaneous injections the peroxisomal β-oxidation decreased, in both liver homogenates and isolated peroxisomes. In contrast to recent proposals, our findings indicate that the amount of erucic acid cannot account for the small increase in peroxisomal β-oxidation observed in the liver of rats adapted to a diet containing 30 cal% rapeseed oil with a high content of this fatty acid. In fact, by the selected criteria erucic acid seems to have a negative modulating effect on both the peroxisomal β-oxidation enzyme system and biogenesis.

Epoxidation reactions catalyzed by rat liver cytochromes P-450 and P-448 occur at different faces of the 8,9-double bond of 8-methylbenz[a]anthracene.

8-Methylbenz[a]anthracene (8-MeBaA) transdihydrodiol metabolites were isolated by reversed-phase and normal-phase HPLCs from incubations of 8-MeBaA with liver microsomes or a reconstituted system containing purified cytochrome P-448 and epoxide hydrolase. Regardless of the enzyme source, the metabolically formed 8-MeBaA trans-3,4- and -5,6-dihydrodiols were found to be enriched in one enantiomeric isomer and differed only in the degree of optical purity. The 8-MeBaA trans-8,9-dihydrodiol formed by liver microsomes from either untreated or phenobarbital-treated rats was enriched with the (+)-enantiomer. In contrast, the 8-MeBaA trans-8,9-dihydrodiol formed either by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted rat liver enzyme system containing cytochrome P-448 and epoxide hydrolase was enriched with the (-)enantiomer. These results indicate that, in catalyzing the formation of 3,4- and 5,6-epoxide intermediates, the interaction with the unsubstituted 3,4- and 5,6-double bonds of 8-MeBaA by the different forms of cytochrome P-450 occur preferentially on the same face of the aromatic plane and they differ only in the degree of stereoselectivity. However, different forms of cytochrome P-450 may interact with different faces of the aromatic plane at the methyl-substituted 8,9-double bond of 8-MeBaA, resulting in the formation of trans-8,9-dihydrodiols enriched in different enantiomeric forms. This demonstrates that different forms of cytochrome P-450 may catalyze the epoxidation reaction preferentially at different sides of the methyl-substituted double bond of a planar polycyclic hydrocarbon molecule. These properties may be used to further classify and to understand the enzyme-substrate interactions of the different forms of cytochrome P-450 in the drug-metabolizing enzyme systems.

Synthesis of delta 5,22-cholestadien-3 beta-ol from delta 5,7,22-cholestatrien-3 beta-ol by a liver enzyme.

The rat liver enzyme system, which catalyzes reduction of delta 5,7,24-cholestatrien-3 beta-ol to cholesterol (delta 5-cholesten-3 beta-ol), converted radiolabeled delta 5,7,22-cholestatrien-3 beta-ol to delta 5,22-cholestadien-3 beta-ol, but not to cholesterol. This enzyme system thus contains membrane-bound delta 7- and delta 24-reductase and no delta 22-reductase. Kinetic and competition studies showed that the enzyme system contains a single delta 5,7-sterol delta 7-reductase, which is not influenced by unsaturation at the delta 22-position of the sterol side chain. The identity of delta 5,22-cholestadienol was established by chromatographic, spectral and chemical analyses. Use of the enzyme system and readily available delta 5,7,22-cholestatrienol provides a facile procedure for specific production of delta 5,22-cholestadien-3 beta-ol in quantity.

ピペリジンN-誘導体のマウスおよびラット肝酵素系によるN-C開裂について

ピペリジンN-誘導体のマウスおよびラット肝酵素系によるN-C開裂について

Conversion of thyroxine to 3,3′,5′-triiodothyronine (reverse-T 3) by a soluble enzyme system of rat liver

The reaction by which thyroxine (T 4) undergoes monodeiodination in the nonphenolic ring to form 3,3′,5′-triiodothyronine (reverse-T 3) has been studied in rat liver. In whole homogenate the conversion of T 4 to rT 3 is obscured by rapid degradation of the product, whereas in cytosol accumulation of rT 3 is linear for 60 min of incubation. In the cytosol system, the rate of rT 3 formation is maximal at pH 8.2, is thiol-dependent, is inactivated by heat, but is not inhibited by anaerobiosis or absence of light. Propylthiouracil is a potent inhibitor of the reaction. The higher pH-optimum of the rT 3-forming system compared to that of T 4-to-T 3 conversion indicates that the former reaction is mediated by an enzyme which is distinct from that controlling 3,5,3′-triiodothyronine (T 3) formation.

Inhibition of in vitro cholesterol synthesis by fatty acids

Inhibitory effect of 44 species of fatty acids on cholesterol synthesis has been examined with a rat liver enzyme system. In the case of saturated fatty acids, the inhibitory activity increased with chain length to a maximum at 11 to 14 carbons, after which activity decreased rapidly. The inhibition increased with the degree of unsaturation of fatty acids. Introduction of a hydroxy group at the α-position of fatty acids abolished the inhibition, while the inhibition was enhanced by the presence of a hydroxy group located in an intermediate position of the chain. Branched chain fatty acids having a methyl group at the terminal showed much higher activity than the corresponding saturated, straight chain fatty acids with the same number of carbons. With respect to the mechanism for inhibition, tridecanoate was found to inhibit acetoacetyl-CoA thiolase specifically without affecting the other reaction steps in the cholesterol synthetic pathway. The highly unsaturated fatty acids, arachidonate and linoleate, were specific inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA synthase. On the other hand, ricinoleate (hydroxy acid) and phytanate (branched-chain acid) diminished the conversion of mevalonate to sterols by inhibiting a step or steps between squalene and lanosterol.

The interaction of folpet with the glutathione-dependent demethylation enzyme system of rat liver

The interaction of folpet with the glutathione-dependent enzyme demethylation of fenitrothion was studied. In a reaction catalyzed by methyltransferase from rat liver in the presence of 0.3 μmol of glutathione, 0.21 μmol of folpet caused a 100% inhibition of the demethylation of 0.2 μmol of fenitrothion. A parallel study was made on the qualitative and quantitative course of the reaction between folpet and glutathione. Phthalimide was formed as the main degradation product of folpet, but the composition of the degradation products of glutathione was dependent on the molar ratio of the reactants. Compounds absorbing at 267 nm (probably derivatives of 2-thiazolidinethiones) were formed when the ratio of glutathione to folpet was 4.5:1. When this ratio was reversed, oxidized glutathione was the main product. From observations on the course of the enzymatic demethylation of fenitrothion and on the degradation of folpet by glutathione, it appears that in the enzyme demethylation system, the reaction between fungicide and glutathione predominates.

Polynucleotides containing 5-mercapto-substituted pyrimidines: inhibition of viral DNA polymerases and the biological implication.

Partially thiolated polycytidylic acids MPC I-III, containing 1.7%, 3.5% and 8.6% 5-mercaptocytidylate units, respectively) inhibited the DNA polymerase of Friend leukemia virus (FLV) in the endogenic reaction as well as in the presence of poly(A)-(dT)14 or poly[d(a-T)] templates; the inhibitory activities were directly related to the percent of thiolation. Various partially thiolated RNA and DNA isolates from Ehrlich ascites cells (containing one 5-mercaptopyrimidine nucleotide/50-100 nucleotide units) also inhibited the DNA polymerases of FLV in the endogenic reaction, and also in the presence of the synthetic templates. The thiolated DNA was the most active, but the thiolated tRNA also showed substantial inhibitory effects, while the thiolated ribosomal RNA was less effective. In a bacterial DNA polymerase (E. coli-K12, using denatured DNA as template), MPC I-III showed no activity. By contrast, MPC III and several partially thiolated nucleic acid isolates significantly inhibited a regenerating rat liver DNA polymerase (I) system; among those tested, the thiolated DNA from Ehrlich ascites cells showed the highest activity. Kinetic analysis of the inhibitory action of this thiolated DNA in the rat liver enzyme system, using as template the corresponding unmodified DNA, demonstrated that the thiolated DNA acts as a competitive inhibitor of the template, with a Ki/Km ratio of 0.5.

Vitamin E, Selenium, and the Membrane-Associated Drug-Metabolizing Enzyme System of Rat Liver

This chapter describes vitamin E, selenium, and the membrane-associated drug-metabolizing enzyme system of rat liver. Polyunsaturated fatty acids are thought to be protected from oxidation by vitamin E, and support for the hypothesis is obtained from the many observations showing that synthetic antioxidants can substitute for α-tocopherol in preventing vitamin E deficiency diseases, and that the autoxidation in vitro of polyunsaturated fatty acids is inhibited by the addition of α-tocopherol. A number of observations have been made that suggest that vitamin E might play a structural role in intracellular membranes. The α-tocopherol functions specifically to prevent the increased heniolytic susceptibility to dialuric acid or hydrogen peroxide that can be demonstrated in erythrocytes derived from vitamin E-deficient rats. In vitamin E-deficient rats, the oxidant-sensitive nonheme iron was replaced by a form of nonheme iron that was not readily oxidizable, and it was further shown that the oxidizable nonheme iron could only be demonstrated when selenium was present in the diet, as well as α-tocopherol.

Sites of X-Irradiation-Induced Damage in the Microsomal Drug Metabolizing Enzyme System of Rat Liver during Development

The effect of x-irradiation at weaning stage on the activity of hexobarbital hydroxylation in male rate liver was studied during development. The increase of activity at the postweaning period was suppressed partially by 200 R irradiation and completely by 400 R, without remarkable change in the microsomal protein content of liver. Activities of microsomal electron transport system were inhibited only slightly at 40 days of age in 400 R irradiated rats, but both content of cytochrome P-450 and spectral change induced by hexobarbital were suppressed considerably. The activity of hexobarbital hydroxylation relative to the magnitude of cytochrome P-450 spectral change was about 60% lower than that of control. Adenosine triphosphatase and inosine diphosphatase activities were rather higher than those of control, while glucose 6-phosphatase activity was lower than that of control at 40 days of age in irradiated rats. Results are discussed in relation to the mechanism of effects of x-irradiation on the differen...

Partial Characterization of the Alkylglycerol Cleavage Enzyme System of Rat Liver

Abstract Subcellular distribution and cofactor requirements of the alkylglycerol cleavage enzyme system of rat liver have been investigated. The bulk of the enzyme system could not be removed from the microsomal fraction by washing procedures. Reduced glutathione (10 mm) increased the extent of the reaction 5-fold. The optimal concentration of ammonium sulfate was 4.8 mm. Ammonium ions increased the extent of the reaction and in addition appeared to activate the enzyme. Several other cations and anions did not stimulate the enzyme. Reduced pteridine was absolutely required for enzyme activity and the optimal concentration was 1.25 mm. The cleavage activity was stimulated 1.2-fold by NAD+ or NADH; NADP+ and NADPH had no effect. In the presence of near optimal concentrations of all known cofactors, rat liver-soluble fraction stimulated the cleavage activity 1.4- to 1.5-fold. The stimulation was produced by a heat-labile, nondialyzable component. The optimum pH for the reaction is approximately 9.0. In the absence of added pyridine nucleotides the long chain products of the cleavage reaction are aldehydes. Fatty acids were formed in the presence of the reduced as well as the oxidized nucleotides in this crude system and fatty alcohols were isolated only in the presence of NADH.

The dietary control of the microsomal stearyl CoA desaturation enzyme system in rat liver

The response of the stearyl CoA desaturation enzyme system of rat liver microsomes to dietary conditions was studied in detail. The activity which had been decreased by starvation was rapidly induced to a very high level on refeeding of the animals. The activity thus induced returned gradually to the steady-state level on continuation of feeding. During this course, the levels of microsomal electron-transport components were not significantly affected, suggesting that the dietary regulation involved the change of mainly the terminal component of the desaturation system, cyanide-sensitive factor. Injection of cycloheximide not only inhibited the induction but also produced a rapid decay of the microsomal desaturation activity with a half-time of 3–4 hr. On continuous refeeding, a rapid increase in the desaturation activity was first detected on the rough microsomal membrane and then on the smooth membrane about 5 hr later. Actinomycn D depressed the increase in the activity on the rough membrane while that in the smooth membrane still continued for about 6 hr after the drug treatment. The significance of the microsomal electron-transport system, with particular attention to cytochrome b 5, is discussed from a viewpoint of the regulatory mechanism of desaturation activity.