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.     

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.     

Isolierung und Rekonstitution des Alkan-Monooxygenase-Systems der Hefe Lodderomyces elongisporus

It is generally assumed that the first step of alkane degradation in several yeast strains and bacteria is catalyzed by a monooxygenase system containing cytochrome P-450 as the terminal oxidase and a NADPH-cytochrome P-450 reductase as the electron transfer component. To prove this hypothesis both proteins were purified to homogeneity from the microsomal fraction of the alkane-assimilating yeast L. elongisporus and reconstituted in vitro. The cytochrome P-450, having a specific content of 12–17 nmoles/mg protein turned out to be a typical member of this class of hemoproteins with respect to the molecular weight (53 500) and spectral properties. The reductase component with a molecular weight of 79000 contains FAD and FMN as prosthetic groups. The recombination of the hemoprotein and the flavoprotein results in an enzyme system which catalyzes the monoterminal hydroxylation of hexadecane. The activity of the reconstituted enzyme system is remarkably stimulated by nonionic detergents, suggesting the requirement of an additional lipid factor. The possible importance of other electron transfer systems and of substrate transport is discussed with respect ot the function of the alkane monooxygenase system under in vivo conditions.     

Turnover of hepatic cytochrome P-450 in experimental cholestasis

In mechanical experimental chllestasis, hypertrophy of smooth microsomal membranes was observed. In contrast to typical induction, the membranes were deficient in cytochrome P-450. The total cytochrome P-450 content of the liver, however, as determined in the liver homogenate remained unchanged. To clarify the mechanism of the development of cytochrome P-450 deficient membranes in cholestasis, the half life of the heme portion of cytochrome P-450, and the initial rate of synthesis of cytochrome P-450 and b₅ hemes were compared in bile duct ligated rats and in control animals after labeling the heme by injection of the precursor δ-[4-¹⁴C]aminolevulinic acid. The half lives were not significantly different, which eliminates the possibility that selective destruction of cytochrome P-450 has occurred. Depression of cytochromal heme synthesis was not observed. During mechanical cholestasis, the relative cytochrome P-450 deficiency is probably caused by proliferation of components of the endoplasmic reticulum other than the hemoprotein.     

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     

Evidence for and characterization of cytochrome P-450 in Neurospora crassa

Cytochrome P-450 was detected in microsomes and presumably in cytosol of Neurospora crassa, and was found to be inducible by progesterone. In the microsomal fraction cytochrome b5 and NADPH-cytochrome c reductase activities were measurable, too. Cytochrome P-450 of Neurospora crassa is inhibited by SKF-525 A and by inhibitors of ergosterol biosynthesis. After induction of cytochrome P-450 with progesterone 11α-hydroxyprogesterone as one metabolite of progesterone was detected in the culture media as well as in the mycelia. After 42 hours about 70% of progesterone were metabolized.     

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.     

The effect of muscular exercise on the microsomal enzyme system of the rat liver

Summary Regular muscular work — in the form of swimming or running — was studied for its effect on hexobarbital sleeping time and on some components of the hepatic monooxygenase system. Sleeping times became shorter after both kinds of exercise. Thus, the causative factor in this type of enzyme induction is regular physical exercise. Antipyrine elimination was also faster in rats exercised by swimming. Regular exertion elicited a rise in the concentration or activity of some components of the hepatic microsomal monooxygenase system (cytochrome P₄₅₀, cytochrome b₅ and NADPH cytochrome reductase EC 1.6.2.3.). The inducing effect of muscular work appears to be most similar to the phenobarbital type of induction. In the development of this exercise-induced rise of enzymatic activity some as yet unidentified humoral and metabolic changes associated with daily physical training are thought to play a role.     

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.     

Activity and inducibility of cytochrome P-450-1A in the liver of mice with different sensitivity to hepatocarcinogenic effect of o-aminoazotoluene

Specific activity of cytochrome P-450-1a is detected in the liver of 7 inbred mouse strains sensitive (SWR, C3HA, DD, and CBA) and resistant (AKR, CC57BR, and C57BL) to o-aminoazotoluene-induced hepatocarcinogenesis: 7-ethoxyresorufin-O-diethylase and 7-methoxyresorufin-O-dimethylase, induced by benzo(a)pyrene and o-aminoazotoluene. Mice sensitive (CC57BR and C57BL) and resistant (AKR) to P-450-1a induction were resistant to induction of liver tumor and, similarly, mice both resistant (SWR and DD) and sensitive (C3HA and CBA) to P-450-1a induction were sensitive to hepatic tumor induction. Therefore, there was no correlation between inducibility of cytochrome P-450-1a and sensitivity to o-aminoazotoluene-induced hepatocarcinogenesis at the initial stages of chemical carcinogenesis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 8, pp. 201–203, August, 1998     

Effects of inhibition and induction of cytochrome P-450 isozymes on hyperoxic lung injury in rats.

Pulmonary oxygen toxicity most likely results from excessive production of reactive oxygen species. The role of the cytochromes P-450 in this process is controversial because these enzymes have been reported both to enhance hyperoxic lung injury and to protect from the damaging effects of 100% oxygen. We sought to further determine the role of the cytochromes P-450 in hyperoxic lung injury by inhibiting and inducing pulmonary cytochrome P-450 isozymes in rats. Treatment with the cytochrome P-450 inhibitor cimetidine or 8-methoxypsoralen did not improve survival or reduce lung edema in rats exposed to 100% oxygen. The activity of cytochrome P-450IIB1, the major pulmonary cytochrome P-450 isozyme in rats, was clearly inhibited by 8-methoxypsoralen. beta-Naphthoflavone (beta NF), a selective inducer of cytochrome P-450IA1, was administered in two-dose and five-dose regimens. The two-dose regimen produced significant and sustained induction of cytochrome P-450IA1 activity, but survival in these rats was not improved when exposed to 100% oxygen. In rats treated with five doses of beta NF, a small increase in survival time was found from 71.1 +/- 8.7 to 88.0 +/- 20.2 h; however, there was no difference in the induction of cytochrome P-450IA1 activity between this five-dose regimen and the two-dose regimen. The small improvement in survival after five doses of beta NF is thus unrelated to cytochrome P-450IA1 induction. We conclude that neither inhibition of cytochrome P-450IIB1 activity nor induction of cytochrome P-450IA1 activity protects adult rats against hyperoxic lung injury.     

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.     

The induction of cytochrome P-450 in Lodderomyces elongisporus

In the alkane-utilizing yeast strain Lodderomyces elongisporus cytochrome P-450 is induced by aliphatic hydrocarbons and to a lesser degree also by some of their derivatives. Cycloheximide and glucose inhibit the induction process, the former by inhibition of cytoplasmic translation, the latter presumably by catabolite repression. Among the nearly 40 checked compounds tetradecane and 1-tetradecene are the most effective inducers. The branching of the alkyl chain as well as the terminal introduction of electrophilic atoms decrease the induction effect.     

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.     

Hepatic microsomal alterations during chronic trypanosomiasis in the field vole, Microtus montanus

The field vole, Microtus montanus, was used as a model system to evaluate the chronic effects of infection by Trypanosoma brucei gambiense on hepatic mixed-function oxidase activity. At day 28 post inoculation there was a 97% increase in liver wet weight per g body weight. A portion of the increase (21%) was accounted for by tissue edema which occurred after day 14 of infection. Total hepatic cytochrome P-450 content and related total tissue mixed-function oxidase activities were decreased to about 60% of control levels at day 28 post inoculation. The decrease in total tissue mixed-function oxidase activity was partly due to a small decrease in microsomal protein per cell, and partly to a large decrease in cytochrome P-450 concentration in the endoplasmic reticulum. Although the decrease in total liver monooxygenase activity in several substrates roughly paralleled the loss in cytochrome P-450 content, several other microsomal enzyme markers not related to cytochrome P-450 monooxygenation were elevated in proportion to total liver microsomal protein content. The results suggest that in M. montanus during trypanosomiasis, there is proliferation of hepatic cells with normal content of endoplasmic reticulum. Furthermore, there appears to be selective toxicity for hepatic cytochrome P-450 and related monooxygenase activities. This may compromise the animals' ability to metabolize and dispose of other drugs to which the animal may be exposed in the course of infection.     

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.

     

Possible mechanisms responsible for the effect of dimedrol on nonsynthetic biotransformation of xenobiotics in rat liver

The ability of dimedrol to inhibit o-demethylation of paranitroanisole, which in rat liver is catalyzed by cytochrome P-450, is demonstratedin vivo andin vitro. The affinity of dimedrol for cytochrome P-450 is higher than that of other xenobiotics. It is suggested that the effect of dimedrol on the metabolism of paranitroanisole and some other xenobiotics is realized during formation of the enzyme—substrate complexes between these substances and cytochrome P-450. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 9, pp. 301–304, September, 1997     

Differences in the cytochrome P-450 enzymes of sterol C-14 demethylase mutants of Saccharomyces cerevisiae

Summary A number of nystatin-resistant strains of S. cerevisiae have been isolated which are defective in lanosterol C-14 demethylation, a reaction normally catalysed by cytochrome P-450. In this paper two of these strains have been compared and found to have differences in their reduced-CO difference spectra indicating different distortions in the enzyme molecule. Nystatin resistance in the C-14 demethylation deficient SG1 in shown to be determined by a single gene, and a sterol 5,6-desaturase defect does not appear to be required for viability of SG1, was reported for the C-14 demethylase deficient isolate JR4 by Taylor et al. (1983). There are at least two discernable mutant phenotypes for the yeast cytochrome P-450 structural gene which give a C-14 demethylase defect.     

Aldehydic products of lipid peroxidation inactivate cytochrome P-450.

The effects of aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), on the structure of rat liver microsomal membrane and cytochrome P-450 was studied. MDA (15-30 microM) similarly to p-chlormercuribenzoate decreased the cytochrome P-450 content by 50 % and lowered microviscosity of lipid surrounding of the spin label OTMB bound to SH-groups of membrane proteins. OTMB was effectively reduced by K3Fe(CN)6 in microsomes preincubated with MDA (20 (M), but not in native microsomes. HNE (10 microM) decreased the cytochrome P-450 content by 90 %. Reduced glutathione and cysteine (5 mM) prevented the decrease of cytochrome P-450 under influence of both MDA or HNE, whereas cytochrome P-420 formation remains unchanged. MDA and HNE decreased activities of NADPH oxidase and NADPH cytochrome c reductase. HNE increased microviscosity of the OTMB lipid environment. The further increase of HNE concentration did not affect this parameter. Both MDA and HNE increased the absorbance at 420 nm, which indicated inactivation of cytochrome P-450 by changes in hydrophobicity of lipid surrounding. We suggest that HNE and aliphatic aldehydes at low concentrations can enter into hydrophobic environment of cytochrome P-450 binding to its SH-groups, which led to inactivation of cytochrome P-450. At the same time, the modification of membrane surface layer and subsequent decrease of hydrophobicity of cytochrome P-450 environment preceded the binding of MDA to SH-groups of cytochrome P-450 to develop its inactivating effect.     

Comparative analysis of family 1 cytochrome P-450 mRNA expression in human intestinal adenocarcinoma and intact portion of the intestine

The expression of mRNA of proteins involved in the transformations of cytostatics (cytochrome P-450 1A1 and 1B1 isoforms) and genes encoding proteins participating in their regulation (Ah receptor, AHRR and ARNT) in intestinal tumors and intact portions of the intestine were studied. The expression of cytochrome P-450 1A1 increased in poorly differentiated tumors in comparison with its expression in intact portions of the intestine (tumor/intact tissue=1.65). The expression of cytochrome P-450 1B1 was higher in well-differentiated tumors (tumor/intact tissue=1.62). The possibility of practical use of high expression of cytochrome P-450 isoforms in tumors in comparison with intact intestinal tissue is discussed. Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 8, pp. 217–220, August, 2008