P450-dependent Mixed-function Oxidase 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

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.

Insecticide resistance may enhance the response to a host-plant volatile kairomone for the codling moth, Cydia pomonella (L.)

The behavioral and electroantennographic responses of Cydia pomonella (L.) to the ripe pear volatile ethyl (2E,4Z)-2,4-decadienoate (Et-E,Z-DD), were compared in insecticide-susceptible and -resistant populations originating from southern France. A dose-response relationship to this kairomonal attractant was established for antennal activity and did not reveal differences between susceptible and resistant strains. Conversely, males of the laboratory strains expressing metabolic [cytochrome P450-dependent mixed-function oxidases (mfo)] or physiological (kdr-type mutation of the sodium-channel gene) resistance mechanisms exhibited a significantly higher response to Et-E,Z-DD than those of the susceptible strain in a wind tunnel experiment. No response of the females to this kairomone could be obtained in our wind-tunnel conditions. In apple orchards, mfo-resistant male moths were captured at significantly higher rates in kairomone-baited traps than in traps baited with the sex pheromone of C. pomonella. Such a differential phenomenon was not verified for the kdr-resistant insects, which exhibited a similar response to both the sex pheromone and the kairomonal attractant in apple orchards. Considering the widespread distribution of metabolic resistance in European populations of C. pomonella and the enhanced behavioral response to Et-E,Z-DD in resistant moths, the development of control measures based on this kairomonal compound would be of great interest for the management of insecticide resistance in this species.

Human Pharmacogenomic Variations and Their Implications for Antifungal Efficacy

Pharmacogenomics is defined as the study of the impacts of heritable traits on pharmacology and toxicology. Candidate genes with potential pharmacogenomic importance include drug transporters involved in absorption and excretion, phase I enzymes (e.g., cytochrome P450-dependent mixed-function oxidases) and phase II enzymes (e.g., glucuronosyltransferases) contributing to metabolism, and those molecules (e.g., albumin, A1-acid glycoprotein, and lipoproteins) involved in the distribution of antifungal compounds. By using the tools of population genetics to define interindividual differences in drug absorption, distribution, metabolism, and excretion, pharmacogenomic models for genetic variations in antifungal pharmacokinetics can be derived. Pharmacogenomic factors may become especially important in the treatment of immunocompromised patients or those with persistent or refractory mycoses that cannot be explained by elevated MICs and where rational dosage optimization of the antifungal agent may be particularly critical. Pharmacogenomics has the potential to shift the paradigm of therapy and to improve the selection of antifungal compounds and adjustment of dosage based upon individual variations in drug absorption, metabolism, and excretion.

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.

NAD(P)H-dependent epoxidation of aldrin in vitro by sea anemone microsomes

Previous studies from our laboratory indicated the presence of a functional P450-dependent mixed-function oxidase system (MFO) in the sea anemone (Phylum: Cnidaria). Further catalytic studies have demonstrated that sea anemone microsomes can metabolize aldrin to dieldrin through an epoxidation reaction in the presence of NAD(P)H. Based upon Nickel 63 ECD gas chromatography, dieldrin was the only metabolite detected. Further, the production of dieldrin was linear with time and protein concentration. Given the low levels of dieldrin produced, immediate and identical extractions were necessary to minimize variation among replicates. Under these conditions, activity was consistently observed in both Bunodosoma cavernata (NADH, 1.1–2.3; NADPH, 0.4–2.1 pmol/min/mg protein) and Anthopleura elegantissima (NADH, 0.5–2.9; NADPH, 0.3–4.5 pmol/min/mg protein). Only 60% of the A. xanthogrammica microsomal preparations were active (NADH, 0–1.0; NADPH, 0–1.5 pmol/min/mg protein). In B. cavernata and A. elegantissima, the rate of aldrin epoxidation was consistent with that observed in other marine invertebrates. [Supported in part by a grant from USDI/MMS.]

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.

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.

Influence of amphotericin B treatment duration of hepatic microsomal enzyme function in rats.

The influence of amphotericin B on various cytochrome P450-dependent mixed-function oxidases, antipyrine clearance and glucose-6-phosphatase was investigated in rats treated daily with deoxycholate amphotericin B (3 mg/kg body weight, intravenously) either for 1 or 4 days. Enzyme activity was measured ex vivo in hepatic microsomes. Following amphotericin B plus deoxycholate application for day 1, ethoxycoumarin-O-deethylase activity decrease significantly whereas microsomal cytochrome P450 concentration, cytochrome c reductase activity, antipyrine clearance and glucose-6-phosphatase activity did not change significantly. In contrast, following application of amphotericin B plus deoxycholate for 4 days the cytochrome P450 concentration was reduced by 50% (p < 0.05) as well as ethoxycoumarin-O-deethylase activity, antipyrine clearance and glucose-6-phosphatase activity: ethoxycoumarin-O-deethylase 232 +/- 68 pmol/mg/min, control 442 +/- 99 pmol/mg/min (p < 0.01); antipyrine clearance 0.56 +/- 0.21 ml/min, control 0.96 +/- 0.18 ml/min (p < 0.01), and glucose-6-phosphatase 193 +/- 28 mU/mg, control 351 +/- 95 mU/mg (p < 0.05). Cytochrome c reductase activity did not decrease significantly. Besides an increase in cytochrome c reductase activity, sodium deoxycholate (a vehicle of amphotericin B) alone induced no significant changes. Microsomal protein related to liver wet weight was significantly reduced by 50% (p < 0.01) only in animals treated for 4 days with amphotericin B plus deoxycholate. The results show that a 1-day treatment of rats with amphotericin B decreases ethoxycoumarin-O-deethylase activity, whereas the hepatic microsomal cytochrome P450 content, cytochrome c reductase and glucose-6-phosphatase activity did not change. Amphotericin B given for 4 days significantly decreases hepatic microsomal enzyme function. The inhibitory effect of amphotericin B on hepatic cytochrome P450 may be due to inhibition of hepatic protein synthesis.

Induction of hepatic CYP1A2 by the oral administration of caffeine to rats: lack of association with the Ah locus

Caffeine was administered to male Wistar albino rats for two weeks at three concentrations, namely 0.1, 0.2 and 0.3%, and hepatic cytochrome P450-dependent mixed-function oxidases determined. Caffeine administration gave rise to a marked, dose-dependent increase in the O-deethylation of ethoxyresorufin and, to a lesser extent, in the O-depentylation of pentoxyresorufin. Erythromycin N-demethylase, p-nitrophenol hydroxylase and lauric acid hydroxylase activities, as well as total cytochrome P450 content were unaffected by this treatment. Immunoblot analysis revealed that caffeine gave rise to a dose-dependent increase in the hepatic CYP1A2, and at the highest dose only, CYP2B apoprotein levels. Apoprotein levels of CYP3A and CYP2E1 were not modulated by the treatment with caffeine at all dose levels studied. Caffeine could not displace [ 3H]TCDD from the rat hepatic cytosolic Ah receptor. Computer analysis showed that caffeine is essentially a planar molecule with an area/depth 2 ratio 4.8, characteristic of CYP1A substrates/inducers. Molecular modelling revealed that the caffeine molecule could orientate itself within the putative CYP1A2 active site so as to facilitate demethylation of the N-1, N-3 and N-7 positions. However, at physiological pH, the N-9 nitrogen atom is likely to be partially protonated, allowing it to participate in an electrostatic interaction with the negatively-charged glutamate 318-residue, favouring N-3 demethylation, the major pathway of metabolism in both humans and animals. In conclusion caffeine, being essentially planar, is an inducer of CYP1A2 in rat liver.

The use of computers in the safety evaluation of drugs and other chemicals.

The toxicity and carcinogenicity of drugs and other chemicals is, in most cases, mediated by highly reactive intermediates which are generated following metabolism catalysed by the enzymic apparatus of the exposed organisms. These reactive intermediates readily interact covalently with vital cellular components to provoke toxicity and carcinogenicity. The ubiquitous cytochrome P450-dependent mixed-function oxidases are the most important enzyme system in the activation of chemicals. This enzyme system comprises a number of families, each of which contains one or more subfamilies. The CYPIA and CYP2E subfamilies are the most closely associated with the production of reactive intermediates and, consequently, the manifestation of toxicity and carcinogenicity. A computer based molecular structure procedure (COMPACT) has been developed which, via a calculation of the molecular and electronic structure of the chemical, determines whether the chemical will interact with either of these two cytochrome P450 subfamilies and hence be metabolised to form reactive intermediates that manifest toxicity. As the basal levels of these two subfamilies are generally low, the ability of a chemical to induce them selectively, on repeated administration, is an important determinant of its toxic and carcinogenic potential. This inductive capability may be determined in short-term studies (ENACT) using only a small number of animals. Thus the combination of COMPACT and ENACT provides a rapid and inexpensive means for the preliminary screening of chemicals for toxicity and carcinogenicity before undertaking the long-term and expensive rodent lifetime bioassays.

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.

Computer modelling in predicting carcinogenicity.

The cytochrome P450-dependent mixed-function oxidases are the most important enzyme system in the oxidation of chemicals to their reactive intermediates which then interact with cellular components to provoke toxicity and carcinogenicity. These enzymes comprise a multifamily of proteins, two families of which, namely CYP1A and CYP2E, activate planar and small molecular weight compounds, respectively. A computer graphic procedure (COMPACT) has been developed which, based on the molecular shape and electronic structure of the chemical, determines whether the chemical will interact with these two particular cytochrome P450 families and thus be metabolized to toxic and carcinogenic intermediates. As the basal levels of these enzyme families are low, the ability of the chemical to induce them selectively, on repeated administration, is an important determinant of its carcinogenic potential. Inductive capability may be determined in short-term experiments (ENACT) utilizing a small number of animals. Thus the combination of COMPACT and ENACT provides a rapid and inexpensive means for the preliminary screening of chemicals, before the long term and expensive rodent lifetime bioassays are undertaken.

Molecular mechanisms of dibromoalkane cytotoxicity in isolated rat hepatocytes

The cytotoxicty of dibromoalkanes to isolated hepatocytes was proportional to the dibromoalkane concentration and increasing chain length of the dibromoalkane C 2-C 6). The rapid hepatocyte glutathione (GSH) depletion which occurred upon addition of the dibromoalkanes was also dependent on the concentration and chain length of the dibromoalkane. When added to hepatocytes, dibromoalkanes also caused a loss in protein sulfhydryl groups. After a lag period, lipid peroxidation occurred before the onset of cytotoxicity. Antioxidants or removing the oxygen from the medium markedly delayed dibromoalkane cytotoxicity. Bromoaldehydic metabolites formed by cytochrome P450-dependent mixed-function oxidases were probably responsible for lipid peroxidation as deuterated 1,2-dibromoethane (d4-DBE) induced less lipid peroxidation and was less cytotoxic even though GSH was depleted as rapidly and as effectively. Hepatocytes were also more resistant to dibromoalkanes if cytochrome P450 isoenzymes were inactivated with SKF 525A or methyl pyrazole. Furthermore, hepatocyte susceptibility to dibromoalkanes was increased markedly if aldehyde dehydrogenase was inactivated with disulnram, cyanamide or chloral hydrate. Cytochrome P450-induced hepatocytes isolated from pyrazole-, phenobarbital- or 3-methylcholanthrene-pretreated rats were also more susceptible to dibromoalkanes. These results suggest that dibromoalkane-induced cell lysis is due to lipid peroxidation as well as cytochrome P450-dependent formation of toxic bromoaldehydic metabolites which can bind with cellular macromolecules. Dibromoethane GSH conjugates also contribute to DBE cytotoxicity as depleting hepatocyte GSH beforehand increased hepatocyte resistance to DBE but not other dibromoalkanes.