Interactions of Water Contaminants I. Plasma Enzyme Activity and Response Surface Methodology following Gavage Administration of CCl4 and CHCl3 or TCE Singly and in Combination in the Rat

Interactions of Water Contaminants. I. Plasma Enzyme Activityand Response Surface Methodology following Gavage Administrationof CCl₄ and CHCl₃ or TCE Singly and in Combination in the Rat.BORZELLECA, J. F., O'HARA, T. M., GENNINGS, C, GRANGER, R. H.,SHEPPARD, M. A., AND CONDIE, L. W., JR. (1990). Fundam. Appl.Toxicol. 14, 477–190. The joint hepatotoxicity of CCUand CHC13 or TCE in male CD rats following simultaneous oraladministration has been investigated. Rats with chronic indwellingarterial cannulas were administered a single oral dose of COl₄,and CHCl₃ or CCl₄ and TCE in 5% Emulphor at doses of 0 to 700mg/kg. Hepatotoxicity was evaluated by measuring the activityof AST, ALT, and SDH in plasma at 0, 3, 6, 12, 24, 36, 48, and72 hr postgavage. Response data were analyzed for interactionusing response surface methodology. CCl₄ alone displayed dose-dependenttoxicity. TCE demonstrated little evidence of hepatotoxicity.In combination, both CCl₄/CHCl₃ and CCl₄/TCE displayed a synergistic(supraadditive) response for peak plasma enzyme activity.     

Chloroform Hepatotoxicity in the Mongolian Gerbil

Chloroform Hepatotoxicity in the Mongolian Gerbil. EBEL, R.E., BARLOW, R., and MCGRATH, E. A. (1987). Fundam. Appl. Toxicol.8, 207–216. CHCl₃ hepatotoxicity was studied in the maleMongolian gerbil and compared to that in the male Sprague–Dawleystrain rat. Based on elevations in serum transaminase activitiesin response to CHCl₃ exposure, control gerbils were more sensitiveto CHCl₃ than were gerbils treated with phenobarbital, chlordecone,mirex, or 3-methylcholanthrene. The increased sensitivity ofthe control relative to the induced gerbil was consistent withearlier observations of CCl₄ hepatotoxicity (Ebel, R. E., andMcGrath, E. A., 1984, Toxicol. Lett., 22, 205–210). Microsomalenzyme concentrations or activities were not decreased in thecontrol or induced gerbils in response to CHCl₃ exposures ofup to 200 µl/kg. At a dose of 500 µl/kg, cytochromeP-450 and its reductase were decreased by about 25% in the chlordecone-inducedgerbil. In contrast, chlordecone- and phenobarbital-inducedrats were sensitive to CHCl₃ as evidenced by marked elevationsin serum transaminase activities, decreases in microsomal enzymeconcentrations or activities, and a transient decrease in hepaticnonprotein sulfhydryl groups. Control rats were insensitiveto CHCl₃. Histopathological changes in the livers of these animalswere consistent with alterations in the biochemical parametersmeasured. The relationship between sensitivity to the hepatotoxiceffects of CHCl₃ and CCl₄ was different for the gerbil and rat.     

Pretreatment with Drinking Water Solutions Containing Trichloroethylene or Chloroform Enhances the Hepatotoxicity of Carbon Tetrachloride in Fischer 344 Rats

Pretreatment with Drinking Water Solutions Containing Trichloroethyleneor Chloroform Enhances the Hepatotoxicity of Carbon Tetrachloridein Fischer 344 Rats. Steup, D. R., Wiersma, D., McMillan, D.A., and Sipes, I. G. (1991). Fundam. Appl. Toxicol. 16, 798–809.Previous studies have demonstrated that various compounds, includingthe common groundwater contaminants trichloroethylene (TCE)and chloroform (CHCl³), can produce a synergistic toxic responsewhen coadministered with the model hepatotoxicant carbon tetrachloride(CCI₄. This phenomenon has not, however, been demonstrated followingadministration of these compounds in drinking water. Initialexperiments indicated that Fischer 344 (F-344) rats were significantlymore sensitive to these effects than the more commonly utilizedSprague–Dawley strain. To establish the suitability ofthis strain as a model, a variety of indicators of hepatotoxicitywas evaluated and compared to histological evidence of injury24 hr after dosing with CCl₄ or a combination of CCl₄ + TCE.Plasma alanine aminotransferase (ALT) activity was the mostreliable indicator of hepatic injury and was well-correlatedwith the histologic data. Dose–response studies utilizingsimultaneous ip dosing confirm the sensitivity of the F-344rat, demonstrating synergistic toxicity at doses as low as 0.165mmol/kg of CCl₄ and 0.6 mmol/kg of TCE. Synergism was also detectedfollowing simultaneous ip administration of 1 mmol/kg CCl₄ and0.5 mmol/kg of CHCl₃. To evaluate the effects of drinking waterexposure, rats were pretreated for 3 days with solutions containingTCE (0–40 mM) or CHCl₃ (0–8 mM) stabilized with1% Emulphor (EL-620P) as their only source of fluids. A single,ip dose of CCl₄ (1 mmol/kg) was then administered and 24 hrlater animals were killed for examination of liver histologyand determination of ALT activity. Although none of the pretreatmentswere detectably hepatotoxic, rats which drank 15 and 40 mm TCEor 8 mm CHCl₃ exhibited an enhanced response to CCl₄     

A CCl4/CHCl3 Interaction Study in Isolated Hepatocytes: Selection of a Vehicle

Emulphor, ethanol, and dimethyl sulfoxide (DMSO) were evaluatedas vehicles in studying the toxicity of CCl₄ and CHCl₃ in isolatedhepatocytes. The appropriateness of the vehicle was determinedby evaluating the following parameters: solubility of CCl₄ andCHCl₃ in the vehicle, cell injury (intracellular K+), cell death(LDH leakage), and lack of interaction (protection or enhancedtoxicity) with CCl₄. and CHCl₃. The relative toxicity of thevehicles according to maximum no effect levels (v/v) was: emulphor(0.125%) > ethanol (1.0%) > DMSO (5.0%). Emulphor at toxiclevels was inadequate to dissolve enough CCl₄ to evaluate inthis system. Ethanol (5.0, 2.5, 1.0, 0.5%) was more toxic thanDMSO and interacted with both CCl₄. and CHCl₃ to enhance toxicity.DMSO (15.0, 5.0, 2.5%) did not significantly alter the toxicityof CCl₄. and CHCl₃ no interaction. These data suggest that DMSOshould be the vehicle for evaluating the toxicity of CCl₄. andCHCl₃ and their mechanisms of action in the isolated hepatocyte.     

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.     

四氯化碳致大鼠肝损伤早期血清总胆汁酸、α-谷胱甘肽S转移酶、嘌呤核苷磷酸化酶和鸟氨酸氨基甲酰转移酶的变化

目的 探讨血清总胆汁酸、α-谷胱甘肽S转移酶(α-GST)、嘌呤核苷磷酸化酶(PNP)和鸟氨酸氨基甲酰转移酶(OCT)对肝损伤早期诊断的应用价值。方法 Wistar大鼠分别ig给予CCl₄ 0.02,0.05和0.08 ml·kg^-1,每天1次,连续10 d。于给CCl₄后第1天和第3天采集血清,采用全自动生化仪测定血清谷丙转氨酶(GPT)、谷草转氨酶(GOT)、碱性磷酸酶、总胆红素以及总胆汁酸的水平,采用ELISA测定血清α-GST, PNP和OCT的水平; 于给CCl₄后第10天,测定血清GPT、 GOT、碱性磷酸酶及总胆红素,计算大鼠的肝指数,观察肝组织病理学变化。通过Logistic回归建立回归模型,分别用ROC曲线分析给CCl₄后第1和第3天总胆汁酸、α-GST、PNP和OCT以及GPT和GOT对CCl₄致大鼠肝损伤早期诊断的意义。结果 与正常对照组同时间点相比,给CCl₄第1天,CCl₄ 0.02,0.05和0.08 ml·kg^-1组PNP均升高(P<0.05, P<0.01),CCl₄ 0.08 ml·kg^-1组GPT和总胆汁酸升高(P<0.05, P<0.01); 给CCl₄第3天,CCl₄ 0.05和0.08 ml·kg^-1组GOT, α-GST和OCT升高(P<0.05, P<0.01); 给CCl₄第10天,CCl₄ 0.02,0.05和0.08 ml·kg^-1组大鼠肝指数均显著升高(P<0.01),肝出现严重的肝细胞脂肪变性(P<0.01)。大鼠血清中GPT、GOT、总胆汁酸、α-GST、PNP和OCT的ROC曲线下面积分别为0.786, 0.728, 0.878, 0.629, 0.850和0.571。GPT和GOT联合检测的ROC曲线下面积为0.871;总胆汁酸、α-GST、PNP和OCT以及总胆汁酸、PNP和OCT联合检测的ROC曲线下面积为0.939,且均高于各指标单项检测。结论 总胆汁酸、α-GST、PNP和OCT可作为CCl₄致肝损伤早期的生物标志物,且总胆汁酸、PNP和OCT联合检测在CCl₄致肝损伤早期具有较高的诊断价值。     

Nephrotoxic Interactions between Ketonic Solvents and Halogenated Aliphatic Chemicals

Nephrotoxic Interactions between Ketonic Solvents and HalogenatedAliphatic Chemicals. Hewitt, W. R., and Brown, E. M. (1984).Fundam. Appl. Toxicol. 4, 902–908. Recent studies haveindicated that (1) ketonic solvents and ketogenic chemicalscan potentiate the nephrotoxic and hepatotoxic effects of oneor more halogenated hydrocarbons; and (2) the relative abilityof ketones to potentiate the liver injury produced by chloroform(CHCl₃) may be influenced by the carbon skeleton length of theketone. Although five ketones (acetone, 2-butanone, 2-pentanone,2-hexanone, (HX), and 2-heptanone) increased CHCl₃-induced kidneyand liver injury in male, Fischer 344 rats, no relationshipbetween ketone chain length and potentiating capacity was observed.HX potentiated the CHCl₃-induced depletion of hepatic glutathionecontent and increased the irreversible binding of ^l4CHCl₃-derivedradiolabel to hepatic constituents. In contrast, CHCl₃ did notalter glutathione content in the renal cortex of either vehicle-or HX-pretreated rats. Although HX increased the binding of¹⁴C from ¹⁴CHCl₃ to renal cortical macromolecules, the magnitudeof the increase was unremarkable, approaching only the extentof hepatic ¹⁴C binding in vehicle-pretreated rats challengedwith ^l4CHCl₃. Since the severity of renal and hepatic injurywas comparable in rats receiving the combination of HX + CHCl₃,it appeared that HX potentiated CHCl₃ nephro- and hepatotoxicityby different mechanisms. Ketone pretreatment did not potentiatethe renal injury produced by potassium dichromate or hexachloro-l,3-butadiene.     

Effect of Adrenalectomy on Chlordecone Potentiation of Carbontetrachloride Hepatotoxicity

Effect of Adrenalectomy on Chlordecone Potentiation of CarbontetrachlorideHepatotoxicity. Agarwal, A.K. and Mehendale, H.M. (19S3).Fundam.Appl. Toxicol 3:507–511. The propensity of chlordecone(CD) to potentiate CCl₄ hepatotoxicity in rats of either sexhas been well documented. The objective of the present studywas to investigate the hepatotoxic effects of CD-CCl₄ interactionin adrenalectomized rats. Adrenalectomized rats were maintainedon 0 or 10 ppm CD and on day 15 they received a single ip injectionof 25 µL CCl₄/kg. Hepatotoxicity was assessed by hepatofunctional,biochemical and histopathological parameters, 24 hrs after CCl₄challenge. CCl₄-induced hepatobiliary dysfunction and elevationof serum enzymes (GPT, GOT, ICD and OCT) were evident. Hepaticdysfunction was most severe in adrenalectomized rats receivingCD-CCl₄ combination treatment. Histopathology of liver exhibitedextensive fatty infiltration in the entire lobular structureaccompanied by some necrosis. These data indicate that the capacityof CD to potentiate CCl₄ hepatotoxicity is unaffected in adrenalectomizedrats.     

Hepatic Failure Leads of Lethality of Chlordecone-Amplified Hepatotoxicity of Carbon Tetrachloride

Hepatic Failure Leads to Lethality of Chlordecone-AmplifiedHepatotoxicity of Carbon Tetrachloride. SONI, M. G., AND MEHENDALE,H. M. (1993). Fundam. Appl. Toxicol. 21, 442–450. Chlordecone (Kepone) amplification of CCl₄ toxicity occurs atsmall, nontoxic levels of chlordecone and CC1₄ and results inhighly increased irreversible hepatotoxicity culminating inlethality. Although it is generally assumed that CCl₄ lethalityis due to hepatic failure, no definitive studies are availablein the literature bridging massive liver failure and death.The present studies were designed to evaluate whether hepaticfailure is the cause of the lethality during chlordecone-amplifiedCCl₄ toxicity. Male Sprague-Dawley rats were maintained on controlor a chlordecone (10 ppm) diet for 15 days and injected withCCl₄ (100 µl/kg, ip) on Day 16. Rats were killed at 0,6, 12, 24, 36, and 48 hr after CCl₄ challenge. Hepatic failurewas evaluated by measuring plasma glucose, ammonia, bilirubin,aspartate trans-aminase (AST), alanine transaminase (ALT), sorbitoldehydrogenase (SDH), hepatic ATP, glycogen, and by histologicaland histomorphometric analyses. Plasma creatinine, urea, andkidney histopathology were also assessed for possible renalinjury. As expected CCl₄ administration to chlordecone-pretreatedrats resulted in 20% lethality by 36 hr, which progressed withtime, and all rats died within 72 hr. A significant and progressivehypoglycemia was observed with a 60% reduction in plasma glucoseat 48 hr. Hepatic glycogen content dropped precipitously. Similarly,hepatic ATP levels remained suppressed (80% of control) at allthe time points studied. Plasma ammonia levels were significantlyelevated, and by 48 hr, a threefold increase was observed. PlasmaALT, AST, SDH, and bilirubin increased progressively until thedeath of rats receiving the chlordecone + CCl₄ combination.Histopathologically, the liver sections revealed a progressiveand irreversible damage evidenced by vacuolation, accumulationof fat, and increase in hepatocellular necrosis resulting indisrupted hepatolobular structure. Administration of the samedose of CCl₄ to rats maintained on a normal diet resulted inonly marginal changes in plasma enzymes, no increase in serumbilirubin, and no significant injury in liver sections at 24hr. CCl₄ administration to chlordecone-pretreated rats resultedin a marginal increase in plasma creatinine and urea only atlater time points. Histopathological examination of sectionsof kidneys from rats receiving either chlordecone + CCl₄ combinationor the same dose of CCl₄ alone did not reveal any evidence ofsignificant renal injury. The clinical end points of hepaticfunction measured in the present study are consistent with thesequel of hepatic failure and hepatic encephalopathy leadingto animal death in chlordecone-amplified CCl₄ toxicity.     

Assessment of the Minima Effective Dose of Acetone for Potentiation of the Hepatotoxicity Induced by Trichloroethylene-Carbon Tetrachloride Mixtures

Assessment of the Minimal Effective Dose of Acetone for Potentiationof the Hepatotoxicity Induced by Trichloroethylene-Carbon TetrachlorideMixtures. CHARBONNEAU, M., PERREAULT, F., GRESELIN, E., BRODEUR,J., AND PLAA, G. L. (1988). Fundam Appl. Toxicol 10, 431–438.Administration of acetone to rats in amounts larger than orequal to a minimal effective dosage (MED) is known to potentiatethe severity of the liver damage produced by CC1₄ alone. Ithas been reported that CCl₄-induced hepatotoxicity is also enhancedby the previous administration of trichloroethylene (TCE). Inaddition, TCE-CCl₄ mixture-induced liver injury is potentiatedby acetone. The present study was undertaken to determine ifthe acetone MED is decreased when the haloalkane challenge isa mixture of TCE-CCl₄ instead of CCl₄ alone. The effect of varyingmixture compositions was also evaluated. In a first series ofexperiments, male Sprague-Dawley rats received corn oil or acetone(0.05–0.25 ml/kg, po); 18 hr later, they received an lpinjection of either CC1₄ (0.1 ml/kg) or [TCE (0.25 ml/kg)–CCl₄(0.1 ml/kg)]. In a second series, rats received corn oil oracetone (0.75 ml/kg), and were challenged with TCE (1.5 ml/kg),CCl₄ (0.25 ml/kg), or a mixture of TCE-CCl₄, where TCE and CC₄dosages were equal to 25–757percnt;, 50–50%, and75–25%, respectively, of those used for the administrationof the solvents alone. In both series, rats were killed 24 hrafter the haloalkane challenge. Liver injury was assessed biochemically(plasma ALT activities and bilirubin concentrations) and morphologically.When TCE was added to a solution of CCl₄, smaller doses of CCl₄were required to produce equally severe liver injury. The MEDof acetone required to potentiate CCl₄-induced liver injurywas at least five times smaller when TCE (0.25 ml/kg) was addedto the challenge solution of CCl₄- The severity of the injuryproduced by TCE-CCU mixtures administered in different proportionswas constant, and potentiated to the same extent by a givendose of acetone. These observations suggest that the presenceof haloalkane mixtures can result in severe liver injury withlower doses of hepatotoxicants. They further illustrate thatprior exposure to acetone may markedly affect the response elicitedby the haloalkane mixture.     

Hepatotoxicity and lethality of halomethanes in Mongolian gerbils pretreated with chlordecone,phenobarbital or mirex

The hepatotoxic and lethal effects of CBrCl₃, CCl₄ and CHCl₃ were investigated in gerbils with or without prior exposure to dietary chlordecone (CD), phenobarbital (PB) and mirex (MX) at 10, 225 and 10 ppm, respectively, for 15 days. Gerbils were quite sensitive to these halomethanes (48 h LD₅₀: 20, 80 and 400 l/kg, respectively). CD, known to potentiate hepatotoxic and lethal effects of halomethanes in rats, failed to potentiate the toxic effects of any of these three halomethanes in gerbils. PB and MX were also ineffective. Since stimulation of early hepatocellular regeneration has been shown to be responsible for the recovery from the toxicity of a low dose of CCl₄, liver cell regeneration and tissue repair were studied in gerbils after CCl₄ administration. The objectives of these studies were to investigate the possible reasons for the high sensitivity of gerbils to halomethane toxicity and to investigate the mechanism for their refractoriness to CD-potentiated halomethane toxicity. A low and a high dose of CCl₄ (15 and 80 l/kg, i.p. respectively) were used to study the time-course of liver injury in gerbils pretreated with or without CD. The low dose of CCl₄ stimulated cellular regeneration as indicated by the increase of³H-thymidine (³H-T) incorporation in hepatic nuclear DNA. The cellular regeneration and tissue repair activities resulted in complete recovery from the limited liver injury in both CD-pretreated and control gerbils. In contrast to rats, however, the process of cell division in gerbils occurred much later, 2 days after CCl₄ administration. Evidence from histomorphometric studies was consistent with serum enzyme and³H-T incorporation data. Significant increase in hepatocyte mitosis did not occur until 42 h after CCl₄ administration. Hepatic injury assessed as hepatocellular necrosis and lipid accumulation was evident as early as 24 h after CCl₄ injection and was maximal at 42 and 72 h after CCl₄ in CD-pretreated and control gerbils, respectively. Administration of a high dose of CCl₄ alone significantly impeded tissue repair. More than 65% of the hepatocytes were necrotic in both CD-pretreated and control gerbils 24 h after the administration of a LD₅₀ dose of CCl₄.³H-T incorporation did not increase up to 48 h after CCl₄ in either group. These findings suggest that the absence of early stimulation of hepatocellular division and tissue repair might be responsible for the very high toxicity of a low dose of CCl₄ in gerbils. Since there is no early tissue proliferative response in gerbils after CCl₄ administration, CD+CCl₄ interactive ablation of liver proliferative response cannot occur, making gerbils refractory to CD-potentiation of CCl₄ toxicity.A preliminary report of these findings was presented at the 29th Annual Meeting of the Society of Toxicology at Miami, Florida. Toxicologist (1990) 10: 209Recipient of the 1988 Burroughs Wellcome Toxicology Scholar Award.     

The Effect of Dietary Exposure to a Mirex Plus Chlordecone Combination on CCl4 Hepatotoxicity

The Effect of Dietary Exposure to a Mirex Plus Chlordecone Combinationon CCl₄ Hepatotoxicity. BELL, A. N., AND MEHENDALE, H. M. (1985).Fundam. Appl. Toxicol. 5, 679–687. The purpose of thesestudies was to investigate the effect of a mirex plus chlordeconecombination on CCl₄-induced hepatotoxicity. Male Sprague-Dawleyrats were maintained on control diet or on diets containing10 ppm chlordecone (CD), 10 ppm mirex (M), or M plus CD (10ppm each; MCD) for 15 days. On Day 15 the rats received a singleip injection of CCl₄ (100 µl/kg) and hepatotoxicity wasassessed 24 hr later. Animals in the control group receivingCCU alone were unaffected. Significant increases in liver-to-bodyweight ratios were observed in all three pretreatment groupsfollowing CCl₄ challenge. Increases in serum enzymes (SGPT,SGOT, and ICD) occurred in all three pretreatment groups withCD = MCD > M > control. While MCD and CD pretreatmentled to significant cholestasis and decreases in PG excretion,no such effect was observed with M. Light microscopic examinationof tissues revealed swollen hepatocytes (balloon cells), hepatocellularnecrosis, and lipid accumulation in the MCD, CD, and M groupsfollowing CCl₄ challenge. In summary, as assessed by serum enzymeelevation, biliary flow and hepatic excretory function, M pretreatmentled to only a slight increase in CCl₄ hepatotoxicity. The MCDcombination pretreatment did not potentiate hepatotoxicity abovethat seen with CD alone. These results provide additional evidencethat CD pretreatment results in a rather specific sensitizationof animals to CCl₄ toxicity in ways independent Of the actionsOf M     

Acetone Potentiation of Rat Liver Injury Induced by Trichioroethylene-Carbon Tetrachionde Mixtures

Acetone Potentiation of Rat Liver Injury Induced by Trichloroethylene-CarbonTetrachloride Mixtures. CHARBONNEAU, M., OLESKEVICH, S., BRODEUR,J., AND PLAA, G. L.(1986) Fundam. Appl. Toxicol. 6, 654–661.Acetone potentiation of haloalkane-induced hepatotoxicity iswell recognized. Others reported that trichloroethylene (TCE)can potentiate the hepatotoxicity induced by CCl₄ The purposeof the present study was to determine if acetone (A) can potentiatethe hepatotoxicity of haloalkane (HA) mixtures. We derived dose-responsecurves for the potentiation of TCE, CCl₄ and TCE-CCl₄ inducedhepatotoxicity by acetone. Male Sprague-Dawley rats receivedacetone (0.25-1.5 ml/kg, po); 18 hr later, they received anip injection of either TCE, 0.25 ml/kg; CCl₄ 0.1 or 0.6 ml/kg;or a mixture of TCE and CCl₄. Rats were killed 24 hr later,the livers excised, and plasma ALT activities and bilirubinconcentrations determined. Histological evaluations were alsoperformed. Neither A, TCE, or both in combination modified thebiochemical indices. Acetone potentiation of CCl₄ liver injurywas greater than TCE-potentiation of CCl₄ Acetone enhanced thehepatotoxic response of the TCE-CCl₄. mixture. Thus, CCl₄ hepatotoxicitywas enhanced by the simultaneous administration of TCE, andthis response was potentiated by the previous administrationof acetone.     

Hepatotoxic interaction between carbon tetrachloride and chloroform in ethanol treated rats

The effect of coadministration of CHCl₃ on CCl₄-induced hepatic damage was investigated at low dose inhalation. Coexposure of CHCl₃ did not influence CCl₄-induced changes in any index of hepatic damage in control rats. Coadministration of CHCl₃, however, enhanced CCl₄ (10 ppm)-induced hepatic damage of ethanol treated rats in a dose- and duration-dependent manner: simultaneous exposure of 50 ppm CHCl₃ potentiated CCl₄-induced increase in plasma GPT activity and number of necrotic hepatocytes; the enhancement of CCl₄-induced hepatic damage by 50 ppm CHCl₃ was found over the 4 h exposure; simultaneous exposure of 10 and 25 ppm CHCl₃ potentiated the CCl₄-induced increase in liver malondialdehyde (MDA) content. In contrast, coadministration of 50 ppm trichloroethylene and 200 ppm 1,1,1-trichloroethane decreased CCl₄-induced increase in plasma GPT activity, though these exposures did not influence the liver MDA content. These results suggest that the concentration of 10 ppm CCl₄ may be significant for CHCl₃ to potentiate the hepatic damage caused by CCl₄ in ethanol-treated rats. Heavy drinkers may have a higher hepatotoxic risk for a mixture of CCl₄ and CHCl₃ than for a single exposure to CCl₄ or CHCl₃, and a particular attention should be therefore given to the joint exposure to CCl₄ and CHCl₃.     

Effect in the Rat of the Interaction of Dichloromaleic Acid and Carbon Tetrachloride on Renal and Hepatic Function

Water purification generates a vanety of chlorinated contaminants,one of which is dichloromaleic acid (DCMA). Exposure to thiscompound is likely to occur in combination with other drinkingwater pollutants, some of which are hepatotoxic. This studywas designed to examine the interactive effects of carbon tetrachioride(CCl₄)a a known hepatotoxin, with DCMA on liver and kidney functionin the Sprague-Dawley rat. Administration of a single dose ofDCMA (200–400 mg/kg, ip) caused modest dose-dependentincreases in alanine aminotransferase (ALT), aspartate aminotransferase(AST), and plasma urea nitrogen, as well as a marked depletionof nonprotein sulfhydryls (NPSH) in the liver, but not the kidney,by 24 hr. Pretreatment with inducers (phenobarbital or 3-methylcholanthrene)or an inhibitor (SKF 525A) of cytochrome P-450 activity failedto alter the response observed with DCMA alone. Alterationsin 24-hr urine volume, osmolality, and water consumption alsowere observed. DCMA-mediated changes in plasma urea nitrogenand NPSH were reduced in magnitude with coadministration ofCCl₄ (1 ml/kg, ip), while anticipated CCl₄-induced increasesin ALT and AST were reduced with coexposure to DCMA. Renal sliceexpenments indicated that DCMA-treated rats were less able toaccumulate the organic anion p-aminohippurate (PAH), whereasDCMA had no effect on accumulation of the organic cation tetraethylammonium(TEA). The combination of CCl₄. and DCMA produced only additiveeffects on organicion accumulation. These results suggest hepaticinteraction possibly related to the metabolism of CCl₄ and DCMA,resulting in renal and hepatic toxicity diminished from thatobserved with exposure to either agent alone.     

Fractional Hepatic Localization of 14CHCl3 in Mice and Rats Treated with Chlordecone or Mirex

Fractional Hepatic Localization of ¹⁴CHCl₃ in Mice and RatsTreated with Clordecone or Mirex. Hewitt, L.A., Hewitt, W.R.,and Plaa, G.L. (1983). Fundam. Appl. Toxicol. 3:489–495.In rodents chlordecone, but not mirex, a nonketonic structuralanalog, significantly augments CHCl₃-induced liver damage, atleast in part, by increasing CHCl₃ bioactivation. To determinewhether the fractional distribution of CHCl₃ was altered inchlordecone-pretreated animals, the irreversible binding of¹⁴CHCl₃ to various liver constituents (a measure of CHCl₃ bioactivation)was examined in vivo in mice and rats. Chlordecone, but notmirex, increased both total and irreversibly bound ¹⁴CHCl₃;furthermore, changes in the ¹⁴Clocalization between lipid, proteinand acid-soluble fractions were noted. Thus, the results suggestthat differences exist between chlordecone and mirex with respectto their capacity to increase the quantity of CHCl₃-derivedreactive metabolite and the eventual distribution of reactivemetabolite.     

First-Order Toxicity Assays for Eye Irritation Using Cell Lines: Parameters That Affect in Vitro Evaluation

First-order toxicity assays can be used to rapidly screen testagents. Investigators in many laboratories have used culturedcell lines to obtain correlations between first-order assayend-points and in vivo eye irritation (Draize test) for a widevariety of compounds. Since validation is a key step in assayacceptance, it is important to understand which factors alterthe responses of cell-line-based assays. In this study we examine:(1) the presence and configuration of a type I collagen gel;(2) the responses of epithelial (Sf-l-Ep) and fibroblast (Sireand 3T3) cell lines; (3) the total glutathione content, ATPcontent, methionine incorporation, and neutral red absorptionendpoint assays; (4) alcohol (C₂-C₈), surfactant (Tween 20),and heavy metal (NiCl) test agents; and (5) test agent exposuretime (1 to 24 hr). The presence of a collagen gel and the celltype did not significantly affect endpoint assay R50 (test agentconcentration that decreases assay response by 50%) values fora 1-hr exposure to hexanol. The ATP and glutathione endpoints(after 1-hr exposure) are able to distinguish between the relativein vivo toxicities of C₂-C₈ normal alcohols. All four endpointassays detected sublethal damage, with the ATP and methionineendpoints being the most sensitive. The type of test agent affectsthe endpoint response, as shown by the lack of a glutathioneR50 value for a 1-hr exposure to Tween 20 or NiCl. Even fora single test agent, endpoint assay R50 values may decreasecontinuously (ATP), decrease and then stabilize (glutathione),or remain unchanged (methionine incorporation) during a 24-hrexposure.     

Assessment of Hepatic Indicators of Subchronic Carbon Tetrachloride Injury and Recovery in Rats

Assessment of Hepatic Indicators of Subchronic Carbon TetrachlorideInjury and Recovery in Rats. ALLIS, J. W., WARD, T. R., SEELY,J. C, and SIMMONS, J. E. (1990). Fundam Appl. Toxicol. 15, 558–570.To determine the course of hepatic recovery from subchronicoral administration of carbon tetrachloride (CCl₄), male F-344rats were gavaged with 0, 20, or 40 mg CCl₄/kg, 5 days/week,for 12 weeks. Exposure to CCl₄ caused dosage-dependent increasesin relative liver weight and the serum levels of aspartate aminotransferase,alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase,and cholesterol as well as a dosage-dependent decrease in hepaticcytochrome P450. Centrilobular hepatocellular vacuolar degeneration,necrosis, and cirrhosis occurred at both 20 and 40 mg/kg, withdosage-dependent severity. Reversibility of these reported effectsvaried with parameter. By Day 8 postexposure, necrosis had disappearedand all serum indicators and cytochrome P450 had returned tocontrol levels. By Day 15 postexposure, the severity of thevacuolar degeneration had decreased. Reversibility of cirrhosiswas dosage dependent; complete recovery occurred in the low-but not the high-dose group by Day 15. The disappearance ofthe increase in relative liver weight was also dependent ondosage; the low- but not the high-dose group had returned tothe control level by Day 22. In an attempt to measure persistenthepatic damage, liver uptake relative to the spleen was determinedfor a sulfur colloid labeled with technetium-99m and for tritiated2-deoxyglucose. Neither method consistently measured hepaticdamage in cirrhotic livers due, in part, to the high degreeof variability in the tracer uptake data.     

Suppression of Humoral and Cell-Mediated Immune Responses by Carbon Tetrachloride

Suppression of Humoral and Cell-Mediated Immune Responses byCarbon Tetrachloride. KAMINSKI, N. E., JORDAN, S. D., HotSAPPIM. P. (1989). Fundam Appl. Toxicol 12, 117–128. The effectsof carbon tetrachloride (CCl₄. following 7 consecutive daysof exposure ip at 500, 1000, and 1500 mg/kg, were determinedon murine humoral and cell-mediated immune responses, body andorgan weights, spleen cell blastogenesis following mitogenicstimulation, and clinical serum parameters for liver injury.In vivo sensitization of CCl₄ B6C3F1 mice resulted in a dose-dependentsuppression of the T-dependent antibody response to sheep redblood cells (sRBC) at all doses-36, 48, and 53%, respectively.The T-independent in vivo antibody response to DNP-Ficoll wassuppressed only at 1500 mg/kg, and only by approximately 16%.This dosing regimen also resulted in a significant decreasein thymus weights; however, there were no significant effectson liver, kidney, lung, or body weights. The serum chemistryprofile indicated a dose-dependent increase in serum glutamic-pyruvictransaminase (SGPT) levels (34-, 47-, and 55-fold) and a non-dose-dependentincrease in serum bilirubin and total protein. Serum glucoseand albumin levels were unaffected. Splenocytes from mice treatedwith 1500 mg/kg and sensitized in vitro with antigen demonstrateda comparably suppressed antibody response to the antigens sRBCand DNP-Ficoll as observed in vivo-66 and 28% respectively.This dose of CCl₄ had no effect on the in vitro antibody responseto the polyclonal antigen lipopolysaccharide. The mixed lymphocyteresponse was dose dependently suppressed following CCl₄ exposure;however, the delayed-type hypersensitivity response was unaffected.Lymphocyte blastogenesis following mitogenic stimulation withlipopolysaccharide or concanavahn A was also inhibited by CCl₄exposure. These studies demonstrate that exposure to CCl₄ resultsin marked suppression in both humoral and cell-mediated immuneresponses at concentrations which also affect the liver as evidencedby the marked increase in SGPT levels.     

The Interactive Toxicity of CHCl3 and BrCCl3 in Precision-Cut Rat Liver Slices

The interactive toxicity of two nontoxic concentrations of chloroform(CHCl₃and bromotricchloromethane (BrCCl₃ was examined in precision-cutrat liver slices. Liver slices were prepared from male Sprague-Dawleyrats (220–250 g) pretreated with phenobarbital for 4 days.Toxicants were administered 1 hr apart. Intracellular K⁺ levelswere similar to untreated controls in slices treated with 0.2mM CHCl₃ or 0.125 µ1 (0.25 mg, 1.26 µmol) BrCCl₃alone, indicating that these concentrations were nontoxic. However,addition of both toxicants, irrespective of order, resultedin a time-dependent loss of intracellular K⁺ which was significantat 9 hr following administration. This was interpreted as evidenceof synergistic toxicity. Cytochrome P450 loss was significantas early as 3 hr following exposure to BrCCl₃ alone or whenadded with CHCl₃ This loss may be attributed to BrCCl₃ suicideinactivation of cytochrome P450. Centrilobular hepatocytes maybe more susceptible to the interactive toxicity of CHCl₃ andBrCCl₃ Activity of enzymes found predominantly in this areawas significantly decreased in slices exposed to both toxicantsrelative to controls. Conversely, activity of enzymes foundpredominantly in the periportal region was similar to that ofuntreated and treated controls. Inter active toxicity of BrCCl₃and CHCl₃ was not a consequence of increased lipid peroxidationor depletion of slice glutathione content. Further studies needto be conducted to elucidate the mechanisms mediating the interactivetoxicity of BrCCl₃ and CHCl₃.