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     

Effect of acute and repeated chlordimeform treatment on rat hepatic microsomal drug metabolizing enzymes

The effect of chlordimeform treatment on the hepatic microsomal drug metabolizing enzymes was examined in male and female rats following either acute or repeated treatment. After acute administration of chlordimeform (100 mg/kg, ip one hr prior to sacrifice) differential effects were observed in various parameters of the hepatic microsomal mixed function oxidase system with significant decreases in ethylmorphine metabolism, cytochrome P-450 content, NADPH cytochrome c reductase, and in the spectral binding of hexobarbital and aniline while no changes were found in the metabolism of aniline or p-nitroanisole. Durations of zoxazolamine-induced paralysis and pentobarbital-induced hypnosis were increased significantly after acute chlordimeform administration. Following repeated administration of chlordimeform (75 mg/kg ip for four days) to adult male rats, a decrease was observed in zoxazolamine-induced paralysis time while pentobarbital-induced hypnosis was not altered. Metabolism studies using isolated hepatic microsomal fractions showed a decrease rate of biotransformation of ethylmorphine and aniline while the activity of p-nitroanisole O-demethylase was not changed. No differences were found in cytochrome P-450 levels whereas microsomal spectral binding of hexobarbital was reduced while that of aniline was not affected. Following acute or repeated administration of chlordimeform to adult female rats, decreases in the hepatic microsomal metabolism of aniline, but not ethylmorphine or p-nitroanisole, were observed. Addition of chlordimeform to microsomal suspensions yielded a Type I spectral binding curve.     

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)     

Oxamniquine inhibits metabolism of caffeine, hexobarbitone and antipyrine in vivo in mice

Inhibition of hepatic metabolism of caffeine (as assessed from expiration of (14)CO(2) resulting from N-demethylation of (14)C-labelled caffeine), hexobarbitone (as assessed from sleeping times) and antipyrine (as assessed from expiration of I4CO2 resulting from the oxidation of "C-labelled antipyrine) was studied in male GB-1 mice administered a single SO mg kg-1 oral dose of the schistosomicidal drug oxamniquine. Metabolism of caffeine, catalysed by cytochrome P-4S0 1A2(CYP1A2), was inhibited most, while hexobarbitone and antipyrine metabolism were inhibited to a lesser, though significant, degree. These results indicate a need for further studies to investigate possible clinically relevant inhibition of hepatic drug metabolism by oxamniquine.     

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.     

Induction of tolerance in mice and rats to the effect of endotoxin to decrease the hepatic microsomal mixed-function oxidase system. Evidence for a possible macrophage-derived factor in the endotoxin effect

Daily administration of low, non-lethal doses of bacterial endotoxin to mice and rats has been shown to induce tolerance to the effect of an acute challenge dose of endotoxin to decrease the hepatic microsomal drug metabolizing activity, the level of cytochrome P-450, and to increase heme oxygenase activity. The serum collected at various times after injection of endotoxin into control animals when injected into untreated animals markedly depressed aniline hydroxylase activity, ethylmorphine N-demethylase activity, and the level of cytochrome P-450. Tolerant animals were not affected by the post-endotoxin serum injection, suggesting the decreased activity caused by the serum in untreated animals was probably due to endotoxin contained in the serum. Injection of tolerant mice and rats with supernatant medium obtained from cultures of peritoneal macrophages incubated with 100 micrograms/ml of endotoxin caused a loss of hepatic microsomal drug-metabolizing activity, and a decrease in the level of cytochrome P-450. These results suggest that peritoneal macrophages release a factor in response to endotoxin that mediates the decreased hepatic mixed-function oxidase activity.     

Pertussis toxin-induced alterations of murine hepatic drug metabolism following administration of diphtheria and tetanus toxoids and pertussis vaccine adsorbed.

Administration of pertussis toxin (PT) in combination with diphtheria and tetanus toxoids adsorbed (DT vaccine) or with acellular pertussis vaccine adsorbed and diphtheria and tetanus toxoids (APDT) elicits dose- and time-dependent alterations in hepatic drug metabolism in mice. Cytochrome P-450 (P-450) levels were inhibited more than 50% at 7 days following a single injection of PT mixed with either vaccine. When combined with DT vaccine, 125 ng of PT was required to produce this effect, while as little as 16 ng of PT combined with APDT vaccine inhibited P-450 levels. The inhibition of P-450 levels is similar to that observed after a single injection of diphtheria and tetanus toxoids and pertussis vaccine adsorbed (DTP). Alterations of P-450 levels were accompanied by increased activities of quinone reductase but not with changes in plasma interleukin-6 or tumor necrosis factor levels. Other Bordetella pertussis virulence factors, such as filamentous hemagglutinin, fimbriae and pertactin, were also tested but had no significant effect on hepatic drug metabolism. Endotoxin or preparations containing endotoxin caused alterations in hepatic drug metabolism within 24 h, concomitant with increased interleukin-6 and tumor necrosis factor levels, but these effects had resolved by 1 week. DTP vaccine and preparations containing PT caused a marked induction of gamma interferon coincident with the maximal inhibition of P-450 levels. This effect was not present with DT or APDT vaccine alone, nor with endotoxin or any combination of factors that did not contain PT. These results demonstrate that PT is a necessary component for the sustained effects of DTP vaccine on hepatic drug metabolism and suggest a role for gamma interferon in this process.     

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.     

Age-dependent alterations in the physicochemical properties of rat liver microsomes

Aging results in a significant decline in liver drug metabolism which is largely attributable to changes in the microsomal mixed function oxidase system. For example, the mixed function oxidase system in the livers of senescent rats is characterized by: (1) a reduced cytochrome P-450 content; (2) a decline in the specific activity of NADPH-cytochrome c (P-450) reductase; and (3) a slower rate of ethylmorphine N-demethylation in comparison to young adult animals. Since several factors intrinsic to the microsomes may influence the efficacy of the mixed function oxidase system, e.g. the phospholipid and cholesterol contents, the saturation index of the fatty acids and the fluidity of the membranes, we conducted a physicochemical analysis of liver microsomes isolated from young adult (3-4 months), mature (12-16 months) and senescent (25-27 months) male Fischer rats. Although the microsomal cholesterol content did not change appreciably between maturity and senescence, there was a marked decline in the total phospholipid content. This resulted in a significant increase in the cholesterol/phospholipid ratio, 0.49 to 0.65 between 16 and 27 months of age. The age-related changes in the total phospholipid content were largely reflected in each of the major fractions, i.e. phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine + phosphatidylserine. Small increases in the relative percentages of highly unsaturated fatty acid species were offset by similar decreases in the more frequent and more saturated species as a function of increased age. As a result, the net change in the fatty acid saturation index was probably minimal. However, the increase in the cholesterol/phospholipid ratio most likely contributes to the significant decline in the order parameter of microsomes isolated from old rats which, in turn, may impair the functional capacity of the hepatic mixed function oxidase system.     

Impairment of hepatic cytochrome P-450-dependent monooxygenases by the malaria parasite Plasmodium berghei

The effect of Plasmodium berghei infection on hepatic monooxygenase activities and cytochrome P-450 contents was investigated in mice. NIH/NMRI or A/J mice infected with active P. berghei showed 30-40% decreases in hepatic cytochrome P-450 contents and the ability to metabolize the test substrates, ethylmorphine and benzo(a)pyrene. These decreases were observed during the erythrocytic stage of the infection, but not during the initial exoerythrocytic stage, or after heat-inactivated sporozoites were injected. These results strongly suggest that malaria infections may significantly impair the capacity of the liver to metabolize drugs, carcinogens, and other foreign compounds.     

An assessment of cadmium toxicity on cytochrome P-450 and flavin monooxygenase-mediated metabolic pathways of dimethylaniline in male rabbits.

Cadmium is an environmental pollutant and its effect on the in vitro metabolism of N,N-dimethylaniline (DMA) using male rabbits was investigated. Activities of cytochrome P-450 and FMO-dependent monooxygenases were studied using hepatic microsomes. Following CdCl2 (i.p.) administration (6 mg/kg/day for 6 days), both DMA-N-oxidation and DMA-N-demethylation decreased by 86%. The effects of CdCl2 on the phenobarbitone (PB)-induced form of P-450 were also studied. Intraperitoneal pretreatment of rabbits with PB (5 mg/kg/day for 5 days) increased N-demethylation by 82%, while N-oxidation decreased by 49%. Both reactions decreased significantly on additional treatment with CdCl2. Promethazine (5 mg/kg/day for 5 days) did not produce any change in the activities of either enzyme. The enzymes remained unaffected by CdCl2 treatment in promethazine-pretreated animals thus confirming its role as a hepatoprotective agent.     

Phenotyping cytochromes P450 with monoclonal antibodies

Monoclonal antibodies (MAbs) to cytochrome P-450 isozymes can be used to phenotype tissues for epitope-specific cytochrome P-450 content. MAbs that inhibit specific cytochrome P-450 dependent drug or carcinogen reactions are useful tools for quantitative measurement of the individual or classes of cytochromes P-450 that catalyze these reactions. This method has been applied successfully to animal as well as human tissues. Radioimmunoassays based on MAbs have been developed and provide a rapid and efficient means for detecting cytochromes P-450 independent of functional enzyme activity. In addition, MAbs coupled to a Sepharose support can be used to immunopurify cytochromes P-450 in a procedure that is more rapid and efficient than conventional purification schemes. MAbs add a new dimension to analyses of cytochrome P-450 multiplicity and will find numerous applications in elucidation of the relationship between cytochrome P-450 phenotype and carcinogen or drug metabolism.     

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.     

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.     

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.     

Effects of thalidomide,cytochrome P-450 and TNF-alpha on angiogenesis in a three-dimensional collagen gel-culture

The anti-angiogenic effects of thalidomide were examined in mouse aortae grown in a three-dimensional collagen gel-culture. In our in vitro model, (+/-)-thalidomide and (-)-thalidomide exhibited no anti-angiogenic effects. On the other hand, when the culture was treated with thalidomide plus cytochrome P-450, both types of thalidomides significantly inhibited angiogenesis. Co-administration of 100 microg/ml thalidomide plus 200 microg/ml cytochrome P-450 inhibited angiogenesis more strongly than thalidomide plus cytochrome P-450 at other concentrations (10 microg/ml + 200 microg/ml and 100 microg/ml + 20 microg/ml). To study the relation between the anti-angiogenic effect and TNF-alpha, we also evaluated the concentration of TNF-alpha in the culture medium. We found that the concentration of TNF-alpha was correlated to the strength of the anti-angiogenic effect. The inhibition of angiogenesis by thalidomide and cytochrome P-450 takes place through a suppression of TNF-alpha and involves the metabolism of the thalidomide.     

Cyclosporine A and Tacrolimus: In vitro investigations on the differential interactions with the cytochrome P450 system in rat and human liver

Species differences in the interactions of cyclosporine A (CSA) and tacrolimus (TAC) with the cytochrome P450 (CYP) system in male rat and human liver were investigated in vitro by assessing effects on a series of model reactions for different CYP isoforms.

CSA and TAC concentration dependently inhibited ethoxyresorufin O-deethylation, ethoxycoumarin O-deethylation and pentoxyresorufin O-depentylation and 7- and 17-testosterone hydroxylation (TH) activities in both species. In rat liver no effect of CSA was seen on ethylmorphine N-demethylation and 2- and 6β-TH activities, but an inhibition due to TAC. Both CSA and TAC, however, distinctly decreased ethylmorphine N-demethylation and 2β- and 6β-TH activities in human liver. The same results were seen with 14- and 15β-TH activities. 2-, 16- and 16β-TH activities were only inhibited in human liver with TAC, whereas only in this case 6-TH activity was left unaffected. p-Nitrophenol hydroxylase activity was not influenced by either substance in both species. Thus, CSA mainly interacts in rat with the CYP isoforms 1A, 2A and 2B and in man with the CYP subtypes 1A, 2A, 2B, 2C and 3A. TAC seems to interfere predominantly in rat with the CYP isoforms 2A, 2B, 2C and 3A and in man with the CYP subtypes 1A, 2B, 2C and 3A.

In summary, our results point to distinct species differences in the interactions with the CYP system with both substances, and although from literature CSA and TAC are known to be metabolized mainly by CYP 3A, according to our findings in rat liver CSA seems not to interact with this CYP subtype.     

Role of interleukin-1 in the depression of liver drug metabolism by endotoxin.

Endotoxin-resistant C3H/HeJ mice were used to test the hypothesis that a macrophage product, possibly interleukin-1, might mediate the depression of liver cytochrome P-450-dependent drug metabolism in endotoxin-treated mice. Depression of liver drug metabolism by endotoxin was observed in normal mice (C3H/HeN) but not in C3H/HeJ mice. Serum transfer experiments demonstrated that a serum factor was responsible for the depression of liver drug metabolism. Experiments of passive transfer of peritoneal macrophages showed that this endotoxin-induced factor might be a macrophage product. In vitro experiments showed that endotoxin-stimulated monocytes produced a factor that depressed cytochrome P-450-dependent metabolism in cultured hepatocytes. Homogeneous human monocyte and recombinant interleukin-1 also depressed liver drug metabolism both in vivo and in vitro, suggesting that this macrophage product might be involved in the regulation of liver function by the immune system.     

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 cytochrome P-4503A (CYP3A) activity in the elderly.

Elderly patients exhibit decreased clearance of multiple drugs biotransformed by the hepatic cytochromes P-450. The cytochromes P-450 are a superfamily of enzymes, which comprise a central component of phase I drug metabolism. Distinct isoforms metabolize specific drugs. In human liver microsomes, the glucocorticoid-inducible cytochrome P-450IIIA, CYP3A, catalyzes the N-demethylation of erythromycin. To examine the activity of hepatic CYP3A in elderly males and females, erythromycin N-demethylation was examined, as reflected by the recently described [14C]erythromycin breath test in 24 healthy volunteers, age 70-88. The [14C]erythromycin breath test was measured in normal elderly males and females to: (a) determine persistence of the gender-related dimorphism (evident in younger subjects) of CYP3A activity in the elderly population, (b) examine the effect of % ideal body weight, age, diet, and medication use on the activity of human hepatic CYP3A, and (c) compare breath test results obtained in normal geriatric volunteers with published results obtained in younger subjects, to determine aging-related alterations in CYP3A enzyme activity. Erythromycin N-demethylation varied fivefold among these patients. Similar to earlier studies examining erythromycin N-demethylation in younger subjects, CYP3A activity was found to vary with gender in the geriatric cohort. [14C]Erythromycin N-demethylation at 60 min was 3.14% +/- 0.75 (n = 13) in females and 2.15% +/- 0.77 (n = 11) in males (P = 0.005). In evaluating the role of % ideal body weight and % dietary fat using multivariable linear regression analyses, [14C]erythromycin N-demethylation, was found to decline significantly as % ideal body weight increased (P = 0.001). This was not confounded by gender. [14C]Erythromycin N-demethylation was not related to dietary fat intake (P less than 0.13). [14C]Erythromycin N-demethylation in the elderly volunteers was similar to values reported for subjects aged 20-60. Performance of a new non-invasive test of the human hepatic glucocorticoid-inducible CYP3A in a geriatric cohort suggests that: (a) the gender-related heterogeneity in function of the glucocorticoid inducible human CYP3A persists during normal aging, (b) that the activity of CYP3A may decrease in obesity, and (c) that the activity of CYP3A is stable throughout normal ageing.