Abstract  One hundred and six compounds, subdivided into 12 chemical classes (5 polycyclic aromatic hydrocarbons, 7 epoxides and N-oxides, 5 nitro aromatics and heterocyles, 12 aromatic and heterocyclic amines, 2 azo compounds, 17 hydrazine derivatives, 16 miscellaneous aliphatics, 5 miscellaneous heterocycles, 11 miscellaneous organics, 11 hexavalent and 7 trivalent chromium compounds, 8 miscellaneous inorganics), were studied in the Salmonella/microsome test. Fifty-eight of them (54.7%) were found to be mutagenic and 4 additional compounds (3.8%) yielded a positive response following nitrosation in human gastric juice. The results are presented in a tabulated form, providing the following information for each test compound: (a) mutagenic response in 5 S. typhimurium his - strains (TA1535, TA1537, TA1538, TA98, TAI00); (b) ranged of activity for positive compounds or maximum dose tested for negative compounds (in nmol/plate); (c) mutagenic potency (in revertants/umol compound), varying over a 6.5 x 106-fold range; (d) effect of S-9 mix containing rat (Aroclor) liver S-9 fractions on the mutagenic response (activation, increase, no change or decrease): (e) remarks concerning the influence of other metabolic systems (up to 10 different rat tissue S-9 fractions, mouse S-9 fractions, human S-9 fractions, cell reparations or biological fluids), the interaction between different compounds, the stability and the formation of mutagenic derivatives in human gastric juice and other experimental details. Overlapping of mutagenicity and carcinogenicity data was not evaluated, since the experimental protocol intentionally included a number of non-carcinogenic mutagens and of non-mutagenic carcinogens with the aim of explaining in some cases the conflicting nature of in vivo and in vitro conclusions.

Abstract  Studies were carried out to compare the effects of CCl4 in vivo and in vitro on adrenal and hepatic microsomal metabolism in guinea pigs. CCl4 administration in vivo decreased adrenal and hepatic microsomal cytochrome P-450 concentrations and lowered benzphetamine (BZ) demethylase and benzo(a)pyrene (BP) hydroxylase activities in both tissues. NADPH-cytochrome c reductase activity was decreased in hepatic but not in adrenal microsomes. Addition of CCl4 to adrenal or hepatic microsomes in vitro produced a type 1 difference spectrum suggesting binding of CCl4 to cytochrome(s) P-450; its magnitude was greater in adrenal than in liver. Incubation of adrenal or hepatic microsomes in vitro with CCl4 alone had little or no effect on mixed-function oxidase activity or on lipid peroxidation. When microsomes were incubated with CCl4 + NADPH, the rates of BZ and BP metabolism decreased, cytochrome P-450 concentrations decreased and lipid peroxidation was increased. The effects of CCl4 + NADPH on enzyme activities were greater in adrenal than in hepatic microsomes. Addition of 1.0 mM EDTA or 0.1 mM MnCl2 to the incubation medium blocked the effects of CCl4 + NADPH on lipid peroxidation in adrenal and liver but had no effect on the decreases in mixed-function oxidase activities. The adrenal cortex in the guinea pig was an active site of CCl4 metabolism. CCl4 metabolism resulted in a loss of microsomal enzyme activities in the adrenal and liver. Lipid peroxidation was not obligatory for CCl4-mediated destruction of microsomal enzymes.

Abstract  Previous studies from this laboratory have shown a concentration-time dependent, site specific cytotoxicity of CCl4 in slices. The present study examined radical formation and lipid peroxidation as a potential mechanism of toxicity. Liver slices from male Sprague-Dawley rats (220– 250 g) were exposed to 0.57 mM CCl4 by vaporization using a roller incubation system. Cytochrome P-450 is responsible for the bioactivation of CCl4 generating the CCl3 radical, which also acts as a suicide inhibitor. In slices exposed to CCl4, cytochrome P-450 loss occurred in a timedependent manner relative to controls (↓ 58% at 9 hr). A 48 hr fast prior to sacrifice both enhanced and accelerated both cytochrome P-450 loss as well as CCl4 toxicity in slices. Unlike cytochrome P-450, glutathione levels were not altered over the course of the experiment. These studies suggest that centrilobular hepatocytes are more susceptible to CCl4 induced injury. Covalent binding studies using 14CC14 confirmed CCl3 radical formation by cytochrome P-450. Binding to slice proteins plateaued as early as 30 min following CCl4 administration whereas lipid binding was saturated by 60 min. Covalent binding of the CCl3 radical was increased two-fold 60 min following phenobarbital pretreatment whereas allylisopropylacetamide caused the converse (50% at 60 min). Conjugated diene formation, an index of lipid peroxidation as detected by 15 min following

Abstract  It was found that the four toxigenic agents, CCl4, CHCl3, CBrCl3, and 1,1-dichloroethylene (vinylidene chloride) all share the property of activating phospholipase A2 (PLA2) of isolated hepatocytes in suspension, as determined over a 60- or 120-min time period. In all cases, PLA2 activation, measured as the appearance of lysophosphatidylethanolamine, preceded the release of lactic dehydrogenase during incubation of the cells at 37 degrees C. It is concluded that for these halogenated hydrocarbons phospholipase A2 activation may be part of the chain of causality leading from initial bioactivation to ultimate cell death.

Abstract  The liver, which is the major organ responsible for the metabolism of drugs and chemicals, is also the primary target organ for many toxic chemicals. Increasing evidence has indicated that inflammatory processes are intimately involved in chemical-induced hepatotoxic processes, and like other inflammatory diseases, such as autoimmunity, are responsible for producing mediators which can effect liver damage or repair. This review will summarize the authors' current understanding of how inflammatory processes influence hepatic pathology and repair following exposure to established hepatotoxic chemicals including carbon tetrachloride (CCl4), an industrial chemical, and acetaminophen (APAP), a widely used analgesic.

Abstract  Investigation of covalent DNA binding in vivo provided evidence for whether a test substance can be activated to metabolites able to reach and react with DNA in an intact organism. For a comparison of DNA binding potencies of various compounds tested under different conditions, a normalization of the DNA lesion with respect to the dose is useful. A covalent binding index, CBI = (mumol chemical bound per mol DNA nucleotide)/(mmol chemical administered per kg body weight) can be determined for each compound. Whether covalent DNA binding results in tumor formation is dependent upon additional factors specific to the cell type. Thus far, all compounds which bind covalently to liver DNA in vivo have also proven to be carcinogenic in a long-term study, although the liver was not necessarily the target organ for tumor growth. With appropriate techniques, DNA binding can be determined in a dose range which may be many orders of magnitude below the dose levels required for significant tumor induction in a long-term bioassay. Rat liver DNA binding was proportional to the dose of aflatoxin B1 after oral administration of a dose between 100 micrograms/kg and 1 ng/kg. The lowest dose was in the range of general human daily exposures. Demonstration of a lack of liver DNA binding (CBI less than 0.1) in vivo for a carcinogenic, nonmutagenic compound is a strong indication for an indirect mechanism of carcinogenic action. Carcinogens of this class do not directly produce a change in gene structure or function but disturb a critical biochemical control mechanism, such as protection from oxygen radicals, control of cell division, etc. Ultimately, genetic changes are produced indirectly or accumulate from endogenous genotoxic agents. The question of why compounds which act via indirect mechanisms are more likely to exhibit a nonlinear range in the dose-response curve as opposed to the directly genotoxic agents or processes is discussed.

Abstract  The in vivo-in vitro DNA repair and DNA replication assay in mouse hepatocytes has promise as a short-term test for detecting potential mouse liver carcinogens. In addition, this assay may provide information on the mode of action of known hepatic carcinogens. The induction of DNA repair is clearly a response to hepatic DNA damage. However, it is unclear whether induction of replicative DNA synthesis (S phase) represents regenerative hyperplasia in response to hepatotoxicity or is a result of direct mitogenic stimulation of the hepatocytes by the test compound. The objective of the present study was to examine the relationship between hepatotoxicity, which was assessed by measuring serum concentrations of glutamic-oxalacetic transaminase (SGOT), glutamic-pyruvic transaminase (SGPT), alkaline phosphatase (AP), and gammaglutamyl transferase (GGT), and induction of S phase following either single or multiple doses of the model mouse hepatocarcinogen carbon tetrachloride (CCl4). Under the experimental conditions in this study, CCl4 elevated SGPT and SGOT but did not affect serum concentrations of AP or GGT. CCl4 did not induce DNA repair. An increase in the percentage of hepatocytes in S phase followed the appearance of elevated SGOT and SGPT in all single-dose studies. The results from the multiple-dose studies showed a similar relationship except that with 20 mg/kg X d the concentrations of SGOT and SGPT decayed to control values after 14 d of dosing whereas the percentage of hepatocytes in S phase remained markedly elevated (greater than 10 X control). The daily dose of CCl4 that gave a no-observed-effect level for induction of S phase was lower with multiple administrations than it was following a single exposure. A single administration of CCl4 at 25 mg/kg did not increase S phase, SGOT, or SGPT, but if 20 mg/kg X d was given for 7 d the number of hepatocytes in S phase and the concentrations of SGOT and SGPT increased more than 10-fold. These data support the hypothesis that induction of replicative DNA synthesis in the mouse liver following CCl4 administration is related to hepatotoxicity. In single-dose studies elevation in S phase was always associated with elevation of SGOT and SGPT. However, in the multidose studies, SGOT and SGPT declined after 14 d of administering 20 mg/kg X d while S phase remained elevated.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract  Background/Aim: The aim of this study was to investigate the effects of S-adenosylmethionine on liver peroxidation and liver fibrogenesis in carbon tetrachloride-induced cirrhosis. Methods: Cirrhosis was induced in three groups of six rats by repeated injections of carbon tetrachloride over 9 weeks. One group of animals was treated with carbon tetrachloride, and the other two received carbon tetrachloride plus S-adenosyl-methionine (10 mg/kg intramuscularly daily) from week 3 to week 9, and from week 6 to week 9 of the study, respectively. Two additional groups of six rats, a control group and a group treated only with S-adenosylmethionine, were also studied. Glutathione concentration, thiobarbituric acid-reactive substances, collagen content, prolyl hydroxylase activity, and procollagen type I mRNA expression were determined in liver samples. Results: All carbon tetrachloride-treated rats had cirrhosis at the end of the study. Cirrhosis was also present in five of the six carbon tetrachloride-treated rats received S-adenosylmethionine for 3 weeks, but in only one of the six rats that received S-adenosylmethionine for 6 weeks. Hepatic glutathione was significantly diminished in carbon tetrachloride-treated rats (2.7±0.3 μmol/g tissue) and returned to normal in rats received S-adenosylmethionine for 3 or 6 weeks (3.7±0.13 and 3.9±0.11 μmol/g tissue, respectively). The hepatic thiobarbituric acid-reactive substances were significantly lower in rats treated with carbon tetrachloride and S-adenosyl-methionine for 6 weeks (98±5 nmol/g) than in rats treated with carbon tetrachloride (134±12 nmol/g) and in those treated with carbon tetrachloride and S-adenosylmethionine for 3 weeks (127±13 nmol/g). There were no differences in either hepatic collagen and prolyl hydroxylase activity between rats that received only carbon tetrachloride and those treated with S-adenosylmethionine for 3 weeks. In contrast, carbon tetrachloride-treated rats received S-adenosylmethionine for 6 weeks had significantly lower collagen and prolyl hydroxylase activity than the other two groups. A much greater increase in procollagen type I mRNA was found in carbon tetrachloride-treated rats than in rats treated with carbon tetrachloride and S-adenosylmethionine for 6 weeks. Furthermore, there was a significant correlation between the hepatic thiobarbituric acid-reactive substances and prolyl hydroxylase activity and hepatic collagen. Conclusions: We conclude that the early administration of S-adenosylmethionine in a model of carbon tetrachloride-induced liver injury restores glutathione levels and reduces lipid peroxidation, resulting in less advanced liver fibrosis.

Abstract  Electron spin resonance spectroscopy has been used to monitor free radicals formed during CCl4 metabolism by perfused livers from phenobarbital-treated rats. Livers were perfused simultaneously with the spin trap phenyl N-t-butylnitrone and with either 12CCl4 or 13CCl4. Perfusate samples and CHCl3:CH3OH extracts of perfusate and liver samples were analyzed for phenyl N-t-butylnitrone radical adducts of reactive free radicals. In the organic extracts, hyperfine coupling constants and 13C isotope effects observed in the ESR spectra indicated the presence of the radical adduct of the trichloromethyl radical. Surprisingly, an additional free radical signal about two orders of magnitude more intense than that of the phenyl N-t-butylnitrone/CCl.3 radical adduct was observed in the aqueous liver perfusate. This adduct was also detected by ESR in rat urine 2 h after intragastric addition of spin trap and CCl4. This radical adduct had hyperfine coupling constants and 13C isotope effects identical with the radical adduct of the carbon dioxide anion radical (CO2-.). Analysis of the pH dependence of the coupling constants yielded a pK alpha of 2.8 for the CO2-. radical adduct formed either in the perfused liver or chemically. Carbon tetrachloride is converted into CCl.3 by cytochrome P-450 through a reductive dehalogenation. The trichloromethyl free radical reacts with oxygen to form the trichloromethyl peroxyl radical, CCl3OO., which may be converted into .COCl and then trapped. This radical adduct would hydrolyze to the carboxylic acid form, which is detected spectroscopically. Alternatively, the carbon dioxide anion free radical could form through complete dechlorination and then react with the spin trap to give the CO2-. radical adduct directly.

Abstract  The 1,N2-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal (HNE-dGp-adducts) were quantitated in tissues of rats treated with trans-4-hydroxy-2-nonenal (HNE) or carbon tetrachloride, respectively, using a 32P-postlabeling method. The method development was based on chemically synthesized HNE-1,N2-propanodeoxyguanosine adduct standard, which was characterized by NMR and mass spectra. The adducts were enriched by Nuclease P1. They were subsequently reacted with γ-32P-ATP to give the respective 3′-5′-bisphosphates, which were two-directionally separated on PEI-cellulose-TLC and quantitated by autoradiography. The labeling efficiency for the adduct standard was 27%, and the recovery of spiked amounts of adduct standard in the enzymatical procedure was about 80%. Internal standard was used to eliminate methodological variations. The determination of the limit of quantitation in DNA from rat tissues by spiking of HNE-dGp-adduct standard revealed a sensitivity of about 20 HNE-dGp-adducts/109 normal nucleotides. Background levels of HNE-dGp-adducts in tissues of rats including liver, kidney, lung, colon and forestomach were found in the range of 18–158 adducts/109 nucleotides with relatively high adduct levels in the liver and low adduct levels in kidney, lung and colon. These background levels were statistically significantly increased by the factor of 2 in liver, lung, colon and forestomach after induction of lipid peroxidation by carbon tetrachloride. The finding that background HNE-dGp-adduct levels may be in context with different metabolic activities of the tissues and the increase of HNE-dGp-adduct levels after application of carbon tetrachloride indicate that HNE-dGp-adducts are an endogenous lesion and that they are probably formed from radical initiated lipid peroxidation.

Abstract  Endogenous DNA adducts may contribute to the etiology of human genetic disease and cancer. One potential source of endogenous DNA adducts is lipid peroxidation, which generates mutagenic carbonyl compounds such as malondialdehyde. A sensitive mass spectrometric method permitted detection and quantitation of the major malondialdehyde-DNA adduct, a pyrimidopurinone derived from deoxyguanosine. DNA from disease-free human liver was found to contain 5400 adducts per cell, a frequency comparable to that of adducts formed by exogenous carcinogens.

Abstract  Free radicals have been implicated in the pathogenesis of alcohol-induced liver injury in humans and carbon tetrachloride (CCl4)-induced liver injury in rats. The most extensively studied aspect of free radical induced liver injury is lipid peroxidation. Recently it has been found that free radicals can cause oxidative damage to cellular proteins and alter cellular function. One such susceptible protein is the enzyme glutamine synthase (GS). The chemical effects of CCl4 on cell proteins and their biological consequences are not known. Hence, in our study, the effect of CCl4 on liver protein oxidation and GS activity were investigated and compared with lipid peroxidation. A significant increase in liver protein carbonyl content (2-3 fold) and a significant decrease in hepatic GS activity (44-57%) were observed. Damage to proteins was rapid in onset and increased with time. Acute exposure of rats to CCl4 resulted in an increase in hepatic protein carbonyl content and a decrease in hepatic GS within 1 h. In cirrhosis of the liver induced by CCl4, the decrease in hepatic GS activity was accompanied by a significant increase in plasma ammonia levels. We conclude that protein oxidation may play a role in the pathogenesis of CCl4 induced liver injury and that the accumulation of oxidised proteins may be an early indication of CCl4 induced liver damage.

Abstract  To study the effect of an acute dose of ethanol on carbon tetrachloride (CCl4) concentration and hepatotoxicity, female rats received ethanol (2.5 ml/kg body wt.) either intragastrically or intraperitoneally following intragastric administration of CCl4 (1.5 ml/kg body wt.). Three hours after acute CCl4 intoxication there was a striking increase in CCl4 concentration in animals treated simultaneously with ethanol intragastrically compared to those receiving ethanol intraperitoneally. This increase was significant (P less than 0.05) and amounted to 211% for blood, 236% for liver and 405% for fat tissue, whereas animals treated with CCl4 alone showed CCl4 concentrations in the range between the two other experimental groups. Serum activities of glutamate oxalacetate transaminase, glutamate pyruvate transaminase and glutamate dehydrogenase were found to be considerably higher in animals treated with the combination of CCl4 and ethanol when compared to those receiving CCl4 alone, showing that ethanol given intraperitoneally or intragastrically enhances CCl4 hepatotoxicity. Since the intraperitoneal administration of ethanol led to a reduction rather than an increase in CCl4 concentration in the early phase of intoxication, additional mechanisms independent of actual levels of CCl4, such as direct effects of ethanol on the CCl4 metabolizing enzyme of the membrane of the endoplasmic reticulum, have to be implicated in the pathogenesis of the potentiation of CCl4 hepatotoxicity by ethanol.

Abstract  An assay is described for the measurement of chemically-induced DNA repair in cultures of primary rat hepatocytes following in vivo treatment with genotoxic agents. Rats were exposed to chemicals then primary hepatocytes were isolated by liver perfusion and cultured with [3H]-thymidine. DNA repair was measured as unscheduled DNA synthesis (UDS) by quantitative autoradiography. Cells from control animals consistently ranged from -2 to -6 net grains (NG). Treatment of rats with 10, 1 or 0.1 mg/kg dimethylnitrosamine (DMN), i.p., yielded 36.6, 6.4 and -0.9 NG, respectively; 10 mg/kg DMN, per os (p.o.), produced 22.2 NG. Oral doses of 50 or 5 mg/kg acetylaminofluorene (AAF) yielded 14.0 and 6.4 NG, respectively. Carbon tetrachloride (CCl4) at 100 or 10 mg/kg, p.o., yielded -3.2 and -5.1 NG, respectively. Thus, dose-related increases in UDS were observed for the hepatocarcinogens DMN and AAF while vehicle controls and the hepatotoxin CCl4 produced no response. An examination of the time-course of DNA repair following DMN treatment shows a linear decline in UDS during the first 24 h post-treatment followed by a slower decline from 24 to 48 h. These results indicate that this assay is a potentially useful system for assessing the genotoxic and potential carcinogenic activity of chemicals in the whole animal.

Abstract  Intracellular accumulation of Ca2+ can occur in the livers of animals poisoned with a toxic dose of CCl4. We have reported even greater accumulation of cytosolic Ca2+ in animals treated with an ordinarily nontoxic dose of CCl4 in combination with prior exposure to chlordecone (CD). Present studies were designed to examine if intact perfused livers obtained from animals receiving either CCl4 (100 μl/kg, ip) alone or in combination with prior dietary exposure to 10 ppm CD for 15 days accumulated 45Ca from the perfusate. Livers obtained at 0, 1, 4, 6, 12, 24, and 36 hr after a single CCl4 injection were perfused with Krebs-Ringer bicarbonate buffer containing erythrocytes, bovine serum albumin, and dextrose. After a 15-min equilibration, 45Ca was added to the perfusate, and the perfusion was continued for 30 min. Hepatic 45Ca accumulation in CCl4-treated animals in the whole liver or in the subcellular organelles obtained from perfused liver was not significantly different from corn oil controls. In the CD + CCl4 combination treatment, 45Ca accumulation followed a biphasic pattern with the first increase at 1 hr and then a progressive rise starting at 12 hr after CCl4 administration. Mitochondrial, microsomal, and cytosolic fractions from perfused liver showed a progressive rise in 45Ca at late time periods, which is indicative of uregulated and irreversible influx of extracellular Ca2+ into these livers. These studies demonstrate that cytosolic Ca2+ progressively increases as a result of the unregulated entry of extracellular Ca2+.

Abstract  OBJECTIVE AND DESIGN: The aim of the present study was to characterize during acute and chronic liver injury induced by CCl4, macrophage phenotypes and whether a change in reactive oxygen intermediates (ROI) and eicosanoids production by Kupffer cells (KC) was observed. MATERIAL AND METHODS: Liver steato-necrosis and cirrhosis were induced in rats after 3 weeks and 9 weeks of CCl4 intoxication, respectively. Monocytes and tissue macrophages were identified by immunohistochemical study using monoclonal antibodies ED-1 and tissue macrophages using the antibody ED-2. The release of ROI and eicosanoids in response to the phorbol ester TPA (protein kinase activator) and to the calcium ionophore A23187 was assessed in cultivated cells. RESULTS: As compared to healthy controls, livers of rats with steato-necrosis or cirrhosis exhibited a significant increase of ED-1 and ED-2 positive cells. Only KC from rats with liver steato-necrosis were found to have higher A23187, TPA + A23187 or opsonized zymosan induced ROI production than healthy controls (p < 0.01). After TPA + A23187 or opsonized zymosan stimulation, KC from both rats with steato-necrosis or cirrhosis produced more TxB2 and leukotrienes and less PGE2 as compared to healthy controls (p <0.05). CONCLUSIONS: These results suggest an influx of monocytes into the liver during acute and chronic injury induced by CCl4. Functional changes of this inflammatory infiltrate have been demonstrated with an increase of ROI production only in the early stage of liver injury whereas a rise in KC leukotriene production and an imbalance between cytoprotective and cytotoxic prostanoids were observed at all stages of liver disease.

Abstract  The volatile solvents carbon tetrachloride and chloroform are carcinogens that are often reported as nonmutagenic in bacterial mutagenicity assays. In this study, we evaluated the mutagenicity of these compounds in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, with and without S9 mix, using a gas exposure method. Tests were also conducted with a glutathione-supplemented S9 mix. Carbon tetrachloride was mutagenic in TA98 without S9 mix, and in WP2/ pKM101 and WP2uvrA/pKM101 with and without S9 mix; carbon tetrachloride was not mutagenic in TA100, TA1535 or TA1537. Chloroform was mutagenic in WP2/pKM101, but only in the presence of glutathione-supplemented S9 mix. Chloroform was not mutagenic in TA98, TA100, TA1535, TA1537, or WP2uvrA/pKM101 with or without S9 mix, and was not mutagenic in TA98, TA100, TA1535, TA1537, or WP2uvrA/pKM101 in the presence of glutathione-supplemented S9 mix. The data indicate that carbon tetrachloride and chloroform are bacterial mutagens when adequate exposure conditions are employed and suggest that a genotoxic mode of action could contribute to the carcinogenicity of these compounds.

Abstract  We examined which human CYP450 forms contribute to carbon tetrachloride (CCl(4)) bioactivation using hepatic microsomes, heterologously expressed enzymes, inhibitory antibodies and selective chemical inhibitors. CCl(4) metabolism was determined by measuring chloroform formation under anaerobic conditions. Pooled human microsomes metabolized CCl(4) with a K(m) of 57 microM and a V(max) of 2.3 nmol CHCl(3)/min/mg protein. Expressed CYP2E1 metabolized CCl(4) with a K(m) of 1.9 microM and a V(max) of 8.9 nmol CHCl(3)/min/nmol CYP2E1. At 17 microM CCl(4), a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl(4) metabolism in three separate human microsomal samples, indicating that at low CCl(4) concentrations, CYP2E1 was the primary enzyme responsible for CCl(4) metabolism. At 530 microM CCl(4), anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl(4) metabolism, suggesting that other CYP450s may have a significant role in CCl(4) metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not contribute significantly to CCl(4) metabolism. Effects of the CYP450 inhibitors alpha-naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CYP3A) were examined in the liver microsome sample that was inhibited only 36% by anti-CYP2E1 at 530 microM CCl(4). Clotrimazole inhibited CCl(4) metabolism by 23% but the other chemical inhibitors were without significant effect. Overall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl(4) bioactivation at lower, environmentally relevant levels. At higher CCl(4) levels, CYP3A and possibly other CYP450 forms may contribute to CCl(4) metabolism.

Abstract  Lanthanum was evaluated for potential genotoxicity using a range of in vitro assays (as the carbonate) in the presence and absence of post-mitochondrial fraction (S9) and in vivo in three independent tests for mutagenicity and clastogenicity (as the carbonate and chloride). The drug was devoid of mutagenic activity in bacterial assays (maximum concentration 5000 microg/plate) using a range of test strains (Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 and TA102 and Escherichia coli WP2 uvrA and WP2 uvrA pkm101). No effects were seen in the hgprt gene mutation assay in Chinese hamster ovary cells in the presence of S9. In the absence of S9, sporadic increases in revertant numbers were not dose-related or reproducible in subsequent experiments and hence were concluded to be chance events. In an in vitro chromosome aberration assay using Chinese hamster ovary cells, chromosome damage in the presence and absence of S9 (concentration 200-5000 microg/ml) was attributed to overt cell toxicity. To confirm this, a comprehensive in vivo evaluation of the drug was performed. Negative results were obtained in two independent rodent micronucleus tests. In the first mice were given oral doses (of carbonate) up to 2000 mg/kg, in the second rats were given a single i.v. bolus injection (of chloride) up to 0.1 mg/kg. Negative results were also obtained in a rat liver unscheduled DNA synthesis assay after treatment for 28 days with i.v. bolus injections (of chloride) up to 0.1 mg/kg/day. In these in vivo studies lanthanum plasma concentrations were >3000 times higher than the steady-state peak plasma concentration observed in dialysis patients given therapeutic doses of lanthanum carbonate. It can be concluded that lanthanum is not genotoxic and that lanthanum carbonate is unlikely to present a latent hazard in therapeutic use.

Abstract  The alteration in protein kinase and phosphatase activities in the liver nuclei of rats administered carbon tetrachloride (CCl(4)) was investigated. Rats received a single oral administration of CCl(4) (1 ml/100 g body wt of 5, 10, and 25% CCl(4) in corn oil), and 5, 24, and 48 h later they were euthanized by bleeding. The administration of CCl(4) (10 and 25%) caused a significant decrease in protein kinase activity in the liver nuclei. The enzyme activity in the liver nuclei from normal and CCl(4)-administered rats was significantly increased by the addition of Ca(2+) (0.5 mM) and calmodulin (10 microg/ml) in the reaction mixture, suggesting that Ca(2+)/calmodulin-dependent protein kinase activation is not suppressed by CCl(4) treatment. Liver nuclear phosphatase activity toward phosphotyrosine, but not phosphoserine and phosphothreonine, was markedly decreased by CCl(4) (5, 10, and 25%) administration. This decrease was seen 5 h after CCl(4) administration. The presence of vanadate (10(-4) M) in the reaction mixture caused a significant decrease in phosphotyrosine phosphatase activity in the liver nuclei from normal and CCl(4)-administered rats, whereas the enzyme activity was not decreased by okadaic acid (10(-5) M) or sodium fluoride (10(-3) M). The effect of anti-regucalcin antibody (100 ng/ml) in increasing phosphotyrosine phosphatase activity was seen in the liver nuclei of CCl(4)-administered rats, suggesting that regucalcin-sensitive phosphatase activity is decreased by CCl(4) administration. The present study demonstrates that CCl(4) administration induces a decrease in protein kinase and tyrosine phosphatase activities, which are involved in signaling factors in the liver nuclei of rats. Copyright 1999 Academic Press.

Abstract  1. Liver cirrhosis was induced in rats by CCl4 administration. We analysed the expression of endothelin receptor subtypes in the renal cortex and medulla using Scatchard analysis and receptor autoradiography, and measured plasma as well as renal-tissue endothelin-1 concentrations using a specific radioimmunoassay. Furthermore, we analysed the effects of the non-selective (A/B) endothelin receptor antagonist, bosentan (6 and 100 mg kg-1 day-1) on mean arterial blood pressure, water and sodium excretion and glomerular filtration rate. 2. Our study revealed an overexpression of the endothelin B receptor (ETB) in the renal medulla of rats with liver cirrhosis (Cir: 2775 +/- 299 fmol mg-1; Con: 1695 +/- 255 fmol mg-1; n = 8; means +/- s.d., P < 0.01), whereas the density of ETB in the cortex and the endothelin A receptor (ETA) in the cortex and medulla were similar in both cirrhotic and control rats. Receptor autoradiography showed that the upregulation of medullary ETB in cirrhotic rats was due to an upregulation of ETB in the inner medullary collecting duct cells. 3. The tissue endothelin-1 concentrations were increased in the renal medulla of cirrhotic rats (Cir: 271 +/- 68 pg g-1wet wt.; Con: 153 +/- 36 pg g-1 wet wt., n = 8; means +/- s.d., P < 0.01). 4. The glomerular filtration rate was slightly decreased in cirrhotic rats but not altered after bosentan treatment in either cirrhotic or control rats. Bosentan decreased sodium excretion to a similar extent in both cirrhotic and control rats, whereas water excretion was significantly reduced by both dosages of bosentan in cirrhotic rats only (Cir + vehicle: 12.5 +/- 0.62 m day-1, Cir + 6 mg kg-1 day-1 bosentan: 8.6 +/- 1.0 ml day-1; Cir + 100 mg kg-1 day-1 bosentan: 7.4 +/- 0.6 ml day-1; n = 10; means +/- s.e.mean). 5. We therefore suggest that the upregulation of the medullary ETB in cirrhotic rats is involved in the regulation of water excretion in rats with CCl4-induced liver cirrhosis.

Abstract  Hepatotoxicants can elicit liver damage by various mechanisms that can result in cell necrosis and death. The changes induced by these compounds can vary from gross alterations in DNA repair mechanisms, protein synthesis, and apoptosis, to more discrete changes in oxidative damage and lipid peroxidation. However, little is known of the changes in gene expression that are fundamental to the mechanisms of hepatotoxicity. We have used DNA microarray technology to identify gene transcription associated with the toxicity caused by the hepatotoxicant carbon tetrachloride. Labeled poly A+ RNA from cultured human hepatoma cells (HepG2) exposed to carbon tetrachloride for 8 hours was hybridized to a human microarray filter. We found that 47 different genes were either upregulated or downregulated more than 2-fold by the hepatotoxicant compared with dimethyl formamide, a chemical that does not cause liver cell damage. The proinflammatory cytokine interleukin-8 (IL-8) was upregulated over 7-fold compared with control on the array, and this was subsequently confirmed at 1 hour and 8 hours by Northern blot analyses. We also found that carbon tetrachloride caused a time-dependent increase in interleukin-8 protein release in HepG2 cells, which was paralleled by a decrease in cell viability. These data demonstrate that carbon tetrachloride causes a rapid increase in IL-8 mRNA expression in HepG2 cells and that this increase correlates with a later and significant increase in the levels of interleukin-8 protein. These results illustrate the potential of microarray technology in the identification of novel gene changes associated with toxic processes.

Abstract  A simplified method for the quantitation of DNA damage in nonlabeled hepatocytes, using a fluorometric technique for the quantitation of DNA in conjunction with a modification of the alkaline elution technique of Kohn et al [1976], following chemical treatment in vitro and in vivo, is described. Freshly isolated hepatocytes were treated in vitro with 2-acetylaminofluorene, aflatoxin B1, and dimethylnitrosamine, then examined for DNA damage. Exposure to each of these compounds resulted in DNA damage. Hepatocytes isolated from rats treated with the hepatocarcinogens 2-acetylaminofluorene, benzidine, azoxymethane, dimethylhydrazine, dimethylnitrosamine, and diethylnitrosamine sustained DNA damage as evidenced by increased alkaline elution. DNA damage in hepatocytes was also observed as a result of treatment with methylmethanesulfonate and azaserine. The hepatotoxin carbon tetrachloride did not induce DNA damage in hepatocytes isolated from treated animals. A comparison of the induction of DNA damage and of unscheduled DNA synthesis in hepatocytes from the same animals revealed that in most cases the extent of elution of DNA from filters was proportional to the induction of DNA repair.

Abstract  Abstract: super(1) super(4)C from super(1) super(4)CCl sub(4) irreversibly bind in vivo) to liver DNA from strain A/J mice and Sprague-Dawley rats. Binding of super(1) super(4)CCl sub(4) to DNA was also observed in vitro) in incubation mixtures containing microsomes and a NADPH-generating system as well as in tissue slices. Chemically induced multiplied by CCl sub(3) (CCl sub(4) + benzoyl peroxide system) intensively binds to DNA. Liver nuclear proteins also irreversibly bind CCl sub(4) metabolites. Nuclear protein fractionation studies revealed that deoxyribonucleoproteins, acidic proteins, histones, and residual proteins are the favorite targets of metabolite interaction. Nuclear sap proteins are less intensively labeled. Nuclear lipids were markedly labeled by CCl sub(4) reactive metabolites. Most of the label is in the phospholipid fraction and diphosphatidylglycerol is most intensively labeled; phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and sphingomyeline are also labeled by metabolites. ds: carbon tetrachloride evised - 2006-11-01 e field[29]: 1,4-Dioxane