Cytochrome P450-dependent Mixed Function Research Articles

Induction of cytochrome P450-dependent mixed function oxidase activities and peroxisome proliferation by chloramine-T in male rat liver

Chloramine-T is an antimicrobial agent recognized for its disinfectant properties widely used in food industry. As an N-chloro-compound, chloramine-T contains electrophilic chlorine and in water it hydrolyses to hypochlorite. Chlorine as hypochlorous acid or hypochlorite is a very reactive chemical which can function as both an oxidant and halogenating agent. Because chloramine-T could be involved in the metabolic activation of drugs, in the present study the effects of chloramine-T on the activities of some drug metabolizing enzymes in rat liver microsomes and peroxisome proliferation were determined in vivo. Rats were treated orally with chloramine-T at doses of 1.25, 2.50, 5 and 10 mg/kg body weight (bw)/day for 6 days. The activities of CYP2E1, CYP1A1/2 CYP2B1/2, CYP3A4 and CYP4A1/2 enzymes significantly increased after treatment with 2.50, 5 and 10 mg/kg bw/day, in a dose-dependent manner as compared to control. This effect was not observed after chloramine-T treatment at dose of 1.25 mg/kg bw/day. Our results suggest that chloramine-T may potentiate the toxicity of many xenobiotics via metabolic activation and/or accumulation of reactive metabolites.

Oxidative stress—implications, source and its prevention

Oxidative stress has been a major predicament of present day living. It has been the product of imbalance between the processes involved in free radical generation and their neutralization by enzymatic and non-enzymatic defence mechanisms. The oxidative stress has been contributed by numerous factors including heavy metals, organic compound-rich industrial effluents, air pollutants and changing lifestyle pattern focussing mainly on alcohol consumption, dietary habits, sun exposure, nuclear emissions, etc. The most common outcome of oxidative stress is the increased damage of lipid, DNA and proteins that resulted in the development of different pathologies. Among these pathologies, cancer is the most devastating and linked to multiple mutations arising due to oxidative DNA and protein damage that ultimately affect the integrity of the genome. The chemopreventive agents particularly nutraceuticals are found to be effective in reducing cancer incidences as these components have immense antioxidative, antimutagenic and antiproliferative potentials and are an important part of our dietary components. These secondary metabolites, due to their unique chemical structure, facilitate cell-to-cell communication, repair DNA damage by the downregulation of transcription factors and inhibit the activity of protein kinases and cytochrome P450-dependent mixed function oxidases. These phytochemicals, therefore, are most appropriate in combating oxidative stress-related disorders due to their tendency to exert better protective effect without having any distinct side effect.

Effects of Sub Chronic Dermal Exposure of Gasoline Blended with Varying Concentration of Benzene on Hepatic Mixed Function Oxidases in Rats

Interaction of chemicals in a mixture, their biotransformation and adverse impact, if any, on environment and workroom atmosphere needs to be evaluated to identify the nature of potential toxic effects elicited by constituent chemical(s) in a mixture. The nature of chemical interaction(s) through which an individual chemical in a mixture undergoes metabolism and/or biotransformation in the target tissue could be helpful in evaluation of its toxic potential. Gasoline is a unique example of such chemical mixture where interaction of aromatics viz., benzene, toluene, ethyl benzene and xylene (BTEX) traverses through a common metabolic pathway involving cytochrome P-450 dependent mixed function oxidases. Travis et al. [1] has quantitatively evaluated that gasoline vapor has an antagonistic effect on benzene metabolism and its biotransformation products. It was reported that 1000 ppm gasoline vapor had the same inhibitory effect as 500 ppm toluene on the metabolism of 290 ppm benzene, indicating that there may be many confounding factors in gasoline which might affect the metabolism of benzene in turn affecting its toxicity at the molecular level. It has been reported that benzene causes myelotoxicity through its bioactive metabolites. Such biotransformation mechanisms involve cytochrome P-450 dependent Phase-I enzymes and the products are further detoxified through Phase-II enzymes. It was therefore considered pertinent to study the impact of gasoline blended with 1, 3 and 5% benzene concentrations. Female albino rats weighing 200–250 gm were exposed by dermal route to gasoline blended with varying benzene concentrations, @ 2 g/kg body wt., six days a week over a period of 90 days. The activities of hepatic mixed function oxidases, viz., aniline hydroxylase and aminopyrene demethylase as a monitor of Phase-I enzymes and Glutathione-S-Transferase for Phase-II enzyme was estimated along with free glutathione levels. Serum creatine kinase was also estimated to assess the extent of muscular dystrophy. The results of this sub chronic study will be discussed in the context of interaction of gasoline with varying concentration of benzene in experimental animals. Keywords: mixed function oxidases, benzene, toxicology, gasoline

Mild hepatitis at recommended doses of acetaminophen in patients with evidence of constitutionally enhanced cytochrome P450 system activity

Acetaminophen (paracetamol) is used throughout the world for pain relief and antipyresis in both children and adults. In many countries, it can be purchased without a medical prescription and it is also a common component of a number of over-the-counter remedies for colds, influenza and the like. Fasting, malnutrition and use of alcohol and/or other drugs are thought to play causal roles in hepatotoxicity associated with recommended doses of acetaminophen although liver injury provoked by therapeutic doses has also been observed in the absence of these factors. We describe two patients who experienced subclinical hepatotoxic reactions after taking acetaminophen at therapeutic doses. The results of an antipyrine metabolism test suggest the presence of constitutional hyperactivity of the cytochrome P450-dependent mixed function oxidative system in both patients. We hypothesize that the latter contributed to the hepatotoxicity and that it may play a role in idiosyncratic reactions to this drug.

Western mosquitofish as a bioindicator of exposure to organochlorine compounds

The evaluation of the alochthonous and cosmopolitan mosquitofish species Gambusia affinis suitability as a bioindicator species and the induction of its liver cytochrome P450-dependent mixed function oxygenase (MFO), measured as the 7-ethoxyresorfin- O-deethylase (EROD) activity, as well as changes in DNA integrity, measured by the Fast Micromethod ®, for the monitoring of organochlorine fresh water pesticide contamination, were the main aims of the study. The test mosquitofish were exposed under laboratory conditions to several doses (0.1, 10 and 100 μg l −1) of lindane in experimental basins for up to 7 days, and a subsequent field study was carried out at five natural ponds in the south-western Istrian peninsula, Croatia, where up to 10 fish were collected from each pond. Results obtained during the studies showed positive correlations between the measured biomarkers in G. affinis liver (EROD activity and DNA integrity status) and lindane (laboratory experiment) or persistent organochlorine pollutant amounts in natural pond sediments (field study). The clear dose–responses of EROD activity and DNA integrity deterioration in G. affinis were recorded after exposure to 0.1–10 μg/l lindane and 96 h exposure to lindane, respectively. The results indicate that the mosquitofish G. affinis, due to its biological–ecological characteristics and the biomarker dose–response, is suitable for the monitoring of fresh water organochlorine pesticide contamination in general and lindane in particular.

Biomonitoring Aquatic Environmental Quality in a Marine Protected Area: A Biomarker Approach

The main aims of the present study, conducted in the framework of the MONIQUA-Egadi Scientific Project, were twofold: first, to make the first step in the development and validation of an ecotoxicological approach for the assessment of marine pollution in coastal environments on the basis of a set of biomarker responses in new sentinel species; and second, to obtain preliminary information on environmental quality in an Italian marine protected area, the Egadi Islands (Sicily). Several cytochrome P450-dependent mixed-function oxidase activities were measured in the following sentinel species: rainbow wrasse Coris julis, gastropod limpet Patella caerulea, and sea urchin Paracentrotus lividus. The results suggest that specimens from the Favignana Harbor may be exposed to P450 inducers, whereas most of the other sites seem to share similar environmental quality. The proposed approach has potential for assessment of environmental quality in marine protected areas.

Antiinflammatory and Analgesic Activities of Two Dicyclic Analogues of Acetaminophen and Its Regioisomer 3-Hydroxyacetanilide

Many of the nonsteroidal antiinflammatory drugs (NSAIDs) currently marketed produce severe gastrotoxic side effects. However, acetaminophen (4-hydroxyacetanilide), which is devoid of gastrotoxicity, is associated with hepatotoxicity and nephrotoxicity. Hepatotoxicity and nephrotoxicity of acetaminophen in man and experimental animals have been attributed to metabolic conversion of the drug into a reactive intermediate by the cytochrome P450 dependent mixed function oxidase system [1]. N-acetyl-p-benzoquinoneimine (NAPQI) is the most likely structure for the ultimate reactive intermediate that depletes cellular glutathione (GSH) and covalently binds to tissue macromolecules [2, 3]. Inasmuch as N-oxidation and attack by tissue nucleophiles on the oxidized aromatic ring of acetaminophen are features that correlate with hepatotoxicity, Harvison and coworkers prepared monomethylated acetaminophen analogues with the expectation that N-oxidation and or reaction with tissue nucleophiles would be hindered [4]. The reported results supported this hypothesis since N-methylation of acetaminophen completely blocks the hepatotoxic reaction [5]. Furthemore, methyl groups ortho to the amide function (2,6-dimethylacetaminophen) were found to block hepatotoxicity [3], whereas 2-methylacetaminophen was found to possess significantly lower hepatotoxicity when compared to acetaminophen [4]. However, the analgesic potency of the above-mentioned analogues seems to parallel their hepatotoxic potential, and both activities parallel the oxidation potentials in this series of compounds. In one sense this is unfortunate, since the authors of these studies had hoped to find a nonhepatotoxic analogue that was active as an analgesic. In research aimed at further understanding the mechanisms of acetaminophen toxicity, it was discovered that the regioisomer 3-hydroxyacetanilide was not hepatotoxic in mice [6 – 8] and also showed analgesic and antipyretic activity. On the other hand, a number of compounds, including the 3,4-dihydrocarbostyril [9], benzoxazolinone [10, 11], pyridinoxazolinone [12], 1,4-benzothiazine [13], pyridin1,4-thiazinone [14], and 1,4-benzoxazinone [15, 16] derivatives, have attracted attention as potential analgesic and or antiinflammatory agents. In view of the above, we considered that it might be of interest to evaluate the analgesic and anti-inflammatory activities of two derivatives of benzoxazine previously synthesized by us [17]. These compounds, since they can be viewed as cyclized versions of substituted 3and 4-hydroxyacetanilides, have frameworks similar to those of acetaminophen.

Liposomal amphotericin B (AmBisome) application and hepatic microsomal enzyme function in the rat.

In the present study we investigated the influence of AmBisome, a lyophilized liposomal amphotericin B formulation on various hepatic cytochrome P450-dependent mixed function oxidases, antipyrine clearance and hepatic glucose-6-phosphatase activity in rats. Animals were treated intravenously for 6 days with AmBisome (15 mg kg-1 body weight). Subsequently, the enzyme activities and cytochrome P450 concentrations were measured ex vivo in hepatic microsomes. Following AmBisome the activity of the microsomal ethoxycoumarin-O-deethylase increased significantly from 333 +/- 77 pmol mg-1 to 459 +/- 125 pmol mg-1, whereas benzpyrenhydroxylase and glucose-6-phosphatase did not change compared with the controls. Accordingly, antipyrine clearance was not affected by AmBisome treatment. Microsomal cytochrome P450 concentrations as well as total microsomal protein concentrations were not changed following treatment with AmBisome and it did not affect either serum levels of liver transaminases or bilirubin. The results show that the application of a high AmBisome dose had no adverse effects on a variety of microsomal hepatic enzymes and the antipyrine clearance in rats. Thus, it seems likely that AmBisome does not seriously impair metabolic liver function.

Isolation from pig liver microsomes, identification by electrospray tandem mass spectrometry and in vitro immunosuppressive activity of a rapamycin tris-epoxide metabolite.

It was demonstrated that rapamycin is metabolized in vitro by pig liver microsomes under the influence of the cytochrome P450-dependent mixed function oxygenase system to a rapamycin tris-epoxide metabolite, as demonstrated by electrospray tandem mass spectrometry. The in vitro immunosuppressive activity of this metabolite was found to be lower than that of rapamycin, probably because the rapamycin effector sector was structurally modified. The effector region of rapamycin was recognized to include the conjugated double bonds of this compound and metabolic reactions affecting this region may change the binding affinity of the rapamycin-FKBP binary complex towards another pharmacological receptor bound to the binary complex. Moreover, metabolic modifications in the effector region are probably able to induce a change in the binding affinities of the rapamycin-FKBP binary complex, including the pipecolic acid moiety and the lactone function of the parent drug.

Cytotoxicity of bile in human Hep G2 cells and in primary cultures of rat hepatocytes.

There has been increasing interest in the development of a hepatocyte bioreactor for the treatment of acute hepatic failure; however, little is known about the effect of hepatocyte byproducts on the viability of the cells in the bioreactor environment. We investigated the effects of increasing concentrations of bile on the growth and viability of the human hepatoma cell line Hep G2 and on the cytochrome P-450 content and dependent mixed function oxidase (MFO) activities, reduced glutathione (GSH) content, and glutathione S-transferase (GST) activity of primary cultures of rat hepatocytes. Our purpose was to determine whether or not it would be necessary to pretreat the plasma from patients with acute liver failure to remove elevated bile concentrations which might be toxic to the hepatocytes in an artificial liver device. Bile was found to inhibit Hep G2 cell growth at concentrations as low as 0.1% and to decrease viability at concentrations above 0.5%. The cytochrome P-450 and GSH contents and the activities of the MFO system and of GST were decreased in the primary cultures of hepatocytes following 24 h treatment with concentrations of bile at and above 0.5%. The MFO activities associated with different cytochrome P-450 isoenzymes decreased to different extents in the presence of bile with the O-dealkylation of pentoxyresorufin being more labile than that of ethoxyresorufin. Our data indicate that elevated bile concentrations are cytotoxic to liver cells, and it may be necessary to pretreat patient plasma to decrease its bile content to protect the cells during the clinical operation of a hepatocyte bioreactor device.

Assessment of Liver Function in Primary Cultures of Hepatocytes Using Diethoxy (5,6) Chloromethylfluorescein and Confocal Laser Scanning Microscopy

A method is presented which can be used to assess the function of hepatocytes in complex culture configurations without disrupting the integrity of the cell environment. It utilises a fluorescent probe for cytochrome P450 dependent mixed function oxidase (MFO) activity diethoxy (5,6) chloromethylfluorescein, and confocal laser scanning microscopy. The MFO activity of individual cells in primary cultures of intact hepatocytes can be detected in situ, and quantified by image analysis. This may be a valuable means of monitoring the effect of culture conditions on the function of bioartificial liver devices, and could be used to assess the need for effective oxygenation of cells, the influence of shear stress and of exposure to patient serum during clinical use.

IN-VITROTESTICULAR BIOACTIVATION OF ACRYLONITRILE

The present work examines the mechanism of testicular toxicity of acrylonitrile. In testicular centrifugal fractions from Sprague Dawley rats, the metabolism of VCN to cyanide (CN−) was highest in the microsomal fraction and required NADPH for maximum activity. This biotransformation of VCN to CN−was characterized with respect to time (30min), microsomal protein concentration (1.5mgml−1), pH (7.5) and temperature (37°C). The Vmaxof the reaction was 65.1pmol CN−mg protein−1min−1and Kmwas 88.6gmmol VCN. Flushing the microsomes with carbon monoxide (CO)(4:1, CO/O2v/v), addition of benzimidazole (1mM) or addition of SKF 525-A (5×10−4M) to incubation mixtures significantly inhibited VCN metabolism by 49%, 54% and 37.4% respectively. Activation of VCN to CN−was markedly increased in microsomes obtained from phenobarbital (PB)-treated rats (128.2%). Addition of glutathione (GSH),L-cysteine,D-penicillamine or 2-mercaptoethanol significantly enhanced the release of CN−from VCN 126%, 247%, 202% and 129% of the control value respectively. These findings indicate that VCN is metabolized in the testis via cytochrome P-450 dependent mixed function oxidase system.

Effects of Cytochrome P450-Interacting Plant Growth Retardants, Fungicides and Related Compounds on Cell Development and Phase-I Biotransformation Capacity of Unicellular Photoautotrophic Green Algae

Fourteen compounds (paclobutrazol, triadimenol, BAS111..W, propiconazole, tetcyclacis, prochloraz, metyrapone, piperonyl butoxide, 1-aminobenzotriazole, fenpropimorph, propham, prohexadione, mepiquat chloride and chlormequat chloride), most of them established inhibitors of cytochrome P450-dependent mixed function oxygenases and used as pesticides, especially plant growth regulators or fungicides, were applied to the non-target organisms Chlorella fusca and Chlorella sorokiniana, two species of photoautotrophic unicellular green algae. The inhibitory properties of these compounds were evaluated by comparing concentration/response relationships for the integral parameters of cell volume growth and cell division with those for the P450-dependent O-dealkylase activity measured in vivo using 7-ethoxycoumarin and 7-ethoxyresorufin as xenobiotic model substrates for phase-I biotransformation. The results obtained indicate a strong algicidal activity for some of these compounds, with differential sensitivity of the order: cell division>O-dealkylation>cell volume increase. EC50 values for cell division of C. fusca ranged from 0·1 to 9·3 μmol litre−1 for prochloraz and paclobutrazol, respectively. Furthermore, in most cases, concentrations around 10 μmol litre−1 limited significantly the capacity for cytochrome P450 O-dealkylase activity.

Involvement of cytochrome P450 3A enzyme family in the major metabolic pathways of toremifene in human liver microsomes

The anti-estrogen toremifen-Fc-1157a or 4-chloro-1,2-diphenyl-1-[4-[2( N, N-dimethylamino) ethoxy]-phenyl]-1-butene is now used for the treatment of breast cancer. This drug is extensively metabolized by cytochrome P450 dependent hepatic mixed function oxidase in man, yielding mainly the N-demethyl-(DMTOR), 4-hydroxy-(4OH-TOR) and deamino-hydroxy- (TOR III) toremifene metabolites. The specific forms of cytochrome P450 involved in these oxidation reactions were examined in 32 human liver microsomal preparations previously characterized with respect to their contents of several known P450 enzymes. Toremifene was demethylated with an apparent K m of 124 μM while it was hydroxylated with an apparent K m of 139 μm. The metabolic rates were 71 ± 56, 13 ± 9 and 15 ± 4 pmol/min/mg microsomal protein, respectively, for DMTOR, 4-OH-TOR and TOR III. The N-demethylation activity was strongly correlated with estradiol 2-hydroxylation ( r = 0.75), nifedipine oxidation ( r = 0.86), tamoxifen N-demethylation ( r = 0.73), testosterone 6β-hydroxylation ( r = 0.78) and erythromycin N-demethylation ( r = 0.84), all these monooxygenase activities known to be supported by CYP3A4 isoform. Furthermore, the CYP3A content of liver microsomal samples, measured by western blot analysis using a monoclonal anti-human CYP3A4 antibody, was strongly correlated with DMTOR formation ( r = 0.80). Compounds such as cyclosporin, triacetyl-oleandomycin and testosterone inhibited the N-demethylation of toremifene metabolism at 80, 89 and 56% vs control, respectively, while the formation of TOR III was inhibited at 78, 82 and 73% vs control and the 4-hydroxylation pathway was inhibited no more than about 50% vs control. Prior incubation of microsomes with 100 μM gestodene, known to be a selective mechanism-based inhibitor of CYP3A4 in the presence of NADPH, led to 76 ± 6 and 76 ± 5% (N = 5 samples) reductions in the N-demethylation and formation of TOR III, respectively. Polyclonal antibody directed against human CYP3A enzymes inhibited formation of DMTOR and TOR III by 60 and 46%, respectively. The metabolism of toremifene was not activated by α-naphthoflavone. Finally, the use of yeasts genetically engineered for expression of human P4501A1, 1A2, 2C9 and 3A4 allowed us to demonstrate that DMTOR and TOR III formations are mediated by P4501A and 3A4 enzymes and by contrast these enzymes are not involved in the 4-hydroxylation pathway. All these results taken together suggest that the major metabolic pathways of toremifene, namely N-demethylation and TOR III formation, are mediated mainly by cytochrome CYP3A4 enzyme in the human liver microsomes owing to the relative hepatic contents in P4501A and 3A.

Comparison of the hepatic effects of coumarin, 3,4-Dimethylcoumarin, dihydrocoumarin and 6-methylcoumarin in the rat

The mechanism of coumarin-induced hepatotoxicity in the rat has been investigated by comparing the effects of coumarin with those of three coumarin derivatives, namely 3,4-dihydrocoumarin (DHC), 3,4-dimethylcoumarin (3,4-DMC) and 6-methylcoumarin (6-MC). Male Sprague-Dawley rats were fed either control diet or diets containing 0.5 or 0.75% coumarin, 0.76% DHC, 0.6 or 0.9% 3,4-DMC or 0.82% 6-MC for 13 wk. The dietary levels of 0.5% coumarin and 0.6% 3,4-DMC, were equimolar (3.43mmol/100g diet), as were the dietary levels of 0.75% coumarin, 0.76% DHC, 0.9% 3,4-DMC and 0.82% 6-MC (5.14mmol/100g diet). All treatments resulted in an increase in relative liver weight, but only coumarin increased plasma alanine aminotransferase and aspartate aminotransferase activities. Morphological examination of liver sections from coumarin treated rats revealed vacuolation of centrilobular hepatocytes and bile duct hyperplasia. Cholangiofibrosis was also observed, particularly in rats given 0.75% coumarin. Treatment with DHC produced no abnormalities, whereas a slight hypertrophy of centrilobular hepatocytes was observed in some 3,4-DMC treated animals and a slight vacuolation of individual hepatocytes was noted in some 6-MC treated rats. DHC, 6-MC and particularly 3,4-DMC treatment resulted in an induction of cytochrome P-450 dependent mixed function oxidase enzyme activities. All treatments induced hepatic GSHS-transferase and γ-glutamyltransferase activities, induction being most marked in rats given coumarin and 6-MC. These results provide further evidence that coumarin-induced hepatotoxicity in the rat is due to the formation of a 3,4-epoxide intermediate.

The effects of inducing agents on cytochrome P450 and UDP-glucuronyltransferase activities in human HEPG2 hepatoma cells

Selective induction in vitro of cytochrome P450-dependent mixed-function oxidase (MFO) and UDP-glucuronyltransferase (GT) activities was observed in the human HepG2 hepatoma cell line. 1,2-Benzanthracene (BA) induced MFO O-dealkylation activities for ethoxyresorufin, methoxyresorufin and benzyloxyresorufin, whereas phenobarbitone (PB) selectively induced pentoxyresorufin O-dealkylation and rifampicin (RIF) selectively induced benzyloxyresorufin O-dealkylation. Antibody inhibition experiments indicated that ethoxyresorufin and methoxyresorufin O-dealkylations were catalysed mainly by the P450 1A subfamily in untreated and BA-induced HepG2 cells, that additional unidentified P450 forms were considerably involved in methoxyresorufin and benzyloxyresorufin O-dealkylations and that the P450 2B subfamily was partially responsible for pentoxyresorufin O-dealkylation in PB-induced cells. Bilirubin GT activity was induced by PB, BA, RIF and dexamethasone, but 1-napthol, morphine and testosterone GT activities were not induced by any of these treatments.

Influence of single and concurrent clofibrate and phenobarbital administration on cytochrome P450‐dependent mixed function oxidase activities and peroxisome proliferation in male rat liver

The influence of both single and concurrent administration of phenobarbital and clofibrate on hepatomegaly, cytochrome P450-dependent mixed function oxidase activities, and peroxisome proliferation in male rat liver have been studied. Both xenobiotics separately increase the liver: body weight ratio and their combined administration results in greater hepatomegaly than either compound alone. Both compounds induce NADPH-cytochrome c(P450) reductase activity and laurate omega- and omega-1-hydroxylase activities, but only phenobarbital induces pentoxyresorufin-O-dealkylase. None of the drug treatments induced microsomal cytochrome b5. Phenobarbital did not cause peroxisome proliferation and inhibited the corresponding clofibrate-dependent proliferation. Taken collectively, our studies have demonstrated that concomitant treatment with phenobarbital and clofibrate are largely permissive with respect to the hepatic mixed function oxidase system but have opposing effects on the phenomenon of peroxisome proliferation in the same tissue.

Alterations in lipid peroxidation, antioxidant enzymes, and carcinogen metabolism in liver microsomes of vitamin E-deficient trout and rat

Feeding rainbow trout for 16 weeks a diet in which the levels of vitamin E were reduced 70-fold resulted in marked depletion (18-fold) of vitamin E levels in liver microsomes from these fish. The susceptibility of hepatic microsomes to lipid peroxidation in vitro and the levels of plasma and liver microsomal lipid hydroperoxides generated in vivo were markedly elevated in vitamin E-depleted trout. No appreciable alterations were observed in the liver microsomal cytochrome P450-dependent mixed-function oxidase system or in the fatty acid composition of trout liver microsomal membranes. Livers from rats fed a vitamin E-deficient diet for 10 weeks also had significantly lower levels of microsomal vitamin E. In addition, total cytochrome P450 levels were depressed (15%) and cytosolic glutathione was enhanced (40%) in livers from rats fed the vitamin E-depleted diet. Covalent binding of [ 3H]-(+)-benzo[ a]pyrene-7,8-dihydrodiol to exogenous DNA in vitro was enhanced with liver microsomes from vitamin E-deficient trout and these fish were much more sensitive to the acute toxicity of this carcinogenic polycyclic aromatic hydrocarbon. These results indicate that trout may be a useful model for studying the significance of peroxidative pathways in carcinogenesis and their manipulation by dietary antioxidants.

The suppression of hepatic cytochrome P4504A mRNA mediated by the interferon inducer polyinosinic acid- poly cytidylic acid

Interferon and interferon inducers are well known to depress the cytochrome P450-dependent hepatic mixed-function oxidase system and cause a decrease in the capacity of the liver to metabolize drugs and xenobiotics. In this study we have shown that the interferon-mediated changes in an induced form of hepatic cytochrome P450 (CYP4A) are mediated via a depression in the levels of mRNA as assessed by Northern blot and slot blot analyses using a 20-base synthetic oligodeoxyribonucleotide hybridization probe. Rats were pretreated with clofibrate to maximize CYP4A mRNA levels prior to the administration of polyinosinic acid-polycytidylic acid (poly 1C), an α/β interferon inducer. Hepatic CYP4A mRNA levels were decreased by 49 and 30% at 6 and 24 hr, respectively, following poly IC administration. In hepatic microsomes cytochome P450 and functional CYP4A as measured by lauric acid hydroxylation, were not affected at 6hr, but were depressed by 39 and 27%, respectively, 24 hr following poly IC administration. These results suggest that interferon depresses induced levels of hepatic drug metabolism by lowering the level of cytochrome P450 mRNAs and subsequent synthesis of cytochrome P450 apoproteins.

Cytochrome P450IA2 in vivo induction: A potential biomarker of polyhalogenated biphenyls and their related chemical's effects on the human

The objectives of our studies have been to test the hypothesis that polyhalogenated biphenyls and related chemicals (PHB) can induce cytochrome P450 dependent mixed function monooxygenase family I enzymes (P450I) in the human, and the degree that a PHB induces P450I activity in the human correlates to the toxicity of the chemical. The P450I family enzyme, P450IA2, activity was monitored in the human by the caffeine breath test. The study subjects were exposed to high amounts of polybrominated biphenyls (PBB), polychlorinated biphenyls with polychlorinated dibenzofurans (PCB/DFs), or mirex. All the PHBs induced the P450IA2 activity of the exposed subjects as compared to the nonexposed control group. The capacity of each PHB to induce P450IA2 activity correlated with the known toxicity of each exposure ( PCB DF PBB >mirex ) in the human. P450IA2 activity as measured by the caffeine breath test may by a most useful biomarker in monitoring and understanding the effects of PHBs on the human population.