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.     

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.     

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)     

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.     

Influence of age-related changes in rodent liver morphology and physiology on drug metabolism--a review

Age-related changes in weight, morphology and physiology of the rodent liver influencing hepatic drug metabolism are reviewed. Next to the changes in liver weight/body weight ratio with age, the spontaneous occurrence of neoplastic and non-neoplastic lesions may be of particular importance. In addition, the decrease in liver blood blow with age diminishes the biotransformation capacity of the total liver. However, the albumin concentration in plasma and drug uptake do not play important roles, since they are unchanged or only slightly lower in old rats or mice. Drugs are generally metabolized by the liver in two phases: the so-called phase I and phase II metabolism. For most drugs, the phase I reaction is an oxidation. This reaction is catalyzed by cytochrome P-450, cytochrome b5 and NADPH-cytochrome c reductase. In microsomes, a decrease with age is generally observed in the cytochrome P-450 concentration and the NADPH-cytochrome c reductase activity, while there is no change in cytochrome b5. In addition, most microsomal drug-metabolizing enzymes decrease with age in male rats but not in females. The changes in enzyme activities in the male and female mouse are more complex. In fact, increases, decreases and no changes were found. Important phase II reactions are glutathione conjugation and glucuronidation; changes in both reactions with age seem to be of minor importance. Studies with hepatocytes isolated from male rats of different ages reveal that the monooxygenase system mediated metabolism of digitoxin and aflatoxin B1 decreases with age. It can be concluded that the observed decrease in the functional capacity of the monooxygenase system greatly determines the decrease in drug metabolism with age. However, it should always be kept in mind that, among others, the age-related changes in drug metabolism in rats are strongly sex dependent, which is not the case in man. Therefore, caution should be exercised in transferring these data to the human situation.     

Effect of Plasmodium yoelii nigeriensis infection and chloroquine on the hepatic mixed function oxidase system of mice

Plasmodium yoelii nigeriensis infection in albino mice significantly altered the hepatic microsomal mixed function oxidase system. Cytochrome P-450 (the terminal monooxygenase) and other monooxygenases, viz. aniline hydroxylase, aminopyrine-N-demethylase and benzo(a)pyrene hydroxylase were significantly lowered while microsomal heme showed 4-fold increase at 80% parasitaemia. Noticeable impairment in the other components like NADH:cytochrome b5 reductase, NADPH:cytochrome c reductase, cytochrome b5 and glucose-6-phosphatase was also observed. Oral treatment of normal and P. y. nigeriensis infected mice with chloroquine (64 mg per kg body weight for 4 days) caused lowering of mixed function oxidase activities which however showed a recovering trend, a week after cessation of treatment.     

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.     

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

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

The effect of 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.     

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.     

The immunocytochemical detection of cytochrome P-450 monooxygenase in the lungs of fetal, neonatal, and adult hamsters

Antibodies against rabbit cytochrome P-450 reductase (reductase), cytochrome P-450 isozyme 2 (P-450 IIB), and cytochrome P-450 isozyme 5 (P-450 IVB) were used to detect homologous enzymes in the developing lung of the Syrian golden hamster. No immunocytochemical labeling was observed on gestational days 11, 12, and 13. On gestational day 14, light immunoperoxidase labeling for reductase and P-450 IIB was observed over cells lining the trachea and cranial portions of lobar bronchi. On gestational day 15, these enzymes were detected in conducting airways at all anatomic levels, and in the media of the pulmonary vein and its branches. Light labeling for P-450 IVB was first observed over cells lining the trachea and lobar bronchi on gestational day 15, but the smallest bronchioles and the media and endothelium of the pulmonary vein did not label for this enzyme until gestational day 16 (neonatal day 1). Type II pneumocytes and the pleural mesothelium first labeled for each of the three enzymes on neonatal day 1. Although the mesothelium no longer labeled for reductase or P-450 IIB in hamsters 3.5 wk old, the other labeling sites persisted in adult hamsters. Because cytochrome P-450 enzymes are associated with the endoplasmic reticulum, an ultrastructural examination of differentiating secretory cells was made to detect its appearance. At each conducting airway level, smooth endoplasmic reticulum was present in the cells 2 d before cytochrome P-450 enzymes could be detected immunocytochemically. The appearance of these enzymes paralleled the development of the hamster lung; they were first present in the trachea and lobar bronchi, then in the bronchioles, and finally in the alveoli.     

Effects of aging and caloric restriction on hepatic drug metabolizing enzymes in the Fischer 344 rat. I: The cytochrome P-450 dependent monooxygenase system

The effects of long-term caloric restriction on the hepatic cytochrome P-450 dependent monooxygenase system were investigated in the 22-month-old Fischer 344 rat. Caloric restriction decreased the age-related changes in hepatic testosterone metabolism, which are associated with demasculinization of the liver. Caloric restriction also increased hepatic microsomal testosterone 6 beta-hydroxylase, lauric acid 12-hydroxylase and 4-nitrophenol hydroxylase activities over corresponding values in both ad libitum fed 22-month and 60-day-old control male rats. This suggests that cytochrome P-450 isozymes, P-450 pcn1&2, P-452 and P450j may be induced by caloric restriction. Such changes in cytochrome P-450 isozyme profiles could result in altered carcinogen activation, radical formation or drug detoxication in the calorically restricted rat.     

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.     

Changes of the level of cytochrome P-450 and NADPH-cytochrome P-450 reductase in rats with hereditary degeneration of the retina

In animals with hereditary degeneration of the retina the level of cytochrome P-450 in the brain microsomal fraction is found to be higher than that in healthy animals. In rats with hereditary degeneration of the retina the activity of NADPH-cytochrome P-450 reductase is unchanged in all tissues examined except for the retina, where it is markedly higher than in healthy animals on postnatal day 90. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 3, pp. 259–261, March, 1994     

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     

In vivo measurement of velocity profiles in arterioles

Measurements of velocity profiles in arterial microvessels (20–103 μ in diameter) of rat mesentery and m. creamaster showed that bloodflow regimen under natural conditions can be similar to that of Newton fluid or pseudoplastic laminar stream, which indicates significant fluctuations in blood viscosity. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 12, pp. 707–709, December, 2006     

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.     

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     

Halonal is a phenobarbital inductor of the monooxygenase system

Experiments on rats demonstrated induction of the hepatic monooxygenase system with halonal. The effects of halonal and phenobarbital on the contents of cytochrome P-450 and its isoform P-450b+e and on the rate of substrate metabolism were similar. This suggests that enzyme-inducing activity of halonal is determined by the effect of its major metabolite. It cannot be excluded that halonal molecules possess intrinsic enzyme-inducing activity.