Carbon Tetrachloride Toxicity Research Articles

The in vivo toxicity of carbon tetrachloride and carrageenan on heart microsomes: analysis by Fourier transform infrared spectroscopy

We investigated the sensitivity of rat heart microsomes to free radical attack using Fourier transform infrared (FT-IR) spectroscopy. This physico-chemical method seemed a valuable technique: quite sensitive to changes in the vibrational spectra. The spectral variations observed between normal and treated rats were in great part due to reactive oxygen species that led to changes in protein conformation involving beta-sheets, aggregation of proteins, and modification of protein synthesis. Carrageenan-induced inflammation slightly enhanced the total lipid content; rearrangement of acyl chains and accumulation of cholesterol esters and phospholipids also occurred in the treated rats. Carbon tetrachloride induced a decrease in both lipid and protein contents. The level of glucidic substrates was diminished with carbon tetrachloride and enhanced with carrageenan; these changes were due to metabolic interactions between cell components and drugs. FT-IR spectroscopy provided an accurate means to monitor, in rat heart, the in vivo effects of inflammatory and peroxidative damages, to discriminate and classify the affected cells, and to correlate the findings with known physiological and biochemical data in close relationship with metabolic disruptions induced by the two xenobiotics.

Pharmacologic application of fourier transform IR spectroscopy: in vivo toxicity of carbon tetrachloride on rat liver.

Microsomal fractions from rat liver were examined by means of Fourier transform IR (FTIR) spectroscopy to study the in vivo toxic effect of carbon tetrachloride administered by intraperitoneal injection. Lipid content was significantly enhanced in the liver of treated rats compared with untreated ones. The level of saturated fatty acids largely increased while that of unsaturated acids slightly decreased as a consequence of lipid peroxidation induced by the xenobiotic compound. The conformational structure of membrane proteins was changed, which was shown by the large decrease in the alpha-helical configuration. In the polysaccharide region we observed an important loss in glucidic structures that could be related to the metabolic changes caused by carbon tetrachloride intoxication. Thus, FTIR spectroscopy appears to be a useful tool to rapidly investigate the chemical alterations induced by this drug in liver microsomes and to correlate them with biochemical and physiological data.

Mechanism of early carbon tetrachloride toxicity in cultured rat hepatocytes.

CCl4 causes liver necrosis in a dose-dependent manner in vivo. However, we found that primary rat hepatocytes in culture were killed after a 2 hr incubation with carbon tetrachloride gas at CCl4 partial pressures above a threshold between 45 and 54 mmHg. Below this threshold concentration no increase in hepatocyte death was observed. We sought to explain the very abrupt CCl4 concentration threshold for hepatocyte death. Two inhibitors of cytochrome P450 2E1, cimetidine and diallyl sulfide, inhibited lipid peroxidation as measured by production of isoprostanes, but did not reduce hepatocyte death from CCl4. At 37 degrees, CCl4 accelerated the mitochondrial permeability transition in vitro, at a threshold CCl4 concentration similar to that which caused hepatocyte death. Phospholipase A2 inhibitors, mepacrine and 4-bromophenacyl bromide, inhibited the increase in mitochondrial permeability, but did not inhibit hepatocyte death caused by CCl4. Rat liver microsomal lipids were used to make liposomes loaded with Ponceau Red (FW 760.6). No leakage of Ponceau red was found at CCl4 concentrations greater than the threshold for cell death. However, CCl4 caused acceleration of liposome fusion, over the CCl4 concentration range spanning the threshold for hepatocyte death. Early hepatocyte death in cell culture is independent of metabolism of CCl4, and may be related to direct effects of CCl4 on intracellular membranes.

Evaluation of sex difference in tissue repair following acute carbon tetrachloride toxicity in male and female Sprague–Dawley rats

Cellular regeneration and tissue repair greatly influence the outcome of acute carbon tetrachloride (CCl 4) hepatotoxicity. This study examined the temporal kinetics of cellular regeneration and tissue repair processes in male and female Sprague–Dawley (SD) rats following an acute CCl 4 exposure (0.8 ml/kg, i.p.). In female rats, hepatic damage peaked at 24 h following the treatment and was ∼2.5-fold (AST 2.7-fold, ALT 2.3 fold) greater than the damage observed in male rats. The hepatic damage in male rats appeared to peak by 3 h post-exposure and did not significantly change through the 36-h time-point. The activity of cytochrome P 4502E1 was 20% greater in male rats and did not correlate with the magnitude of hepatic damage. Morphometric analysis of cell cycle indices revealed that cellular regeneration was significantly greater in female rats as compared to male rats at 48 h and corresponded proportionally to the extent of liver damage. This study demonstrated that female SD rats respond more severely to acute CCl 4 hepatotoxicity than male SD rats and the extent of tissue repair and cellular regeneration was greater in female rats. Furthermore, our results suggest that tissue repair is unlikely to result in accounting for the different responses exhibited by male and female SD rats to CCl 4 hepatotoxicity.

Metallothionein-independent hepatoprotection by zinc and sakuraso-saponin

Hepatoprotective activities of zinc and sakuraso-saponin against toxicity of carbon tetrachloride were investigated in metallothionein (MT)-deficient mice. Pretreatment of control 129/Sv mice with zinc or sakuraso-saponin blocked carbon tetrachloride-induced elevation of plasma transaminase activities. Quantitatively equivalent protection against carbon tetrachloride-induced hepatic damage was also observed in MT-deficient mice. Zinc and sakuraso-saponin caused elevation of hepatic MT levels in control 129/Sv mice, whereas hepatic MT was undetectable in MT-deficient mice. To examine the possibility that sakuraso-saponin-induced hepatoprotection is mediated by endogenous zinc, the hepatic concentration of zinc was analyzed. Hepatic zinc concentration in MT-deficient mice was not changed by the treatment of sakuraso-saponin. Injection of sakuraso-saponin caused a decrease of activity of aniline hydroxylation. The suppression of cytochrome P450 appears to be a mechanism by which sakuraso-saponin protects mice from the hepatotoxic effects of carbon tetrachloride. These findings indicate that the hepatoprotective activity of zinc or sakuraso-saponin is not dependent on their MT-inducing activity.

Protection against Hepatotoxicity by a Single Dose of Amphetamine: The Potential Role of Heat Shock Protein Induction

Amphetamine has been shown previously to increase levels of the inducible 70-kDa heat shock protein (hsp70i) in mouse liver. In the present study, the hepatic concentrations of a variety of hsps in livers of mice pretreated with amphetamine (15 mg/kg, ip) were evaluated, and the time course of hsp induction was examined. Amphetamine treatment caused an acute rise in core body temperature to 40°C for at least 1 hr and increased hsp25 and hsp70i levels, as measured by Western blotting, at 6, 24, 48, and 72 hr with no apparent induction of other hsps (hsp60, hsc70, or hsp90). A 72-hr amphetamine pretreatment lowered the hepatotoxicity of an acute dose of acetaminophen (350 mg/kg, ip) or bromobenzene (0.45 ml/kg, ip), but had no effect on the toxicity of carbon tetrachloride (0.04 ml/kg, ip) or cocaine (50 mg/kg, ip), as measured by serum alanine aminotransferase activity and histopathological analysis. No protection from acetaminophen or bromobenzene hepatotoxicity was observed when hepatotoxicant administration was delayed until hsp levels had returned to control values (144 hr after amphetamine pretreatment). Amphetamine pretreatment did not reduce in vivocovalent binding to proteins of radiolabeled [ 3H]acetaminophen, [ 14C]bromobenzene, [ 14C]carbon tetrachloride, or [ 3H]cocaine, indicating that the protective effects were not due to inhibition of reactive metabolite formation from these toxicants. These results suggest that elevated levels of hsp25 and hsp70i provide protection against acetaminophen and bromobenzene hepatotoxicity.

Effect of dosing vehicle on the developmental toxicity of bromodichloromethane and carbon tetrachloride in rats.

Several halocarbons have been shown to cause full-litter resorption (FLR) in Fischer-344 rats when administered orally in corn oil. Since halocarbons often occur as contaminants of drinking water, we sought to determine the influence of the vehicle, aqueous versus lipid, on the developmental toxicity of two of these agents. In separate assays, bromodichloromethane (BDCM) and carbon tetrachloride (CCl4) were administered by gavage to Fischer-344 rats on gestation days (GD) 6-15 at 0, 25, 50, or 75 mg/kg/day in either corn oil or an aqueous vehicle containing 10% Emulphor EL-620. Dams were allowed to deliver and the litters were examined postnatally. Uteri of females that did not deliver were stained with 10% ammonium sulfide to detect FLR. Effects of both agents on maternal weight gain were slightly more pronounced in the aqueous vehicle at lower doses, but at the highest dose, CCl4 was more maternally toxic in corn oil. Developmentally, both agents caused FLR at 50 and 75 mg/kg in both vehicles. At 75 mg/kg, dams receiving corn oil had significantly higher rates of FLR (83% for BDCM, 67% for CCl4) compared to their aqueous-vehicle counterparts (21% for BDCM, 8% for CCl4). Blood concentrations of BDCM following GD-6 gavage revealed a shorter elimination half-life in the aqueous dosing vehicle (2.7 h) compared to the oil vehicle (3.6 h). Benchmark doses of CCl4 were similar for the oil (18.9 mg/kg) and aqueous (14.0 mg/kg) vehicles. For BDCM, the corn oil vehicle yielded a less conservative (i.e., higher) value (39.3 mg/kg) than the aqueous vehicle (11.3 mg/kg), reflecting different confidence intervals around the estimated 5%-effect dose levels.

Correlation and Reproducibility Data on a Replacement Agent for Carbon Tetrachloride Testing of Organic Vapor Cartridges

Abstract Carbon tetrachloride has been used for years as the standard compound for evaluating the adsorption capacity of charcoal. The National Institute for Occupational Safety and Health is mandated under 42 CFR 84 to test organic vapor/gas cartridge and canister breakthrough characteristics against carbon tetrachloride. However, due to carbon tetrachloride's toxicity and potential lack of commercial availability, a substitute test agent is desirable. Screening tests employing ethyl acetate, pentane, n-hexane, and heptane have been completed and published on negative-pressure organic vapor respirator cartridges. Two test conditions (550 ppm pentane and 1000 ppm n-hexane) were selected for limited side-by-side testing with the 42 CFR 84 test criterion of 1000 ppm carbon tetrachloride. This article presents previously unpublished cartridge breakthrough time data on negative-pressure and powered air-purifying respirator organic vapor cartridges. The data were generated to establish a correlation between possible substitute test agents and carbon tetrachloride. Reproducibility data not previously reported are also presented. The results show that both pentane and n-hexane are potential replacement compounds for carbon tetrachloride. Additional work with less-adsorbent charcoals still needs to be performed.

Toxicity of chloroform and carbon tetrachloride in primary cultures of rainbow trout hepatocytes

The toxicity of two chlorinated hydrocarbons, carbon tetrachloride (CCl 4) and chloroform (CHCl 3) was studied in primary cultures of rainbow trout ( Oncorhynchus mykiss) hepatocytes. The aim was to study the cytotoxicity of these two chemicals in vitro in fish hepatocytes and to evaluate the sensitivity of the model system in comparison to mammalian systems. Both chemicals showed a steep dose response in this system with CCl 4 being five times more toxic than CHCl 3 measured as LDH release. Glutathione levels were rapidly depleted in the presence of CCl 4 and CHCl 3. There was a difference, however, in the pattern of GSH depletion. CCl 4-induced GSH depletion followed the release of LDH, while CHCl 3 caused a significant decrease in the GSH content prior to LDH release. The cytochrome P450 inhibitor, SKF-525A (12 μM) decreased the toxicity of 5 mM CCl 4 and of doses ranging from 10 to 25 mM CHCl 3. The addition of the antioxidant DPPD (20 μM) decreased the toxicity of both CCl 4 and CHCl 3. The protection was more pronounced with CHCl 3 than with CCl 4. When the time course of GSH depletion with EC 50 concentrations of each chemical was studied, DPPD effectively protected the cells from CHCl 3-induced GSH depletion, while no significant maintenance of GSH was seen in cells treated with DPPD and CCl 4. The relationship between DNA single strandbreaks (SSB) and cytotoxicity may provide some insight into potential carcinogenicity of non-genotoxic chemicals. In cells treated with CHCl 3, DNA SSB were seen only concommitant with high toxicity. With CCl 4, however, DNA SSB were seen in the absence of LDH release at 2.5 mM. This study indicates that the toxicity of CCl 4 and CHCl 3 in trout hepatocytes is due to metabolism and the formation of free radicals at doses causing low toxicity and to a combination of metabolism dependency and solvent effect at doses causing high toxicity. This study also indicates that there are differences in the mechanisms of toxicity of CHCl 3 and CCl 4 in rainbow trout hepatocytes. Furthermore, the model system based on primary cultures of rainbow trout hepatocytes is a sensitive tool in toxicological studies and is comparable to model systems based on mammalian hepatocytes.

Dose- and Route-Dependent Alterations in Metabolism and Toxicity of Chemical Compounds in Ethanol-Treated Rats: Difference between Highly (Chloroform) and Poorly (Carbon Tetrachloride) Metabolized Hepatotoxic Compounds

The dose and route dependency of the metabolism and the toxicity of chloroform (CHCl3) and carbon tetrachloride (CCl4) was investigated in ethanol-treated rats. Rats that had been kept on either an ethanol (containing ethanol at 2 g/rat/day) or a control (containing no ethanol) liquid diet for 3 weeks were challenged with CHCl3or CCl4by inhalation (0, 50, or 500 ppm × 6 hr), or by po or ip administration (0, 0.105, or 1.675 mmol/kg). Ethanol consumption, which increased thein vitrometabolism of both compounds six- to sevenfold, affected the metabolism and toxicity of CCl4differently from those of CHCl3. Ethanol increased the metabolism and toxicity of CHCl3at 500 ppm only, whereas it increased the metabolism and toxicity of CCl4at either 50 or 500 ppm. In addition, the effect of ethanol consumption differed between CHCl3and CCl4depending on the route of administration (po or ip) and the dose (0.105 or 1.675 mmol/kg). For the po route, ethanol increased the metabolism and toxicity of both compounds at either dose. For the ip route, however, ethanol increased the metabolism and toxicity of the high dose of CHCl3only, but of both CCl4doses. The dose- and route-dependent differences in the effect of enzyme induction on the metabolism and toxicity between CHCl3and CCl4can be explained by a supposition that the hepatic blood flow rate-limits the metabolism of a low dose of CHCl3(perfusion-limited metabolism), whereas the metabolic capacity of the liver limits the metabolism of CCl4(capacity-limited metabolism) irrespective of dose.

Effect of Dosing Vehicle on the Developmental Toxicity of Bromodichloromethane and Carbon Tetrachloride in Rats

Effect of Dosing Vehicle on the Developmental Toxicity of Bromodichloromethane and Carbon Tetrachloride in Rats

Alterations in susceptibility to carbon tetrachloride toxicity and hepatic antioxidant/detoxification system in streptozotocin-induced short-term diabetic rats: effects of insulin and Schisandrin B treatment.

The streptozotocin-induced short-term (2 week) diabetic rats showed an increase in susceptibility to carbon tetrachloride (CCl4)-induced hepatocellular damage. This diabetes-induced change was associated with a marked impairment in the hepatic glutathione antioxidant/detoxification response to CCl4 challenge, as indicated by the abrogation of the increases in hepatic reduced glutathione (GSH) level, glucose-6-phosphate dehydrogenase and microsomal glutathione S-transferases (GST) activities upon challenge with increasing doses of CCl4. While the hepatic GSH level was increased in diabetic rats, the hepatic mitochondrial GSH level and Se-glutathione peroxidase activity were significantly reduced. Insulin treatment could reverse most of the biochemical alterations induced by diabetes. Both insulin and schisandrin B (Sch B) pretreatments protected against the CCl4 hepatotoxicity in diabetic rats. The hepatoprotection was associated with improvement in hepatic glutathione redox status in both cytosolic and mitochondrial compartments, as well as the increases in hepatic ascorbic acid level and microsomal GST activity. The ensemble of results suggests that the diabetes-induced impairment in hepatic mitochondrial glutathione redox status may at least in part be attributed to the enhanced susceptibility to CCl4 hepatotoxicity. Sch B may be a useful hepatoprotective agent against xenobiotics-induced toxicity under the diabetic conditions.

Schisandrin B protects against carbon tetrachloride toxicity by enhancing the mitochondrial glutathione redox status in mouse liver

Previous studies in our laboratory have demonstrated the effect of Schisandrin B (Sch B), an active ingredient of the fruit of Schisandra chinensis, on enhancing the hepatic glutathione antioxidant system in mice, as evidenced by the hepatoprotection against carbon tetrachloride (CCl 4) toxicity. In the present study, the mechanism involved in the hepatoprotection afforded by Sch B treatment was investigated. Treating female Balb/c mice with 1,3-bis(2-chloroethyl)-l-nitrosourea, an inhibitor of glutathione reductase (GRD), at a dose of 2 mmol/kg (i.p.) did not abrogate the hepatoprotective action of Sch B in CCl 4-treated mice. The result indicates that the increased activity of hepatic GRD is not ascribable to the hepatoprotective action of Sch B. In control mice, the same Sch B treatment regimen caused an enhancement in hepatic mitochondrial glutathione redox status, as indicated by the significant increase and decrease in reduced and oxidized glutathione levels, respectively. While the CCl 4 intoxication greatly impaired mitochondrial glutathione redox status, the beneficial effect of Sch B treatment became more evident after CCl 4 challenge. Our results strongly suggest that the mechanism of hepatoprotection afforded by Sch B treatment may involve the enhancement of mitochondrial glutathione redox status.

Pretreatment with Aluminum and Gallium Relieved the Acute Lethal Toxicity of Carbon Tetrachloride in Mice (Proceedings of the 21st Symposium on Toxicology and Environmental Health)

Pretreatment with Aluminum and Gallium Relieved the Acute Lethal Toxicity of Carbon Tetrachloride in Mice (Proceedings of the 21st Symposium on Toxicology and Environmental Health)

Effect of schisandrin B on hepatic glutathione antioxidant system in mice: protection against carbon tetrachloride toxicity.

Pretreating female Balb/c mice with schisandrin B (Sch B) at increasing daily doses (1-4 mmol/kg) for 3 days caused dose-dependent increases in hepatic glutathione S-transferase (GST) and glutathione reductase (GRD) activities. However, the activities of glucose-6-phosphate dehydrogenase (G6PDH), Se-glutathione peroxidase (GPX), and gamma-glutamylcysteine synthetase (GCS) were down-regulated to varying degrees in a dose-dependent manner. While there were biphasic changes in hepatic reduced glutathione (GSH) level as well as susceptibility of hepatic tissue homogenates to in vitro peroxide-induced GSH depletion, a gradual decrease in hepatic malondialdehyde content was observed. The beneficial effect of Sch B on the hepatic GSH anti-oxidant system became more evident after CCl4 challenge. The same Sch B pretreatment regimen caused a dose-dependent protection against carbon tetrachloride (CCl4)-induced hepatotoxicity. The hepatoprotection was associated with significant enhancement in hepatic GSH status, as indicated by the substantial increase in tissue GSH levels and the corresponding decrease in susceptibility of tissue homogenates to GSH depletion. Where the activities of GST and GRD were increased linearly over non-CCl4 control values, there was also a gradual elevation in G6PDH activity upon administration of increasing doses of Sch B. In contrast, GPX activity was moderately down-regulated. The ensemble of results suggests that the hepatoprotection afforded by Sch B pretreatment may mainly be attributed to the enhancement in the functioning of the hepatic GSH anti-oxidant system, possibly through stimulating the activities of GSH related enzymes.

Further Evidence for Zinc as a Hepatoprotective Agent in Rat Liver Toxicity

The present study was envisaged to investigate the protective effects of zinc treatment on the activities of various drug metabolizing enzymes, reduced glutathione content, and extent of lipid peroxidation in the livers of male albino rats subjected to long-term carbontetrachloride (CCl 4) toxicity. It was observed that the activities of cytochrome P450, cytochrome b 5. NADPH-cytochrome- e-reductase, and glutathione concentrations were significantly depressed following treatment with CCl 4 for 6 weeks. At the same time, a marked elevation was seen in the extent of NADPH-dependent lipid peroxidation after CCl 4 toxicity. Moreover, significantly depressed concentrations of zinc (Zn 2+) were observed in both serum and liver after 6 weeks of the CCl 4 administration. However, simultaneous zinc treatment to CCl 4-intoxicated rats resulted in significant improvement in the activities of most of the drug metabolizing enzymes as well as the glutathione levels. Similarly, zinc treatment also normalized the CCl 4-induced increase in the extent of NADPH-dependent lipid peroxidation. We conclude that zinc supplementation considerably attenuates the liver injury induced by chronic CCl 4 treatment to rats.

Evaluation of Carbon Tetrachloride Replacement Agents for Use in Testing Nonpowered Organic Vapor Chin-Style Canisters

Abstract For years, charcoal's ability to adsorb organic compounds has been evaluated employing carbon tetrachloride. The breakthrough characteristics for chin-style canisters against organic vapors mandate the use of carbon tetrachloride as per 30 CFR, Part 11. However, due to carbon tetrachloride's toxicity and the lack of commercial availability, a substitute organic vapor test agent is needed. This article deals with the evaluation of four potential substitute agents (ethyl acetate, pentane, n-hexane, and heptane) for testing chin-style canisters. Screening tests identified replacement agent challenge concentrations that gave breakthrough characteristics similar to 5000 parts per million (ppm) carbon tetrachloride. Side-by-side correlation testing was done between 4000 ppm pentane at 64 L/min, 80 percent relative humidity (RH), and 25°C with the most critical 30 CFR, Part 11 test condition (85% precondition followed by testing at 50% RH, 32 L/min, and 25°C). Finally, a reproducibility study was perfor...

Toxicology studies of a chemical mixture of 25 groundwater contaminants: hepatic and renal assessment, response to carbon tetrachloride challenge, and influence of treatment-induced water restriction.

Because groundwater contamination is an important environmental concern, we examined the hepatic and renal effects of repeated exposure to a mixture of 25 chemicals frequently found in groundwater near hazardous-waste disposal sites and the effect of such exposure on carbon tetrachloride (CCI4) toxicity. Adult male F-344 rats received ad libitum deionized water and feed (Ad Lib Water) or ad libitum 10% MIX (referring to 10% of a technically achievable stock mixture) and feed for 14 d. Because exposure to the 25-chemical mixture via the drinking water resulted in decreased water and feed consumption, restricted deionized water and feed controls (Restricted Water) were included. On d 14, rats were gavaged with 0, 0.0375, 0.05, 0.075 or 0.15 ml CCl4/kg, and hepatic and renal toxicity assessed 24 h later. Little or no hepatic and renal toxicity was observed in rats exposed to 10% MIX alone. No hepatic or renal lesions occurred that could be attributed to 10% MIX alone. Slight but statistically significant alterations, of uncertain biological significance, resulted from the water treatments: 10% MIX increased alanine aminotransferase, urea nitrogen (BUN), and BUN/creatinine ratio; Restricted Water increased 5'-nucleotidase and decreased alkaline phosphatase. Relative kidney weight was increased by both 10% MIX and Restricted Water. CCI4 resulted in significant dosage-dependent hepatotoxicity in all three water treatment groups but had little or no effect on renal indicators of toxicity. Relative to Ad Lib Water, significantly greater hepatotoxicity occurred in both 10% MIX and Restricted Water rats. The response to CCI4 in the Restricted Water rats was similar to that of 10% MIX rats, indicating that a substantial portion of the effect of 10% MIX on CCI4 hepatotoxicity is due to decreased water and feed intake.

Role of metallothionein in zinc(II) and chromium(III) mediated tolerance to carbon tetrachloride hepatotoxicity: evidence against a trichloromethyl radical-scavenging mechanism.

The .CCl3 radical generated during the metabolism of CCl4 is readily spin trapped in vivo and in vitro by phenyl N-tert-butylnitrone (PBN) to form the stable PBN/.CCl3 radical adduct, which can then be extracted into organic solvents and detected by ESR spectroscopy. We have used this technique to examine the proposed protective roles of Zn(II), Cr(III), and metallothionein (MT) against carbon tetrachloride toxicity in vivo. Hepatic MT, which is induced by Zn(II), has been proposed to protect against CCl4-induced cellular damage by scavenging the free radical metabolites formed. CCl4-induced hepatotoxicity was significantly suppressed in male Sprague-Dawley rats pretreated with a single dose of 5 mg/kg Zn(II) or Cr(III) according to standard serum assays for liver-specific enzymes, and hepatic MT was elevated after pretreatment with either Zn(II) or Cr(III). In vitro, no difference was detected in either the amount of CCl4-derived free radical metabolites formed or the rate at which they were formed by microsomes from rats pretreated 24 h in advance with 5 mg/kg Zn(II) or Cr(III). Extraction of rat liver with 2:1 chloroform/methanol 1 h after the administration of a 0.8 mL/kg intraperitoneal or intragastric dose of CCl4 also revealed no difference in the amount of trichloromethyl radical spin trapped in vivo following pretreatment with either Zn(II) or Cr(III). These results suggest that pretreatment with either Zn(II) or Cr(III) does not affect CCl4 metabolism nor does the MT significantly scavenge the trichloromethyl free radical metabolite.

The Involvement of Kupffer Cells in Carbon Tetrachloride Toxicity

Carbon tetrachloride (CCl 4) is a classical pericentral hepatotoxicant; however, precise details of its mechanism of action remain unknown. One possibility is that Kupffer cells participate in this mechanism since CCI4 elevates calcium, and the release of toxic eicosanoids and cytokines by Kupffer cells is calcium-dependent. Therefore, these studies were designed to evaluate the role of Kupffer cells in CCl 4 toxicity in the rat in vivo. Kupffer cells were destroyed selectively with gadolinium chloride treatment (10 mg/kg GdCl 3 iv) 1 day prior to administration of CCl 4 (4 g/kg ig). Twenty-four hours after CCl 4 treatment, rats were anesthetized, blood samples were drawn for aspartate aminotransferase (AST) determination, which is indicative of parenchymal cell damage, and trypan blue was infused into the liver to stain the nuclei of dead hepatocytes. AST levels were in the normal range and trypan blue staining was negligible in livers from vehicle- or GdCl 3-treated rats. As expected, CCl 4 treatment alone elevated AST levels to values over 4000 U/liter and caused massive cell death (60-90 trypan blue-positive cells/ pericentral field). In dramatic contrast, the elevation in AST and cell death due to CCl 4 were almost completely prevented by GdCl 3 treatment. In attempts to understand this phenomenon, metabolic and detoxification pathways were assessed. CCl 4 is metabolized via cytochrome P450 II.E. 1; however, GdCl 3 treatment did not alter this pathway as assessed from p-nitrocatechol formation from the selective substrate, p-nitrophenol. GdCl 3 treatment also had no effect on hepatic glutathione levels. On the other hand, GdCl 3 treatment significantly reduced infiltration of neutrophils resulting from exposure to CCl 4. These data clearly support the hypothesis that Kupffer cells participate in the mechanism of toxicity of CCl 4 in vivo, possibly by release of chemoattractants for neutrophils.