The antitoxic function of the liver in D-galactosamine poisoning

Two intragastric administrations of 500 mg/kg of D-galactosamine reduce the RNA and the cytochrome P-45, and b5 content in the hepatic microsomes of rats; inhibit the activity of aminopyrine-N-demethylase, hexobarbital hydroxylase, aniline-p-hydroxylase, and glutathione-S-transferase; reduce the rate of NADP.H and NAD.H oxidation; accelerate inactivation of cytochrome P-450 to cytochrome P-420; reduce the number of points of hexobarbital binding with N-octilamine, though increase the hemoprotein affinity to these substrates. Destruction of the nucleus, endoplasmic reticulum, and mitochondria occurs in the hepatocytes of D-galactosamine poisoned rats.     

Enzym-Induktion in der Hefe Lodderomyces in Gegenwart von n-Alkanen

The utilization of n-alkanes is connected with extensive modifications of the yeast cell, especially of the cytochrome P-450-containing membrane. Beside the cytochrome P-450 the NADPH cytochrome P-450 reductase, the cytochrome b₅, long-chain alcohol and long-chain aldehyde dehydrogenases are induced. The activity of the alkane-hydroxylating enzyme system grows more than the concentration of its terminal oxidase. The induction of the cytochrome P-450 is inhibited by cycloheximide. A low concentration of oxygen in the culture medium amplifies the induction both of the alkane-hydroxylating enzyme system and of catalase and cytochrome oxidase, which are localized in the peroxisomes and mitochondria, respectively.     

Correction of biotransformation of xenobiotics by α-tocopherol in combination with nicotinamide and methionine in the liver damaged by ultrasound

Six day after rat liver sonication, the content of cytochrome P-450, rate of NADPH oxidation, activity of NADPH—cytochrome P-450 reductase, and rate of aniline hydroxylation in the microsomal fraction decrease. After 12 days, the rate of ethylmorphine N-demethylation also decreases. Intragastral administration of methionine, nicotinamide, and vitamin E for 6 and 12 days activates these enzymes and uridine 5′-diphosphate glucuronyl transferase. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 123, No. 4, pp. 420–423, April, 1997     

Characterization of the cytochrome P-450 monooxygenase system in nonciliated bronchiolar epithelial (Clara) cells isolated from mouse lung

The nonciliated bronchiolar epithelial (Clara) cell of the mouse is highly susceptible to toxicants that undergo metabolic activation, presumably because this cell type has high levels of cytochrome P-450 monooxygenases. As a first step in further defining the role of Clara cells in pulmonary xenobiotic activation and detoxication, we have isolated Clara cells (75 to 80% purity) and characterized them morphologically and biochemically. The identity of Clara cells, confirmed by transmission electron microscopy, was based on several features, including abundant agranular endoplasmic reticulum, large mitochondria, and dense secretory granules. Immunocytochemistry of isolated mouse cells showed that the majority were positive with antibodies against three major components of the pulmonary cytochrome P-450 monooxygenase system, cytochrome P-450 isozymes 2 (IIB), 5 (IVB), and NADPH cytochrome P-450 reductase, purified from rabbit lung. The isolated cells also showed a positive reaction with an antibody against the cytochrome P-450 isozyme that is active in the stereoselective metabolism of naphthalene, cytochrome P-450 mN (mN). Immunocytochemistry using the antibody against cytochrome P-450 isozyme 6 (IA1), purified from rabbit lung, showed no reaction in the isolated cells. The presence of intact cytochrome P-450 protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots of homogenates of isolated cell preparations. The N-demethylation of benzphetamine and epoxidation of naphthalene occurred at easily measurable rates in incubations of isolated Clara cells. In contrast, diols, quinones, and monohydroxylated benzo(a)pyrene metabolites, analyzed by high performance liquid chromatography, were undetectable in extracts of Clara cells incubated with 3H-labeled substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of hydrogen peroxide generation in cultured endothelial cells.

Endogenous hydrogen peroxide (H2O2) release from aortic endothelial cells was studied in the presence of antioxidant enzyme inhibitors, mitochondrial inhibitors, a microsomal cytochrome P-450 inhibitor, and after oxidative stress induced with H2O2 or menadione. Extracellular H2O2 generation was determined spectrofluorometrically using 3-methoxy-4-hydroxy phenylacetic acid, and intracellular H2O2 production (in or near peroxisomes) was measured indirectly using aminotriazole, which inactivates catalase in the presence of H2O2. Extracellular H2O2 release was 0.079 +/- 0.005 nmol/min/mg protein in Hanks' balanced salt solution, was constant during a 120-min incubation period, and was not affected by the cell passage number. The half-life for catalase inactivation with aminotriazole was 23 min. Inhibition of catalase, glutathione reductase, or gamma-glutamylcysteine synthetase did not change the rate of extracellular release of H2O2. Furthermore, inhibition of the mitochondrial respiratory chain (rotenone, antimycin A) or microsomal cytochrome P-450 (8-methoxypsoralen) did not change extracellular H2O2 release or intracellular H2O2 production (at peroxisomes) by endothelial cells or cells in which glutathione reductase was inactivated. When the cells were exposed to exogenous H2O2 (30 microM), extracellular H2O2 was scavenged primarily by the glutathione redox pathway. Exogenously added H2O2 (100 microM) changed intracellular H2O2 production (in or near peroxisomes) only when the glutathione redox cycle was inactivated. Menadione (20 microM), which undergoes intracellular redox cycling, increased extracellular H2O2 release almost 4-fold to 0.3 nmol/min/mg protein. Furthermore, menadione increased peroxisomal H2O2 levels and decreased the half-life for catalase inactivation in the presence of aminotriazole to 13 min. Catalase inhibition increased extracellular H2O2 release during menadione treatment, indicating that H2O2 can diffuse across the plasma membrane during oxidant stress.(ABSTRACT TRUNCATED AT 250 WORDS)     

Administration of testosterone alleviates the constitutive sex difference in rat brain cytochrome P-450

Sex-related difference was observed in the levels of total cytochrome P-450 (P-450) and the mono-oxygenase activity mediated by P-450(b,e), namely, aminopyrine N-demethylase and morphine N-demethylase activity in rat brain microsomes. Male rat brain had higher activity of the above enzymes as compared to the female rat brain. On the other hand, P-450(c,d) mediated 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase activity showed no sex-related difference in rat brain. Administration of testosterone elevated the levels of total P-450, aminopyrine N-demethylase and morphine N-demethylase in female rat brain to levels comparable with that of the male rat brain. No significant change was observed in the levels of 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase and NADPH cytochrome c reductase. All of the above enzyme levels were unaffected in the male rat brain following the treatment with testosterone. These results indicate that testosterone may regulate the forms of cerebral P-450 that are associated with the sex-related difference observed in rat brain.     

Changes in the prolactin-binding capacity of mouse hepatic membranes with development and aging

The objective of this study was to document the developmental changes in the prolactin-binding capacity of hepatic membranes of the female mouse. Prolactin-binding capacity was measured in the microsomal membranes of liver obtained from C3H female mice at various ages. Binding capacity of these membranes remained low until 21 days of age after which it increased and reached the adult levels by 44 days of age. Additional studies were made to observe this parameter in male and female mice during aging. Both female and male mice at 450-470 days of age had values of prolactin binding that were 66% and 79% that of the 77-day-old animals, respectively. A significant increase in membrane lipid microviscosity was observed in animals from both sexes at 450-470 days of age. This was in agreement with earlier studies that showed that prolactin receptors of hepatic membranes are modulated by changes in the membrane lipid microviscosity. These changes suggest that such modifications of cellular membranes are interrelated and that changes in the membrane microviscosity with aging may be a factor modulating cellular responses in older animals.     

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.     

Effects of phospholipid-containing hepatoprotectors on cytochrome P-450-dependent antitoxic function of the liver during experimental toxic hepatitis

Phospholipid-containing hepatoprotectors essentiale and eplir inhibited conversion of cytochrome P-450 into cytochrome P-420 and restored aminopyrine N-demethylase and anilinen-hydroxylase activities of cytochrome P-450 in rats during acute hepatitis induced by CCl₄ and allyl alcohol. The polyphenol phytopreparation legalon did not prevent degradation of cytochrome P-450. Differences in the effects of hepatoprotectors on the impaired antitoxic function of the liver are probably associated with the abilities of essentiale and eplir to provide phospholipids for regeneration of endoplasmic reticulum membranes of hepatocytes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 4, pp. 392–394, April, 1999     

Leukotriene B4 omega-oxidation by human polymorphonuclear leukocytes is inhibited by pyocyanin, a phenazine derivative produced by Pseudomonas aeruginosa.

Human polymorphonuclear leukocytes (PMNL) metabolize the potent chemotaxin leukotriene B4 (LTB4) by omega-oxidation to 20-hydroxyl-LTB4 and 20-carboxy-LTB4. The ability of unstimulated human PMNL to metabolize exogenous LTB4 was found to be inhibited by pyocyanin, a phenazine derivative produced by Pseudomonas aeruginosa, in a dose-dependent manner. 1-Hydroxyphenazine (1-OHP), a metabolite of pyocyanin, was not inhibitory under identical conditions. The initial enzymic step in the conversion of LTB4 is catalyzed by an NADPH-dependent cytochrome, P-450. Reduction of the phenazine derivatives by NADPH was measured spectrophotometrically. Pyocyanin was reduced by NADPH in vitro in a pH-dependent manner, while 1-OHP was poorly or negligibly reduced under similar conditions. Formation of NADP+ was 20.3 +/- 1.8 nmol min-1 for pyocyanin (10 microM) at pH 5.5, compared with 0.6 +/- 0.2 nmol min-1 for 1-OHP (10 microM), while at pH 7.5 a value of 2.2 +/- 1.3 nmol min-1 was obtained for pyocyanin, with no detectable activity for 1-OHP. This indicates that inhibition of LTB4 omega-hydroxylase activity by pyocyanin might be achieved by competition for NADPH. Incorporation of exogenous 5-hydroxyeicosatetraenoic acid by PMNL into lipid pools was not affected by either phenazine derivative. The ability of bacterial pyocyanin to limit the omega-oxidation of LTB4 may have important implications for PMNL LTB4 receptor status and chemotaxis in vivo.     

Subcellular organization of alkane oxidation in the yeast Candida maltosa

After spheroplast lysis and differential centrifugation the alkane monooxygenase system consisting of cytochrome P-450 and the NADPH-cytochrome P-450 reductase of alkane-grown Candida maltosa cells is enriched in the microsomal fraction. This membrane fraction is nearly free of intact mitochondria (cytochrome oxidase) and peroxisomes (catalase), but contains considerable amounts of plasma membrane fractures (azide insensitive, vanadate-sensitive Mg²⁺-ATPase) as demonstrated by biochemical an electron microscopic examinations. By means of sucrose density gradient centrifugation it was possible to separate the cytochrome P-450 containing membranes ( = 1,11 g/cm³) from the plasma membranes ( = 1,18 g/cm³). Therefore the cytochrome P-450 alkane monooxygenase system is most likely localized in the endoplasmic reticulum of the yeast cells. For the following enzymatic steps of terminal alkane oxidation to the corresponding fatty acid a quite different subcellular distribution was observed. The fatty alcohol oxidase and aldehyde dehydrogenase activities are mainly localized in the mitochondrial peroxisomal membrane fraction. During the oxidation of n-alkanes by yeast cells the fatty alcohol should be regarded as an intracellular transport from between the cytochrome P-450 containing endoplasmic reticulum and the sites of its further oxidation in peroxisomes and mitochondria.     

Thymus influences liver functions through hypothalamus-pituitary-gonad axis in rats

The aim of the review is to summarize our recent studies on the influence of the thymus on liver functions and its intermediary pathway in rats. Young adult thymectomized rats were used as a model in the experiments, and either thymic peptides or sex hormones were supplemented to these animals. Liver microsomal cytochrome P-450 and aminopyrine-N-demethylase (ADM) activities were decreased in thymectomized rats, and the change in the male was more significant than that in female rats. An increase of liver malondialdehyde (MDA) and a decrease of liver glutathione (GSH) and superoxide dismutase activity were observed in the female thymectomized rats, but not in the males. Accompanied by the increase of MDA, a decline of membrane fluidity of liver microsomes and mitochondria and a decrease of Ca²⁺ uptake by liver microsomes were exhibited in the female thymectomized rats. Subcutaneous injection of thymic peptides decreased MDA level, and increased GSH content, membrane fluidity and Ca²⁺ uptake by microsomes in the liver of thymectomized rats. On the other hand, male thymectomized rats showed a decrease of hypothalamic luteinizing hormone-releasing hormone (LHRH), plasma luteinizing hormone (LH) and testosterone levels. Subcutaneous injection of testosterone propionate to these animals restored their liver P-450 and ADM activities to normal levels. Female thymectomized rats exhibited a decline of hypothalamic LHRH and plasma estradiol levels. Supplementation of estradiol benzoate reversed the increase of liver MDA in these animals. The data suggest that the thymus may influence liver functions through the hypothalamus-pituitary-gonad axis. Thus, a new ‘thymus-neuroendocrine-liver pathway’ is proposed, which may account for the significance of the thymus in maintaining homeostasis and integrative functions in the body.     

小儿紫绀型先天性心脏病血浆MDA和血液流变学变化

测定３５例青紫型先天性心脏病患儿红细胞、血浆丙二醛（ＭＤＡ）含量、红细胞膜微粘度及血液流变学指标。结果患儿红细胞、血浆ＭＤＡ、膜微粘度、全血粘度、经细胞比积、红细胞聚集指数、刚性指数均显著高于正常组；ＭＤＡ含量，膜微粘度、红细胞刚性指数、聚集指数及全血粘度之间均存在正相关关系。提示患儿红细胞脂质过氧化反应增强，导致膜流动性降低、红细胞变形性差，聚集性增加，可能是血液粘度增加较为主要的原因。     

Zonation of the adrenal cortex. III. Distribution of cytochrome P-450 in the zona glomerulosa of the bovine adrenal cortex.

A microsomal fraction was obtained from the zona glomerulosa of the bovine adrenal cortex. Glucose-6-phosphate activity of the fraction was found to be much lower than that of the liver. Contents of RNA and phospholipids, besides electron microscopic findings, of the fraction also indicate that it is rich in smooth-surfaced endoplasmic reticulum. Distribution of cytochrome P-450 in the zona glomerulosa was studied using various fractions including the microsomal fraction described above. The amount of cytochrome P-450 in mitochondria and that in microsomes were determined to be 0.73 and 0.32 nmoles/mg protein, respectively. The CO-difference spectrum was affected not only by the concentration of added deoxycholate but also by the incubation time after addition. Approximately 40-50% of cytochrome P-450 in the samples were converted to cytochrome P-420 within 20-30 seconds of incubation with deoxycholate.     

Isolation of a human cytochrome P-450 reductase cDNA clone and localization of the corresponding gene to chromosome 7q11.2

We have isolated and sequenced cDNA clones that code for rat and human NADPH dependent cytochrome P-450 reductase. The cDNA coding for the human protein was used to analyse, by Southern blot hybridization, DNA isolated from a panel of 8 independent humanrodent somatic cell hybrids. The results indicate that cytochrome P-450 reductase is encoded by a single gene ( POR ) located on human chromosome 7(pter-q22). Analysis of human metaphase chromosomes by hybridization in situ confirmed the results and refined the localization to 7q11.2. Northern blot hybridization revealed that in human liver the expression of the gene varies by less than 3-fold between different individuals.     

Membrane fluidity of trophoblast cells and susceptibility to natural cytotoxicity

This study examines the relationship between membrane lipid microviscosity and susceptibility of villous trophoblast to lysis by natural cytotoxic cells. Trophoblast-enriched cell suspensions prepared from term human placentae were treated with cholesteryl hemisuccinate (CHS)--a modulator of membrane lipid microviscosity. CHS-treated cells were more susceptible targets for natural lymphocyte cytotoxicity than were untreated controls. In binding experiments, increased binding of lymphocytes to CHS-treated target cells was found. Preincubation with progesterone prevented membrane rigidification by CHS. Progesterone, cortisol, and estriol restored the impaired resistance of CHS-treated trophoblast cells to lysis. We determined microviscosity and progesterone concentration in villous surface membranes, prepared from placentae from idiopathic spontaneous abortions and normal first-trimester pregnancies. An inverse relationship was found between progesterone content and microviscosity of the membranes. Microviscosity of the membranes from abortion placentae was significantly higher (P less than .01) and progesterone concentration was significantly lower (P less than .001) than those in the membranes of normal first trimester placentae.     

Hepatotoxicity of vinyl chloride and 1,1-dichloroethylene. Role of mixed function oxidase system.

Vinyl chloride, an occupational carcinogen, produces acute liver injury in rats pretreated with phenobarbital or Aroclor 1254. Injury appears related to morphologic changes in the endoplasmic reticulum. The degree of injury, as indicated by elevation of serum enzymes derived from the liver, correlates with the magnitude of induction of cytochrome P-450 and its reduction by NADPH. Hepatic injury following 1,1-dichloroethylene exposure differs strikingly from that caused by vinyl chloride and appears to involve plasma membranes, mitochondria, and chromatin and spares endoplasmic reticulum. Induction of cytochrome P-450 appears to protect against 1,1-dichloroethylene but not vinyl chloride.     

Drug metabolism in rats with arthritis induced by 6-sulfanilamidoindazole (6-SAI)

Hexobarbital sleeping time was prolonged and ethylmorphine N-demethylation was inhibited after a single dosage or seven administrations of 6-SAI to old rats. These effects were independent of the development of arthritis. Changes in cytochrome P-450 concentration after 6-SAI treatment were insignificant and thus not responsible for the decrease in drug metabolism.In vitro 6-SAI inhibited ethylmorphine N-demethylation; the inhibition was of a mixed type. 6-SAI bound to cytochrome P-450 and induced a type II spectrum. The magnitude of hexobarbital-induced type I spectral changes was diminished by 6-SAI.It is concluded that 6-SAI inhibits cytochrome P-450-dependent drug metabolism by binding to cytochrome P-450.     

Modulation of Human T-Lymphocyte Plasma Membrane Ca +2 Permeability by Imidazole Antimycotics

The role of cytochrome P-450 in the regulation of plasma membrane Ca⁺² permeability of human peripheral T-lymphocytes by intracellular Ca⁺² was examined. We assessed the effect of imidazole inhibitors of cytochrome P-450 on the intracytoplasmic free Ca⁺² ([Ca⁺²]i) response generated using the microsomal ATPase inhibitor thapsigargin (THG) to deplete the intracellular Ca⁺² stores. Econazole, miconazole and clotrimazole dramatically inhibited the THG mediated increase in [Ca⁺²]i and indud an increase in [Ca⁺²]i themselves. This inhibitory effect was previously observed in other cell systems and was attributed to inhibition of cytochrome P-450 by these agents. However, we evaluated a variety of structurally dissimilar P-450 inhibitors and found that none affected [Ca⁺²]i, indicating that the mechanism of imidazole action does not involve P-450.     

Resistance of different forms of cytochrome P-450 of rat liver to factors destabilizing the microsomal membrane

The effect of factors destabilizing the membrane of the liver microsomes on the spectral properties of cytochrome P-450 (P-448) was investigated in intact rats and rats receiving phenobarbital (PB) or 3-methylcholanthrene (MC). Considerable resistance of microsomes induced by PB and MC to enzymic and nonenzymic peroxidation of polyunsaturated fatty acids of membrane phospholipids was discovered. A clear difference was shown in the sensitivity of cytochrome P-448 and cytochrome P-450 of intact rats and rats receiving PB to in vitro treatment with sodium deoxycholate. The results indicate structural changes in the microsomal membrane during induction by PB and MC, which are two different types of inducers of the monooxygenases of the liver.Institute of Clinical and Experimental Medicine, Siberian Branch, Academy of Medical Sciences of the USSR, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 5, pp. 553–555, May, 1977.