Covalent binding of food carcinogens MeIQx, MeIQ and IQ to DNA and protein in microsomal incubations and isolated rat hepatocytes.

The metabolic activation of 14C-labelled food carcinogens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to macromolecular bound species was studied in microsomal and hepatocellular incubations. Several data indicated that the covalent binding was dependent on P450 enzymes: It was dependent on NADPH, it was induced many times by the P450 IA1 and IA2 upregulators beta-naphthoflavone and polychlorinated biphenyls, and was inhibited by the P450 IA1 and IA2 inhibitor alpha-naphthoflavone. In both hepatocellular and microsomal incubations the three compounds bound with similar efficiency, with IQ being somewhat more potent compared to MeIQx and MeIQ. The binding appeared to follow saturation kinetics with Km values less than 20 microM. In incubations with hepatocytes the compounds bound to both cellular DNA and to bovine serum albumin in the medium. The fact that 13-26% of total adducts were formed with bovine serum albumin, indicates that reactive metabolites of the compounds may be transported and react at distant sites from their formation without any further activation.     

Combined Effects of Ethanol and pH-Change on Protein Synthesis in Isolated Rat Hepatocytes

Abstract: Rat liver parenchymal cells were isolated and incubated for 80 min. in buffered salt solutions. ¹⁴C-valine incorporation in the presence of a high concentration of unlabelled valine (4.2 mM) was taken as a measure of protein synthesis. The pH dependence of the synthesis of cell and medium proteins was studied at pH values varying from 6.6 to 7.9. The results showed that maximum protein synthesis occurred close to the physiological pH value. Protein synthesis was reduced at both lower and higher pH-values. Protein synthesis was inhibited by the addition of ethanol (30 mM). The relative inhibition caused by ethanol got more pronounced as the pH of the hepatocyte suspensions was lowered. The metabolism of ethanol resulted in lowering of the pH in cell suspensions. It is suggested that the depression of protein synthesis by ethanol could be mediated by the fall in pH due to ethanol metabolism.     

Effect of Warfarin on ATP Content,Viability, Glycosylation and Protein Synthesis in Isolated Rat Hepatocytes

Abstract: The effect of warfarin on ATP content, viability and incorporation of labelled glucosamine and leucine into protein was studied in suspensions of rat hepatocytes. When incubated with warfarin the synthesis of total protein and glycoprotein was equally inhibited, and the viability and ATP content of the cells were also lowered. These observations suggest that warfarin is not an inhibitor of glycosylation of proteins in isolated hepatocytes, however, the presence of warfarin leads to reduced ATP content of the cells which causes a decrease in protein synthesis and viability.     

The Metabolism of Acetonitrile to Cyanide by Isolated Rat Hepatocytes

The Metabolism of Acetonitrile to Cyanide by Isolated Rat Hepatocytes.FREEMAN, J. J., and HAYES, E. P. (1987). Fundam. Appl. Toxicol.8, 263–271. The metabolism of saturated nitriles, includingacetonitrile, has been assumed to occur by a cytochrome P-450-dependentoxidation at the -carbon, yielding a cyanohydrin intermediatewhich may spontaneously degrade to hydrogen cyanide and an aldehyde.However, results of studies in our laboratory suggest that formaldehydeis not a metabolite of acetonitrile. Since acetonitrile is structurallysimilar to iodomethane, a substrate for glutathione (GSH) S-transferases,we hypothesized that the metabolism of acetonitrile to cyanidemight also occur by a nucleophilic substitution reaction involvingGSH. The present studies were conducted to investigate thesehypotheses and to further our study of the effects of acetoneon acetonitrile metabolism. Female Sprague–Dawley ratswere pretreated with buthionine sulfoximine BSO (4 mmol/kg ip,at –4 and –2 hr), cobalt heme (90 µmol/kgsc, at –48 hr), acetone (1960 mg/kg po, at –24 hr),or vehicle, and hepatocytes were isolated after collagenaseperfusion of the liver. BSO reduced the cellular GSH contentby >80%, but did not appear to affect the metabolism of acetonitrile:the liberation of cyanide correlated with cytochrome P-450,and not GSH, concentrations. Cobalt heme depleted hepatocellularcytochrome P-450 (–45%) content, decreased cell yieldand viability, and resulted in a marked reduction in the metabolismof acetonitrile to cyanide. Cobalt heme did not affect the recoveryof sodium cyanide from hepatocyte suspensions. Pretreatmentof rats with acetone resulted in a twofold increase in the metabolismof acetonitrile to cyanide. Addition of acetone in vitro inhibitedacetonitrile metabolism, with an IC50 of 319 µM. Theseresults suggest that the metabolism of acetonitrile to cyanideoccurs by a cytochrome P-40-dependent pathway, and not by anucleophilic substitution reaction with GSH.     

The Role of Glutathione and Protein Thiols in CBrCl3-Induced Cytotoxicity in Isolated Rat Hepatocytes

Abstract: The role of glutathione (GSH) and protein thiols in the pathobiochemical process of CBrCl₃ cytotoxicity was investigated in isolated hepatocytes. Administration of 0.5, 1.0 and 1.5 mmol/1 CBrCl₃ affected cellular viability as assessed by trypan blue exclusion, release of lactate dehydrogenase and loss of intracellular potassium in a dose-dependent manner. Intracellular glutathione and the capacity to reduce 3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazolium bromide (MTT, thiazolyl blue) decreased almost independently of the CBrCl₃ concentration. Protein thiols were not markedly oxidized in the presence of CBrCl₃. However, compromising cellular defence mechanisms by either inhibition of glutathione regeneration or depletion of glutathione enhanced the cytotoxicity of CBrCl₃ and induced a loss of protein thiols in the late phase of cellular injury. Under these conditions the thiol-dependent Na⁺, K⁺ ATPase revealed high sensitivity towards CBrCl₃. Thus, glutathione proved to exert effective cytoprotection, and sulfhydryl groups of particular proteins were supposed to be an important target of radical attack.     

Dose-Dependent Cytotoxicity of Chlorinated Hydrocarbons in Isolated Rat Hepatocytes

Dose-Dependent Cytotoxicity of Chlorinated Hydrocarbons in IsolatedRat Hepatocytes. DAHLSTROM-KJNG, L., COUTURE, J., LAMOUREUX,C, VAILLANCOURT, T., AND PLAA, G. L. (1990). Fundam. Appl. Toxicol.14, 833–841. The aim was to determine if isolated suspendedhepatocytes could differentiate between the effects of fourchlorinated hydrocarbons that are hepatotoxic In vivo and fourthat are not. Membrane integrity was assessed by measuring alanineaminotransferase (ALT) release after 30- to 180-min incubationsin vitro. From the results, the chlorinated hydrocarbons fellinto three groups: tetrachloroethylene and 1,1,2,2-tetrachloroeth-anewere the most potent cytotoxicants; CCl₄, 1,1,2-trichloroethane,and trichloroethylene exhibited intermediate cytotoxicity; andlow cytotoxicity was observed with CHCl₃, 1,1,1 -trichlo-roethane,and 1,1-dichloroethylene. Cytotoxicity ranking correlated poorlywith the reported In vivo hepatotoxicity of these agents. Theeffect of adding SKF-525A on the cytotoxicity of tetrachloroethyleneand CCI4 was also assessed. In addition, hepatocytes from ratspretreated with 2,5-hexanedione were used to determine if theywere more susceptible to the effects of CHCl₃, CCl₄ or tetrachloroethylene.SKF-525A decreased the cytotoxicity of both CO, and tetrachloroethylene,whereas pretreatment with 2,5-hexanedione enhanced their effect.The effects of both SKF-525A and 2,5-hexanedione on CCl in vitroare consistent with In vivo findings. However, tetrachloroethyleneis not hepatotoxic In vivo, suggesting that SKF-525A might actby stabilizing plasma membranes rendering the hepatocyte moreresistant to lysis. Overall, the results cast doubts on theuse of ALT release from isolated hepatocytes as an appropriatein vitro model for assessing hepatotoxic properties of chlorinatedhydrocarbons.     

Cytotoxic Effects of N-Hydroxyparacetamol in Suspensions of Isolated Rat Hepatocytes

Abstract: The cytotoxicity of N-hydroxyparacetamol (N-OH-pHAA), a postulated proximate metabolite of the hepatotoxic and nephrotoxic analgesic paracetamol, was studied in suspensions of hepatocytes isolated by collagen-perfusion of livers of male rats. Incubation of cells with 0.25–2.0 mM N-OH-pHAA led after 3–5 hours to increased cell permeability measured by increased trypan blue uptake, increased NADH penetration or leakage of prelabelled ⁵¹Cr. N-OH-pHAA rapidly depleted cellular glutathione, 16% of initial levels were seen after 30 min. incubation. ³H-N-OH-pHAA bound covalently to cellular proteins in a time- and concentration-dependent manner, considerably higher binding rates were seen with boiled cells compared to intact cells. Pretreatment of animals with the cytochrome P-450 inducer phenobarbital did not affect N-OH-pHAA cytotoxicity or covalent binding, whereas the cytochrome P-450 inhibitor metyrapone inhibited both cytotoxicity and binding. Lipid peroxidation in hepatocytes could be seen as a late event after a limited range of N-OH-pHAA concentrations. In contrast, lipid peroxidation was an early event in cells exposed to carbon tetrachloride. A minimal exposure time of 30 min. of the hepatocytes to N-OH-pHAA was sufficient to elicit cellular damage occurring after 3–5 hours.     

Modulation of N-Hydroxyparacetamol Cytotoxicity in Suspensions of Isolated Rat Hepatocytes

Abstract: The effect of inhibitors of toxicity of N-hydroxyparacetamol(N-OH-pHAA), a postulated proximate metabolite of paracetamol, was studied in isolated rat hepatocytes. Additions of ascorbate, menadione, thiol-containing amino acids and glutathione (GSH) led to an increased stability of N-OH-pHAA, reduced the covalent binding of N-OH-pHAA to cellular protein and decreased GSH depletion caused by N-OH-pHAA. Two to three hours elapsed after a 30 min. exposure of the cells to N-OH-pHAA before the cells responded with increased cell permeability. Ascorbate, acetylcysteine, GSH and promethazine were capable of inhibiting this second phase of N-OH-pHAA cytotoxicity in addition to their effects during the initial exposure phase. In contrast, the anti-oxidant tocopherole and phenacetin were only effective during the second phase. Increasing the incubation medium pH during the second phase of N-OH-pHAA mediated cellular damage resulted in decreases in cytotoxicity. Lipid peroxidation, as measured by accumulation of thiobarbituric acid reactive metabolites, did not seem to be directly correlated with cytotoxicity, since cysteamine or higher concentrations of N-OH-pHAA inhibited lipid peroxidation without decreasing cellular damage.     

Drug Metabolism Activities of Isolated Rat Hepatocytes in Monolayer Culture

Abstract: The levels of cytochrome P-450 in hepatocytes cultured as monolayers for 22 hrs in Dulbecco's modified Eagle medium supplemented with serum and insulin was reduced to approximately 40% of initial values of freshly isolated hepatocytes. In correspondance with this the activities of the cytochrome P-450 monooxygenases aryl hydrocarbon (benzo(a)pyrene) hydroxylase (AHH) and ethylmorphine (EM) N-demethylase were reduced to 40 and 22% of their initial activities, respectively. Modifying the culture medium through omission of cysteine and cystine, and adding dexamethazone and delta-amino levulinic acid, increased the content of cytochrome P-450 to 59 % and EM N-demethylase to 46 % of initial values, but was without effect on AHH activity. However, further modifications by adding high concentrations of asparagine and leucine increased AHH activity to 62% of initial values, but did not further enhance the total content of cytochrome P-450 or the EM N-demethylase activity. The activities of cytochrome P-450 reductase, flavin containing monooxygenase, epoxide hydrolase and glutathione S-transferase decreased less (to about 70–80% of initial values) than cytochrome P-450 associated monooxygenase activities, whereas UDP-glucuronyl transferase decreased to about 50% of initial values. In contrast to what was observed regarding cytochrome P-450 and associated monooxygenase activities, modification of the incubation conditions did not affect the non-cytochrome P-450 enzymatic activities.     

Paracetamol Metabolism in the Rat: Relationship to Covalent Binding and Hepatic Damage

1. The degree of liver damage observed 48 h after administration of ¹⁴C ring-labelled paracetamol (3–23 mmol/kg) to rats was proportional to the amount of a highly reactive metabolite retained in the liver, bound covalently to hepatocellular proteins.

2. With increasing doses of paracetamol, urinary excretion of the glucuronide and sulphate conjugates reached a plateau, whereas the output of cysteine and mercapturic acid conjugates increased markedly.

3. The degree of covalent binding at 48 h was proportional to the rate of urinary elimination of these two latter conjugates in the first 24 h after dosing.     

Ameliorative Effects of Taurine Against Methimazole-Induced Cytotoxicity in Isolated Rat Hepatocytes

Methimazole is used as an antithyroid drug to control the symptoms of hyperthyroidism and maintain patients in a euthyroid state. Administration of this drug is associated with agranulocytosis and hepatotoxicity, which are the two most significant adverse effects. The present investigation was conducted to study the protective role of taurine against cytotoxicity induced by methimazole and its proposed reactive intermediary metabolite, N-methylthiourea, in an in vitro model of isolated rat hepatocytes.At different points in time, markers such as cell viability, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and hepatocyte glutathione content were evaluated.Treating hepatocytes with methimazole resulted in cytotoxicity characterized by the reduction in cell viability, an increase in ROS formation and lipid peroxidation, mitochondrial membrane potential collapse, and a reduction in cellular glutathione content. Furthermore, a significant amount of oxidized glutathione (GSSG) was formed when rat hepatocytes were treated with methimazole. N-methylthiourea toxicity was accompanied by a reduction in cellular GSH content, but no significant changes in lipid peroxidation, ROS formation, GSSG production, or changes in mitochondrial membrane potential were detected. Administration of taurine (200 μM) effectively reduced the toxic effects of methimazole or its metabolite in isolated rat hepatocytes.     

Modulation of Glucose Metabolism in Isolated Rat Hepatocytes by 1,1,1,2-Tetrafluoroethane

Modulation of Glucose Metabolism in Isolated Rat Hepatocytesby 1,1,1,2-Tetrafluoroethane. OLSON, M.J., REIDY,C. A., ANDJOHNSON, J.T. (1990). Fundam Appl Toxicol. 15,270–280.The thennodynamic behavior and lack of ozone-depleting potentialof 1,1,1,2-tetrafluoro-ethane (R-134a) suggest it as a likelyreplacement for dichlorodifluoromethane (R-12), now used asthe refrigerant in many air-conditioning systems. To furtherthe presently incomplete toxicological analysis of R- 134a,the effects of R-134a on cell viability and functional competenceof glucose metabolism were evaluated in suspension culturesof hepatocytes derived from fed or fasted rats. R-134a concentrationsup to and including 75% (750,000 ppm) in the gas phase of sealedculture flasks did not produce evidence of cytolethality (LDHleakage) following 2 hr of exposure; in contrast, halothane(l,l,l-trifluoro-2-bromo-2-chloroethane) caused cell death ata gas phase concentration of only 1250 ppm. In hepatocytes isolatedfrom fed rats, R-134a at concentrations of 12.5 to 75% increasedglycolysis (production of lactate + pyruvate) in a concentration-dependentmanner, no effect was observed at 5%. At 25%, R-12 and 1,1,2,2-tetrafluoro-l,2-dichloroethane(R-l14) were of equal potency to R-134a in stimulating glycolysis;l,l,l,2,2-pentafluoro-2-chloroethane (R-115) depressed glycolysisslightly. Halothane, at concentrations as low as 300 ppm, markedlyincreased rates of glycolysis. Glucose production by hepatocytesof fed rats was decreased by R-134a, R-12, and R-114 only atconcentrations of 25% or more. On the other hand, halothane(>300 ppm) potently decreased glucose production by hepatocytes.In cells isolated from livers of fasted rats, R-134a exposureinhibited gluconeo-genesis in a concentration-dependent manneralthough this effect was not significant until R-134a concentrationsreached 12.5%. Comparative potency studies showed that R-l 34a,R-12, or R-l 14 (25% gas phase) inhibited gluconeogenesis aboutequally while as little as 300 ppm halothane was effective andR-115 (25%) was without effect. Considering that the thresholdfor alteration of the rate of glucose metabolism in this invitro paradigm is about 12.5% R-134a, we conclude that toxicologicallysignificant alteration of glucose-linked bioenergetics is unlikelyat the levels of R-l34a exposure anticipated in workplace orenvironment     

Uptake of Colchicine,a Microtubular System Disrupting Agent,by Isolated Rat Hepatocytes

The possible mechanism of hepatic uptake of colchicine (CLC), a microtubule system disrupting agent, was examined using isolated rat hepatocytes. The existence of a carrier-mediated active transport system for CLC was demonstrated. This transport system is saturable, is affected by metabolic inhibitors (dinitrophenol, KCN) and a SH-group blocker (p-hydroxymercuribenzoic acid but not N-ethylmaleimide), and is sensitive to temperature. Ouabain, an inhibitor of Na⁺, K⁺-ATPase, does not affect the transport system of CLC.     

Dose Dependence of Covalent Binding of Acrylonitrile to Tissue Protein and Globin in Rats

The dose dependence of acrylonitrile (AN) covalent binding totissue protein, following a single acute exposure over a 100-foldrange in dose, was measured. Covalent binding was a linear functionof AN dose in the lower dose range (0.0–0.95 mmol AN/kg).The slopes of the dose-response curves indicated that tissuesvaried by nearly 10-fold in their reactivity with AN. The relativeorder of covalent binding was as follows: blood > kidney= liver > forestomach = brain > glandular stomach >muscle. Similar dose-response behavior was observed for globintotal covalent binding and for globin N-(2-cyanoethyl)vallne(CEValine) adduct formation. The latter adduct was found torepresent only 0.2% of the total AN adduction to globin. Regressionof tissue protein binding versus globin total covalent bindingor globin CEValine adduct indicated that both globin biomarkerscould be used as surrogates to estimate the amount of AN boundto tissue protein. At higher AN doses, above approximately 1mmol/kg, a sharp break in the covalent binding dose-responsecurve was observed. This knot value is explained by the nearlycomplete depletion of liver glutathione and the resultant terminationof AN detoxification. The toxicity of AN is known to increasesharply above this dose. The data suggest that a comparisonof specific tissue proteins labeled by AN above and below thisthreshold dose may provide some insight into the mechanism ofAN-induced toxicity.     

HPLC Assay for FK 506 and Two Metabolites in Isolated Rat Hepatocytes and Rat Liver Microsomes

Despite the current use of a standard two-step enzyme immunoassay in the clinical monitoring of the immunosuppressant FK 506, the lack of specificity for the parent drug in this assay renders it unsuitable for drug metabolism studies. An HPLC assay has been developed for studying the metabolism of FK 506 in isolated hepatocytes and microsomal mixtures. This assay allows simultaneous measurement of the parent drug and two of its time dependent metabolites. Metabolism of this drug was studied in intact rat liver cells and rat liver microsomes. We have shown that the metabolites observed are products of phase 1 oxidation reactions. Correlation of the 6-testosterone hydroxylase activity with the FK 506 metabolite (Ml) initial formation rate is consistent with the belief that CYP 3A isozymes are involved in FK 506 metabolism in male rats.     

Comparative Metabolism of Methyl Parathion in Intact and Subcellular Fractions of Isolated Rat Hepatocytes

Metabolism of the widely used insecticide methyl parathion byisolated hepatocytes and various subcellular fractions was comparedto determine the effects of cellular integrity on the metabolicprofile observed. A reverse-phase ion-pair high-performanceliquid chromatographic method was developed to separate andquantify methyl parathion and six of its hepatic biotransformationproducts: methyl paraoxon; desmethyl parathion; desmethyl paraoxon;p-nitrophenol; p-nitrophenyl glucuronide; and p-nitrophenylsulfate. Most compounds exhibited linear responses and limitsof detection below 1 nmol. The chromatographic method was usedto determine metabolic profiles of methyl parathion in isolatedrat hepatocytes, sonicated hepatocytes, postmitochondrial fraction,microsomes, and cytosol. Isolated hepatocytes produced significantlymore desmethyl parathion and p-nitrophenyl sulfate than thesubcellular preparations, demonstrating that cellular integritysignificantly affects the quantitative metabolic profile observed.     

In Vitro Covalent Binding of New Brain Tracer,Para 125 I-Amphetamine,to Rat Liver and Lung Microsomes

ABSTRACT

Butachlor action on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities in central nervous tissue of the snail Pita globosa was assayed following the method of ELLMAN et al¹, in vitro by adding butachlor directly (10–100 μ moles), to tissue homogenates and in in vivo by exposing the snails to sub-lethal concentration (26.6 ppm) and taking out the tissue for experimentation at different intervals (3, 6,12, 24 and 48 h) of exposure. The enzyme activities decreased in a dose-dependent manner in vitro, and up to 12–24 h in vivo after which they showed recovery towards the control. The inhibition of cholinesterases by butachlor in vitro indicates a direct action of the herbicide on these enzymes. Presumably butachlor exercises its neurotoxic effects through cholinergic impairment in a way similar to that of organophosphates and carbamates.     

Microsomal Cytochrome P450 Levels and Activities of Isolated Rat Livers Perfused with Albumin

Purpose. We recently showed that the perfusion of isolated rat livers with perfusates containing bovine serum albumin (BSA) would significantly stimulate the release of tumor necrosis factor (TNF)-. Here, we hypothesize that BSA-induced increase in the release of TNF-, and possibly other cytokines, would affect cytochrome P450 (CYP)-mediated drug metabolism. Methods. Rat livers were perfused ex vivo for 1, 2, or 3 h with a physiologic buffer containing or lacking 1% BSA (n = 4-5/group). At the end of perfusion, liver microsomes were prepared and analyzed for their total CYP, CYP2E1, CYP3A2, and CYP2C11 protein contents and the activities of cytochrome c reductase, CYP2E1, CYP3A2, CYP2C11, CYP2E1, CYP2D1, CYP1A1, and CYP2B1/2. In addition, the concentrations of various cytokines and nitric oxide were quantified in the outlet perfusate. Results. In the absence of BSA, the perfusate levels of all measured cytokines and nitric oxide were low. However, when the perfusate contained BSA, the levels of TNF-, interleukin-6, and nitric oxide increased significantly (p < 0.005). Perfusion of the livers for 3 h with the BSA-containing perfusate resulted in significant (p < 0.05) decreases in the total CYP (41%), CYP2E1 (59%), CYP3A2 (68%), and CYP2C11 (50%) protein contents and activities of cytochrome c reductase (31%), CYP2E1 (66%), CYP3A2 (54%), and CYP2C11 (51%). In contrast, perfusion of livers for 1 or 2 h with the BSA perfusate did not have any significant effect on CYP-mediated metabolism. The CYP1A2, CYP2D1, and CYP2B1/2 activities were not affected by BSA, regardless of perfusion time. Conclusion. Addition of BSA to perfusates, which is a routine practice in isolated rat liver studies, can reduce CYP-mediated drug metabolism by a mechanism independent of protein-binding effect.     

Foetal Exposure to Food and Environmental Carcinogens in Human Beings

Abstract: Exposure to many different chemicals during pregnancy through maternal circulation is possible. Transplacental transfer of xenobiotics can be demonstrated using human placental perfusion. Also, placental perfusion can give information about the placental kinetics as well as metabolism and accumulation in the placenta because it retains the tissue structure and function. Although human placental perfusion has been used extensively to study the transplacental transfer of drugs, the information on food and environmental carcinogens is much more limited. This review deals with the foetal exposure to food and environmental carcinogens in human beings. In particular, human transplacental transfer of the food carcinogens such as acrylamide, glycidamide and nitrosodimethylamine are in focus. Because these carcinogens are genotoxic, the functional capacity of human placenta to induce DNA adduct formation or metabolize these above mentioned CYP2E1 substrates is of interest in this context.