Regulation of heme metabolism during senescence: activity of several heme-containing enzymes and heme oxygenase in the liver and kidney of aging rats

The activities of several heme-containing enzymes plus succinate dehydrogenase, the content of mitochondrial cytochromes, the amount of microsomal cytochrome P-450, and the activity of heme oxygenase, the major enzyme of heme degradation, have been compared in young, mature and senescent rats. Measurements were made in liver, a tissue previously reported to have an age-related decrease in δ-aminolevulinic acid synthetase, and in kidney, a tissue previously reported to have no age-related change in this enzyme, the rate-limiting enzyme of heme biosynthesis (Paterniti, Lin and Beattie, Arch. Biochem. Biophys., 191 (1978) 792–797). The activity of cytochrome oxidase in liver mitochondria did not decrease with age while this activity in kidney mitochondria was highest in animals one year old and decreased in the two-year-olds. By contrast, succinate dehydrogenase of both kidney and liver mitochondria decreased markedly in the aging rats. No age-related change in the content of cytochromes b, c or aa₃ was observed in liver mitochondria; however, a marked age-related decrease in cytochrome aa₃ was observed in kidney mitochondria. Similarly no change in cytochrome P-450 levels was observed in either tissue obtained from aging animals. In the liver, catalase activity increased while in the kidney it decreased in senescent as compared to mature animals. Heme degradation does not decrease with age as the activity of heme oxygenase increased in both liver and kidney of two-year-old rats as compared to one-year-olds. These results suggest that the lower activity of δ-aminolevulinic acid synthetase observed in the aging rat may not be correlated with a decrease in the activity of heme-containing proteins and that the regulation of the heme pool used for the synthesis of various intracellular hemo-proteins may be complex.     

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     

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.     

The damage of the hepatic mixed functional oxygenase system by CCl4: significance of incorporation of 14CCl4 metabolites in vivo

The relation between incorporation of ¹⁴C from ¹⁴CCl₄ into liver lipids and damage to the mixed functional oxygenase system of liver microsomes was investigated in rats of different sex or differently pretreated. Under the respective conditions of pretreatment the ¹⁴C incorporation rate and the relative decrease of the cytochrome P-450 level as the parameters most influenced by CCl₄ were compared with those for untreated female rats. Induction by 3-methylcholanthrene does not after both parameters. Induction by sodium phenobarbital enhances the ¹⁴C incorporation rate as well as the cytochrome P-450 decrease but the latter is more affected. In male rats only the ¹⁴C incorporation rate increased. Vitamin D₃-pretreated rats showed a smaller decrease of cytochrome P-450 but an enhanced ¹⁴C incorporation rate. Therefore, there is no correlation between the cytochrome P-450 decrease and ¹⁴C incorporation rate. Reasons for the lack of that correlation are discussed with regard to the mechanism of liver cell damage by carbon tetrachloride.     

Depression of cytochrome P-450 in mouse liver induced by fractions from Nocardia opaca

We measured the liver cytochrome P-450 content of mice 24 h after they had been injected with the following immunoadjuvants: Nocardia opaca derivatives and peptidoglycans from several bacterial strains. The cell wall fraction was not active, the others diminished liver cytochrome P-450 levels. The dose-response activity varied with the bacterial origin of the peptidoglycans. These findings indicate that the toxicity and efficiency of immunochemotherapeutic protocols can be modified by altering drug metabolism.     

The quantitation of liver cytochrome P450—LM2 mRNA in rabbits of different ages and after phenobarbital treatment

Using very young (neonate or 0 day), 50-, 100-, 300- and 830-day-old rabbits we have studied changes in liver drug-metabolizing activities. Total liver microsomal cytochrome P450 content, benzphetamine N-demethylase and 7-ethoxycoumarin 0-deethylase activities all decline with increasing age. When the rabbits of different age groups were pretreated with phenobarbital, liver microsomal enzyme activities increased in each group as compared with its control, but the total induced activities still decreased with increasing age. Using a specific assay for translatable cytochrome P450-LM₂ mRNA, we show that the age-dependent decrease in control and phenobarbital-induced enzyme activities is due to a decrease in the levels of translatable mRNA specific for cytochrome P450-LM₂. This is the first report on an age-dependent decrease in the level of a specific translatable mRNA. We do not know whether this decrease is due to decreased mRNA synthesis, or increased mRNA degradation.     

Rat model of acute tetracycline hepatosis and its dynamic predictors

It is demonstrated that intraperitoneal administration of tetracycline hydrochloride in a dose of 125 mg/kg leads to the development of acute fatty hepatosis in rats within a 24-h period, by which time the maximum accumulation of lipids and triacylglycerides is observed in the liver. In addition, a direct dependence is established between the severity of fatty hepatosis and a decrease in the cytochrome P-450 content. The cytochrome P-450 content is a dynamic predictor of tetracycline fatty hepatosis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp. 603–605, December, 1994     

Untersuchungen über den zeitlichen Verlauf der Induktion der delta-Aminolävulinsäuresynthetase,des Hexobarbital-abbauenden Enzyms und von Cytochrom-P450 in der Rattenleber nach Phenobarbital

Zusammenfassung Die Aktivität der delta-Aminolävulinsäuresynthetase, des Hexobarbital-abbauenden Enzyms, sowie die Konzentration von Cytochrom P-450 in der Rattenleber wurden in verschiedenen Zeitabständen nach einer einmaligen Injektion von 80 mg/kg Phenobarbital-Natrium bestimmt. Die Aktivität der delta-Aminolävulinsäuresynthetase erreicht ein Maximum bereits 8 Std nach der Verabreichung von Phenobarbital. Die Aktivität des hexobarbitalabbauenden Enzyms und die Konzentration von Cytochrom P-450 steigt bis zur 46. Std nach der Phenobarbitalinjektion an. Es wird hieraus geschlossen, daß nach Phenobarbital neben Cytochrom P-450 noch andere Hämoproteine oder Häm vermehrt gebildet werden.

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Summary The activity of delta-aminolevulinic acid synthetase and of the hexobarbital oxidizing enzyme and the concentration of cytochrome P-450 was estimated in rat liver at different periods after an injection of 80 mg per kg phenobarbital. An increase of the activity of delta-aminolevulinic acid synthetase was demonstrated with a maximum 8 hours after the injection of phenobarbital followed by a decrease to nearly the norm during the next 8 hours. The activity of the hexobarbital oxidizing enzyme and the concentration of cytochrome P-450 in liver microsomes increased more slowly to a maximum 46 hours after the injection of phenobarbital. This observations indicate that phenobarbital may not only induce drug metabolizing enzymes but also may increase the snythesis of free heme or other hemoproteins in the liver.

     

A cytochrome P-450 gene family mapped to human chromosome 19

We have recently isolated a cloned cDNA coding for a cytochrome P-450 of human liver microsomal membranes, which corresponds to a major phenobarbital-inducible cytochrome P-450 of rat liver. This human cytochrome P-450 is encoded by a member of a multigene family. DNA extracted from a panel of 12 independent human-rodent somatic cell hybrids was analysed by Southern blot hybridization with the cloned cDNA. The results indicate that all components of this cytochrome P-450 gene family are located on chromosome 19. Evidence from hybrids derived from an individual carrying a balanced translocation suggests a regional localization of 19p13.2→qter. Analysis of human metaphase chromosomes by in situ hybridization localizes this cytochrome P-450 gene family further to the long arm of chromosome 19 in the region q13.1→qter. We propose the designation P450PB for this locus.     

The effects of cimetidine,ranitidine and famotidine on rat hepatic microsomal cytochrome P-450 activities

It has been questioned whether the interaction of H₂-antagonists with cytochrome P-450 that is observedin vitro is also relevant for thein vivo situation. Until now the possibility that cytochrome P-450 may function with different modes of action has been neglected in this respect. We studied the effect of cimetidine, ranitidine and famotidine on the monoxygenase, the oxidase and the peroxidase action of cytochrome P-450. Biotransformation catalyzed by the monoxygenase and oxidase action of cytochrome P-450 was affected by cimetidine (probably via its ligand interaction with cytochrome P-450), whereas metabolism by the peroxidase mode of action of cytochrome P-450 was hardly influenced. Ranitidine and famotidine (both pharmacodynamically more potent than cimetidine) only slightly affected cytochrome P-450 activities.     

Immunohistochemical localization of cytochrome P-450 in rat brain

The presence of cytochrome P-450 in rat brain was studied by immunohistochemistry, using antibodies to cytochrome P-450 purified from livers of phenobarbital- or 3-methylcholanthrene-treated rats. Immunoreactive nerves were observed only in brain sections incubated with immunoglobulin-G to 3-methylcholanthrene-induced cytochrome P-450. This immunoreactivity was abolished by preabsorption of the antibody with highly purified rat liver cytochrome P-450c, the major cytochrome P-450 isozyme induced by 3-methylcholanthrene, but was not affected by other cytochrome P-450 isozymes induced by phenobarbital, isosafrole or pregnenolone-16-carbonitrile.

The most abundant concentration of nerve fibers with cytochrome P-450 immunoreactivity was observed in the globus pallidus. Immunoreactive fibers were also observed in the caudate putamen, amygdala, septum, ventromedial nucleus of the hypothalamus, medial forebrain bundle, ansa lenticularis, and ventromedial portion of the internal capsule and crus cerebri. Cell bodies with cytochrome P-450 immunoreactivity were observed in the caudate putamen and in the perifornical area of the hypothalamus. The cytochrome P-450 immunoreactive fibers in the globus pallidus and caudate putamen do not appear to emanate from cell bodies in the substantia nigra, since there was no reduction in the density of these fibers after unilateral stereotaxic electrolytic destruction of the substantia nigra (zona compacta and reticulata). Our data suggest that these striatal nerve processes are derived from cell bodies within the caudate putamen itself.

The present results indicate that rat brain contains a form of cytochrome P-450 with antigenic relatedness to the hepatic 3-methylcholanthrene-inducible cytochrome P-450c. This cytochrome P-450 isozyme was detected in brain areas which metabolize morphine and convert estradiol and estrone into catecholestrogens, which suggests an important role for this enzyme in the metabolism of both ex´ogenous and endogenous compounds in brain.     

Antagonism by chloramphenicol of carbon tetrachloride hepatotoxicity: Examination of microsomal cytochrome P-450 and lipid peroxidation

Administration of chloramphenicol early in CCl₄ intoxication prevents lipid peroxidation of endoplasmic reticulum membranes. Conversely, a sulfamoyl analog, Tevenel, was ineffective in preventing the lipoperoxidative process. Likewise, in an in vitro microsomal system chloramphenicol inhibited a lipid peroxidation process and Tevenel did not. However, both compounds bind to cytochrome P-450. Chloramphenicol did not maintain cytochrome P-450 levels after CCl₄ administration nor did it depress cytochrome P-450 levels in untreated animals. The data obtained indicate that chloramphenicol may prevent lipid peroxidation either by inhibiting CCl₄ metabolism or by acting as a free radical sequestering agent.     

Factors involved in the down-regulation of cytochrome P450 during Listeria monocytogenes infection

The activation of host defense mechanisms has been shown to cause a depression in hepatic cytochrome P450-mediated metabolism in rodents and humans. In a previous study, it was demonstrated that the Gram-positive bacteria Listeria monocytogenes causes a down-regulation of hepatic cytochrome P450 and related substrate metabolism as a result of a pretranslational depression of apoprotein synthesis. The objectives of this study were to determine whether the effect of listeria on hepatocyte cytochrome P450 involves hepatic nonparenchymal cells and whether the hemolysin, secreted only by hemolytic forms of the bacteria, plays any part in mediating this effect. Total cytochrome P450 levels as well as ethoxyresorufin-O-dealkylase (EROD) and benzyloxyresorufin-O-dealkylase (BROD) activities were significantly reduced in hepatic microsomes isolated from mice infected in vivo for 48 h with 15U listeria, whereas the same dose of the avirulent non-hemolytic M3D strain had no effect. Listeria (15U) significantly depressed hepatocyte EROD and BROD activities after 24 h incubations with liver cell cultures containing hepatocytes and nonparenchymal cells, as the result of both a direct effect on the hepatocyte and an interaction of listeria with hepatic nonparenchymal cells. The M3D strain of listeria had no effect on cytochrome P-450-mediated metabolism in isolated cells, confirming that hemolysin is an essential component of the mechanism responsible for the down-regulation of cytochrome P450 during listeria infections.     

Hepatic gene expression in protoporphyic Fech mice is associated with cholestatic injury but not a marked depletion of the heme regulatory pool

BALB/c Fech(m1Pas) mice have a mutated ferrochelatase gene resulting in protoporphyria that models the hepatic injury occurring sporadically in human erythropoietic protoporphyria. We used this mouse model to study the development of the injury and to compare the dysfunction of heme synthesis with hepatic gene expression of liver metabolism, oxidative stress, and cellular injury/inflammation. From an early age expression of total cytochrome P450 and many of its isoforms was significantly lower than in wild-type mice. However, despite massive accumulation of protoporphyrin in the liver, expression of the main genes controlling heme synthesis and catabolism (Alas1 and Hmox1, respectively) were only modestly affected even in the presence of the cytochrome P450-inducing CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. In contrast, in BALB/c mice exhibiting griseofulvin-induced hepatic protoporphyria with induction and destruction of cytochrome P450, both Alas1 and Hmox1 genes were markedly up-regulated. Other expression profiles in BALB/c Fech(m1Pas) mice identified roles for oxidative mechanisms in liver injury while modulated gene expression of hepatocyte transport proteins and cholesterol and bile acid synthesis illustrated the development of cholestasis. Subsequent inflammation and cirrhosis were also shown by the up-regulation of cytokine, cell cycling, and procollagen genes. Thus, gene expression profiles studied in Fech(m1Pas) mice may provide candidates for human polymorphisms that explain the sporadic hepatic consequences of erythropoietic protoporphyria.     

Dexamethasone modulation of in vivo effects of endotoxin, tumor necrosis factor, and interleukin-1 on liver cytochrome P-450, plasma fibrinogen, and serum iron

Treatment of mice with endotoxin (lipopolysaccharide, LPS) and the two LPS-induced monokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1), caused a depression of liver cytochrome P-450 and related drug-metabolizing enzymes, as well as other acute-phase changes including increase in plasma fibrinogen levels and hypoferremia. However, only IL-1, not TNF or LPS, depressed cytochrome P-450 in cultured hepatocytes, suggesting that the effect of TNF in vivo might be mediated by a second mediator. TNF- or LPS-stimulated monocytes released a factor capable of depressing cytochrome P-450 in cultured hepatocytes. This factor was inhibited by anti-IL-1 antiserum, and its synthesis, like that of IL-1, was inhibited by dexamethasone (DEX). Pretreatment of mice with DEX protected against the depression of liver cytochrome P-450 by LPS or TNF but not by IL-1, suggesting that IL-1 directly depresses cytochrome P-450 and that DEX acts by inhibiting IL-1 synthesis in vivo induced by LPS or TNF. However, DEX did not inhibit two other effects of LPS and TNF in vivo: increase of plasma fibrinogen levels and decrease of plasma iron, suggesting that these might not be mediated by IL-1. Therefore, the effect of DEX in vivo, although supporting the hypothesis that depression of liver cytochrome P-450 by LPS and TNF is mediated by IL-1, indicates the existence of IL-1-independent pathways in the acute-phase response.     

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.     

Effect of cis-hydroxyproline on collagen and other proteins in skin wounds, granuloma tissue, and liver of mice and rats

Acute and chronic pharmacologic effects of cis-hydroxyproline (cis-Hyp) were studied in mice and rats. Given as a single dose intraperitoneally, cis-Hyp is relatively non-toxic and the LD₅₀ approaches that of l-proline.Chronic administration of cis-Hyp (200 mg/kg/day) to mice did not affect breaking strength of dermal wounds or content and specific activity of noncollagenous protein in the liver. Although cis-Hyp produced a significant decrease tn noncollagenous protein content of polyvinyl alcohol sponge induced granuloma tissue, collagen content and rate of synthesis in granuloma tissue and liver were increased significantly.Rats given cis-Hyp (200 mg/kg/day subcutaneously in divided doses every 12 hr) and control animals demonstrated identical hepatic microsomal protein, cytochrome P₄₅₀ and cytochrome b₅ content. Breaking strength of dermal wounds was increased significantly in treated animals. Ultrastructural analysis of rat liver failed to demonstrate any structural alterations of cellular organelles due to cis-hyp treatment.We conclude that chronic in vivo administration of cis-Hyp does not inhibit collagen synthesis or accumulation in normal or repaired tissues and does not decrease the breaking strength of skin wounds. Other effects on protein metabolism could result from nonspecific, chronic toxicity of cis-Hyp.     

Heterogeneity of cytochrome P-450 distribution in the hepatic lobule revealed by carbon tetrachloride

The concentration of cytochrome P-450 was compared with the histological picture of development and elimination of necrotic foci in the liver of CBA mice after inhalation of CCl₄ vapor. After 2 h the cytochrome P-450 content was reduced by 49%, and after 17, 24, and 48 h by 84%; only about 40% of the area of the hepatic lobule underwent necrosis under these circumstances. It is suggested that the selectivity of injury by CCl₄ to the central lobular hepatocytes is connected with the sharp difference in cytochrome P-450 content in different zones of the lobule.Laboratory of Cytology, N. K. Kol'tsov Institute of Developmental Biology, Academy of Sciences of the USSR. Laboratory of Physical Chemistry of Biomembranes, M. V. Lomonosov Moscow State University. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 4, pp. 364–366, April, 1979.     

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.     

Factors involved in the depression of hepatic mixed function oxidase during infections with Listeria monocytogenes

A number of infections are capable of depressing the capacity of the liver to metabolize drugs. We have studied a number of factors which could be involved in the depression of cytochrome P-450 and related drug biotransformation enzymes during infections with Listeria monocytogenes. During the course of the infection, drug metabolism and heme content of hepatic microsomes were depressed but heme oxygenase was elevated. A free radical scavenger alpha-tocopherol did not prevent the loss and xanthine oxidase activities did not correlate with the time course of the loss. Infections in susceptible (balb/c) mice produced a larger loss in drug metabolism than in resistant (C57BL/6) mice, and an avirulent strain of the bacteria was without effect. A preparation of hemolysin isolated from Listeria monocytogenes produced a dose-dependent loss of cytochrome P-450 in isolated hepatocytes. These experiments indicate that the loss of drug metabolism during Listeria infections is most likely due to hemolysin released by the bacteria.