Document evZnEK3BYwovKDJMLeJ3zyg0E

effects, there are no pplication of Quil A ) ng/ml in an iscom vith the iscom preparein etal. (1984) the reparation tested had 60 jtg/ml). NCES ^ants Saponines. II. Influnine sur l'effet d'irritation t 77, 1297-1301. nin adjuvants. Arch Ges54. ith, K. R., Gaunt, J. R, , and Gangolli, S. D, tudy ofQuillaia extract in 20. 15-23. tatus and toxicity of sapo3,85-91. ira Pharmacopoeia, 28th >p. 325. The Pharmaceuti- .. Hogland. S.. Dals\us, A. (1984). Iscom, a .ic presentation of memped viruses. Nature (Lon- . Uytdehaag, R, Jar\nd Morein, B. (1985). nune response in cats by .emia virus iscom. J. Im- TH. K. R., Gaunt, J. R, P. (1979). Long-term toxct in mice. Food Cosmet. iWEiG, L. (1975). On the olvsis. II. Inhibition of hehem. Pharmacol. 22, 77- j. (1973). Chemie und Bi'tschntie der Chemie Orlerz., H. Grisebach, and \ pp. 461 --496. Springer- -ralization of "Student's" ent population variances . 28-35. $ FUNDAMENTAL AND APPLIED TOXICOLOGY 10, 431-438 (1988) Assessment of the Minimal Effective Dose of Acetone for Potentiation of the Hepatotoxicity Induced by Trichloroethylene-Carbon Tetrachloride Mixtures1 Michel Charbonneau,* Francois Perreault,* Erminio Greselin* Jules BRODEUR,f and Gabriel L. Plaa*-2 *Departement de Pharmacologic et iDepartement de Medecine du Travail et d'Hygiene du Milieu, Faculte de Medecine, Universite de Montreal, Montreal, Quebec, Canada H3C SJ7 Received May 5.1987; accepted November 16,1987 Assessment of the Minimal Effective Dose of Acetone for Potentiation of the Hepatotoxicity Induced by Trichloroethylene-Carbon Tetrachloride Mixtures. Charbonneau, M., Per reault, R, Greselin, E., Brodeur, J., and Plaa, G. L. (1988). Fundam. Appl. Toxicol, 10, 431 -438. Administration of acetone to rats in amounts larger than or equal to a minimal effec tive dosage (MED) is known to potentiate the severity of the liver damage produced by CCL alone. It has been reported that CCL-induced hepatotoxicity is also enhanced by the previous administration oftrichloroethylene (TCE). In addition, TCE-CCL mixture-induced liver injury is potentiated by acetone. The present study was undertaken to determine ifthe acetone MED is decreased when the haloalkane challenge is a mixture ofTCE-CCl, instead ofCCL alone. The effect of varying mixture compositions was also evaluated. In a first series of experiments, male Sprague-Dawley rats received com oil or acetone (0,05-0.25 ml/kg, po); 18 hr later, they re ceived an ip injection of either CCL (0.1 ml/kg) or [TCE (0.25 ml/kg)-CCL (0.1 ml/kg)]. In a second series, rats received com oil or acetone (0.75 ml/kg), and were challenged with TCE (1.5 ml/kg), CCL (0.25 ml/kg), or a mixture of TCE-CCL, where TCE and CCL dosages were equal to 25-75%, 50-50%, and 75-25%, respectively, of those used for the administration of the sol vents alone. In both series, rats were killed 24 hr after the haloalkane challenge. Liver injury was assessed biochemically (plasma ALT activities and bilirubin concentrations) and morphologi cally. When TCE was added to a solution ofCCL, smaller doses ofCCL were required to produce equally severe liver injury. The MED ofacetone required to potentiate CCL-induced liver injury was at least five times smaller when TCE (0.25 ml/kg) was added to the challenge solution of CCL. The severity of the injury produced by TCE-CCL mixtures administered in different proportions was constant, and potentiated to the same extent by a given dose of acetone. These observations suggest that the presence of haloalkane mixtures can result in severe liver injury with lower doses of hepatotoxicants. They further illustrate that prior exposure to acetone may markedly affect the response elicited by the haloalkane mixture, 1988 Society ofToiicoiogy. Haloalkanes are extensively present in the industrial environment, and several ofthem are; known to produce liver injury in humans and1 in animals. Ketonic (e.g., acetone) or keto-genic compounds can potentiate haloalkane-induced hepatotoxicity (Hewitt et al., 1980; 1 Supported by IRSST Quebec and an NSERC Strate gic Grant. 2 To whom all correspondence should be addressed. Pilon et ai, 1986); trichloroethylene (TCE) and carbon tetrachloride (CC14) figure among the haloalkanes whose toxicity is potentiated. The minimal effective dosage (MED) of acetone required to potentiate a challenge of CCU (0.1 ml/kg) in rats was previously re ported to be 0.25 ml/kg (Plaa et ai, 1982; Charbonneau et al. 1986a). Pessayre et ai (1982) reported that rats treated intraperitoneally with a mixture of nontoxic dosages of 43, 0272-0590/88 $3.00 Copyright <> 1988 by the Society ofToxicology. All rights of reproduction in any form reserved. SL 035048 432 CHARBONNEAU ET AL. TCE and CC14 exhibited a moderate to severe liver injury. Their findings were consistent with the contention that the liver damage ob served after the administration of TCE-CCLt mixtures resulted from a TCE enhancement of CCU hepatotoxicity. They also showed that when the TCE or CCU dose is kept con stant, the severity of the liver injury is depen dent on the dose of the other haloalkane in the mixture. More recently, Charbonneau et al. (1986b) demonstrated that acetone strongly potentiates the hepatotoxicity in duced by a mixture of TCE and CCU. The aim of the present study was to define more precisely the interaction between ace tone, TCE, and CCU. Two objectives were pursued: (a) To determine ifthe MED of ace tone required to potentiate CCU liver injury is smaller when the challenge is a mixture of TCE and CCU instead of CCU alone, (b) To determine if the severity of the acetone-po tentiated TCE-CCl4-induced liver injury was altered when the dosage of CCU is decreased but accompanied by a concomitant increase of the TCE dosage in the mixture. Liver in jury was assessed by biochemical indices and a semiquantitative morphological analysis. METHODS Animal treatments. Male Sprague-Dawley rats < 175-- 200 g) were purchased from Charles River Canada, Inc. (St. Constant, Quebec), and maintained on Charles River rat chow No. 5012 and water ad libitum. Animals were used after a 4-day acclimation period in animal quarters maintained on a 12-hr light/dark cycle. The rats were housed (six per cage) in stainless steel open-bottom cages. The experimental unit consisted ofsix rats. During the treatment period, animals received a single po injec tion (3:00 pm) ofcom oil (10 ml/kg) or acetone solution; the solutions were prepared such that 10 ml of solution/ kg was administered. In a first series ofexperiments (vari ation of the MED), the acetone dosages employed were 0.05,0.10,0.15, 0.20, and 0.25 ml/kg (0.7, 1.4, 2.0, 2.7, and 3,4 mmol/kg, respectively). Rats in the second series of experiments were dosed with acetone (0.75 ml/kg) (10.2 mmol/kg). After an 18-hr treatment period, the rats were challenged with an ip injection of haloalkane in both experiments. Plaa and Hewitt (1982) reported that acetone potentiation of CHClj-mduced liver injury was maximal when the challenge was administered 18 hr later. Traiger and Plaa (1971) showed that the potentia tion ofCCU-induced liver injury by isopropanol, a ketogenic substance producing acetone, was maximal when the challenge was administered 18-24 hr later. The CCU solutions in com oil were prepared such that the rats re ceived 4 ml of solution/kg. Rats in the first series were challenged with either (a) com oil (4 ml/kg), (b) TCE (0.25 ml/kg) (2.8 mmol/kg), (c) CCU (0.1 ml/kg) (1.0 mmol/kg), (d) TCE (0.25 ml/kg)-CCU (0.1 ml/kg). Rats in the second series were challenged with TCE-CCU mix tures prepared by using different percentages (25, 50, or 75%) of a weakly toxic dosage determined for each halo alkane; the 100% dosages selected were 1.5 ml/kg (16.7 mmol/kg) for TCE and 0.25 ml/kg (2.6 mmol/kg) for CCU. The different mixtures were (A) 25% TCE-75% CCU, (B) 50% TCE-50% CCU, (C) 75% TCE-25% CCU. Animals were weighed 24 hr later and lightly anesthe tized with ether, and blood was removed via the abdomi nal aorta with heparin as the anticoagulant. Biochemical analyses. Alanine aminotransferase (ALT) activity was determined in aliquots of plasma by the method ofReitman and Frankel (1957), using a Dade Chemical Co. reagent kit. Plasma total bilirubin concen trations were determined by the Jendrassik method (Jendrassik and Grof, 1938; Nosslin, 1960), using an Ameri can Monitor kit. Morphologicalanalyses. After fixation in 10% buffered Formalin, sections of the lateral median lobe ofthe liver were dehydrated and embedded in paraffin. Coronal sec tions (5 jim) from the paraffin-embedded tissue were cut and stained with hematoxylin-eosin. Quantitative analy sis was performed by observing 10 fields for each slide (one slide per rat; six rats per group) at 100x magnifica tion, and measuring the distance occupied by ballooning, necrotic, and normal hepatocytes between the central vein and the portal triad as previously described by lijima etal. (1983). Statistical analysis. A log (X) transformation was ap plied to ALT values prior to statistical analysis to nor malize response variables and reduce treatment vari ances, as evaluated by Levene's test for homogeneity of variance. Biochemical data used to determine the MED were submitted to a 6 x 4 (potentiator, challenge) facto rial analysis of variance followed by a Newman-Keuls multiple range test; a = 0.05 was used as the level of sig nificance. RESULTS Variation ofAcetone MED Table 1 depicts the results obtained for corn oil--or acetone (0.05-0,25 ml/kg)-- pretreated rats challenged with corn oil, TCE, CC14, or a TCE-CCU mixture. No significant increases in plasma ALT activities and biliru bin concentrations were observed in the * 4T X \ **; 4 5 t FFECTOF a ireatment (ml/kg) rn oil clone (0.05) clone (0.10) ;etone (0.15) cetone (0.20) cetone (0.25) Sole. Ratsw 4 ml/l^Urich 'lasn^^kple " U^Prrow b Lower row *' Significant! an identical ch groups chal ml/kg. Coi with a TC plasma Al < 0.05) th plasma bili nificantly ( two group; those prev (1982)and The ace the haloal termined ; statistical creased (/ comparisc receiving The MEL (0.1 ml/k (Table 1). SL 0350A9 ry by isopropanol, a ketotone, was maximal when I 18-24 hr later. The CCL, tired such that the rats reits in the first series were n oil (4 ml/kg), (b) TCE c) CCU (0.1 ml/kg) (1.0 g)-CCU (0.1 ml/kg). Rats iged with TCE-CCU mix* nt percentages (25, 50, or letermined for each haloted were 1.5 ml/kg (16.7 ml/kg (2.6 mmol/kg) for were (A) 25% TCE-75% (C) 75% TCE-25% CCU. ater and lightly anestheremoved via the abdomi:ticoagulant. anine aminotransfeAse in aliquots of plasma by nkel (1957), using a Dade aa total bilirubin concen' Jendrassik method (Jen1, 1960), using an Ameri- r fixation in 10% buffered I median lobe of the liver 1 in paraffin. Coronal secmbedded tissue were cut osin. Quantitative analyg 10 fields for each slide oup)at 100X magnifica occupied by ballooning, tes between the central ously described by lijima ) transformation was apatistical analysis to norreduce treatment vari' test for homogeneity of J to determine the MED ntiator. challenge) facto r'd by a Newman-Keuls s used as the level of sig- rs 0 :sults obtained for 05-0.25 ml/kg)-- with corn oil, TCE, ture. No significant ictivities and biiiru- observed in the ACETONE-POTENTIATED TCE-CCU TOXICITY 433 TABLE 1 ffect of Acetone Dosage on Plasma ALT activity and Bilirubin Concentration Measured 24 hr after a Challenge of Corn Oil, TCE, CCU, or a Mixture of TCE-CCU reatment ml/kg) i oil one (0.05) ;one (0.10) tone (0.15) tone (0.20) jtone (0.25) Com oil 36 4" 0.29 + 0.01* 40+ 1 0.25 + 0.01 39 + 2 0.27 + 0.04 38 + 5 0.25+0.01 34+1 0.25+0.02 36 + 3 0.28 0.02 TCE Challenge ecu 45 4 0.29 0.02 31 2 0.26 0.02 45 2 0.30 0.04 42 9 0.25 0.02 36 1 0.22 0.02 40 3 0.25 0.02 51 6 0.29 0.03 81 + 11 0.15 0.01 79 9 0.32 0.03 94 13 0.31 0.02 65 10 0.36 0.02 172 35f 0.16 0.02 TCE-CCU 144+ 17 0.25 + 0.03 395 + 90* 0.38 + 0.02 523 70f 0.21 0.02 699 200* 0.45 + 0.04` 736 276c 0.49 0.06 ` +706 76r 0.48 0.051 'ute. Rats were treated orally with com oil (10 ml/kg) or acetone and were challenged (ip) 18 hr later with com oil ml/kg), trichloroethylene (TCE, 0.25 ml/kg), CCU (0.1 ml/kg), or a [TCE (0.25 ml/kg)-CCU (0.1 ml/kg)] mixture, isma samples were obtained 24 hr after the challenge. J Upper row values represent mean SE ofplasma ALT activity U/ml (six animals/group). ' Lower row values represent mean SE of plasma bilirubin concentration mg/dl (six animals/group). 1 Significantly larger (p < 0.05, Newman-Keuls multiple range test) than the com oil-pretreated group receiving .1 identical challenge. groups challenged with corn oil or TCE 0.25 ml/kg. Com oil-pretreated rats challenged with a TCE-CCU mixture yielded a mean plasma ALT activity three-fold higher (p < 0.05) than CCl4-challenged rats, whereas plasma bilirubin concentrations were not sig nificantly different (p > 0.05) between these two groups. These results are in accord with those previously reported by Pessayre et al. (1982) and Charbonneau el al. (1986b). The acetone MED required to potentiate the haloalkane-induced liver injury was de termined as the smallest dose for which the statistical analysis revealed a significantly in creased (p < 0.05) plasma ALT activity in comparison to the corn oil-pretreated group receiving an identical haloalkane challenge. The MED of acetone for a challenge of CC14 (0.1 ml/kg) alone was equal to 0.25 ml/kg (Table 1). This agrees with the findings pre viously reported by this laboratory (Plaa et al., 1982; Charbonneau et al. 1986a). When the haloalkane challenge was a TCE-CCU mixture, the MED of acetone, however, was equal to or lower than 0.05 ml/kg (Table 1). Variations in the response of the TCE-CCU group prompted us not to test smaller ace tone dosages. Therefore, the addition of TCE (0.25 ml/kg) to the CCU solution resulted in at least a five-fold decrease of the acetone MED required for potentiation of CC14 liver injury. Furthermore, the resulting plasma ALT activities and bilirubin concentrations of the [acetone (0.05 ml/kg) + TCE-CCU]treated group were significantly higher (p < 0.05) than the value obtained for the [ace tone (0.25 ml/kg) -I- CCl4]-treated group. The morphological analysis was performed by measuring the distance occupied by ne crotic, ballooning, and normal hepatocytes in the hepatic lobule; the ratio of necrotic to normal hepatocytes was calculated. This ra tio is a useful parameter to quantify the major changes in treated rats, since both types of SL 035050 434 CHARBONNEAU ET AL. TABLE 2 Histological Evaluation of the Effects of acetone Dosage on CCL and TCE-CCL Liver Injury in Male Rats Dosage of acetone CCU TCE-CCL (ml/kg) Nec Bal Nor Nec/Nor Nec Bal Nor Nec/Nor 0.00 0.05 0.10 0.15 0.20 0.25 0 173 256 26 83 217 35 138 316 24 148 239 29 183 209 41 173 200 0.00 0.12 0.11 0.10 0.14 0.21 80 194 205 119 178 159 131 175 165 114 181 162 99 151 174 132 130 157 0.39 0.75 0.79 0.70 0.57 0.84 Note. Rats were pretreated orally with acetone (0.05, 0.10, 0.15, 0.20, or 0.25 ml/kg) and were challenged (ip) 18 hr later with CCU (0.1 ml/kg) or a [TCE (0.25 ml/kg)-CCU (0.1 ml/kg)] mixture. Liver samples were obtained 24 hr after the challenge. Results are expressed in distance (>m) occupied by cell type from the portal triad to the central vein; values represent the mean of 10 observations/animal for six rats/group. Nec, necrotic hepatocytes; Bal, balloon ing ofhepatocytes; Nor, normal hepatocytes. acute alterations (necrosis with inflammatory cell infiltration and ballooning cells including vacuolization) are evaluated in a unique measurement. For a constant number of necrotic cells, a larger number of balloon cells leads to a smaller number of normal cells; therefore the calculated ratio is larger. The data in Table 2 show that the [necro sis/normal] ratio was nearly two-fold higher in CCl4-challenged rats pretreated with ace tone (0.25 ml/kg = MED) than in acetone (0.05-0.20 ml/kg)-pretreated animals, and represented a mild increase in comparison to com oil-pretreated rats (no necrosis). Com oil-pretreated rats challenged with a mixture of TCE-CCL, yielded a ratio higher than that of those challenged with only CC14. The ace tone (0.05-0.25 ml/kg)-pretreated groups challenged with a mixture of TCE-CCL, yielded ratios that were 3.5-4.0 times larger than those of the rats challenged with only CCU and receiving the MED dosage of ace tone (0.25 ml/kg) (Table 2). Therefore, the morphological findings confirmed that the MED of acetone is decreased when TCE was added to the solution of CC14. Mixture Composition on TCE-CClrInduced Liver Injury The addition of TCE to a CC14 solution (0.1 ml/kg), as discussed above, reduces the MED of acetone required for potentiation of the haloalkane mixture-induced liver injury. Thus, it appeared important to determine whether the CCU dose could also be lowered when larger doses of TCE are used. To ad dress this problem, three mixtures ofdifferent proportions of TCE and CCU were prepared. CCU (0.25 ml/kg) alone was first selected as the 100% dose. Plasma ALT activities mea sured for these rats was slightly increased above normal [Fig. 1 vs Table 1 (com oil + com oil)], indicating a mild hepatotoxic re sponse. TCE (1.5 ml/kg) was used as the 100% TCE dose and was shown to be a very weak hepatotoxicant [Fig. 1 vs Table 1 (com oil + com oil)]. Mixtures were prepared using CCU dosages equal to 75, 50, and 25% of the 0.25-ml/kg dosage; the decreases in the CCU dosage were accompanied by a concomitant increase of the TCE dosage equal to 25, 50, and 75% of the 1.5 ml TCE/kg dosage, (Figs. 1-3 and Ta ble 3). Plasma ALT activities measured in com oil-pretreated rats challenged with (dos ages in ml/kg) [TCE (0.38)-CCU (0.19)], [TCE (0.75)-CCU (0.13)], or [TCE (1.11)CCL (0.06)] were similar to each other, but approximately 13 times higher than the activ ity obtained for CCU (0.25 ml/kg)-treated rats (Fig. 1). Histological analysis showed that the SL 035051 i I ecu t 3-CCU Nor Nec/Nor 205 0.39 159 0.75 165 0.79 162 0.70 174 0.57 157 0.84 1 were challenged (ip) 18 pies were obtained 24 hr ortal triad to the central jpatocytes; Bal, balloon- for potentiation of tduced liver injury, rtant to determine uld also be lowered E are used. To adnixtures ofdifferent XI4 were prepared, vas first selected as iLT activities mea- slightly increased Table 1 (com oil riild hepatotoxic re) was used as the shown to be a very 1 vs Table 1 (com $ l 1 " using CCU dosages of the 0.25-ml/kg e CCI4 dosage were mitant increase of 25, 50, and 75% of . (Figs. 1-3 and Tavities measured in allenged with (dos.38)-CCl4 (0.19)], . or [TCE (1.11)to each other, but 'her than the activml/kg)-treated rats sis showed that the ACETONE-POTENTIATED TCE-CCU TOXICITY QCCI4 or TCE alone ^ TCE-CCI4 mixture 435 PLASMA ALT ACTIVITY ( U/ml ) Fig. 1. Plasma ALT activities measured in com oil-pretreated rats challenged with TCE-CCU mixtures of different proportions. Rats were pretreated orally with com oil (10 ml/kg) and were challenged (ip) 18 hr later with CCU (0.25 ml/kg), TCE (1.5 ml/kg), or a TCE-CCU mixture: (dosages in ml/kg) [TCE (0.38)-- CCU (0.19)], [TCE (0,75)-CCU (0.13)], or [TCE (1.11 )-CCU (0.06)]. Plasma samples were obtained 24 hr after the challenge. Results represent the mean SE for six animals/group. necrosis/normal) ratio was slightly smaller vhen the CCl4 dosage in the mixture was dereased (Table 3); the (necrosis/normal) raios for the TCE-CC14 mixtures were 1.9-2.4 ;imes higher than the ratio measured in rats challenged with only CCl, (0.25 ml/kg). Ad ministration of a high dose of TCE (0.11 ml/ kg) in a mixture with a low dose of CCL, (0.06 ml/kg) yielded a mean plasma ALT activity and a (necrosis/normal) ratio that was mark edly increased when compared to the values obtained with a small dose (0.1 ml/kg) of CCI4 given alone (1355 104 vs 51 6 and 0.71 vs 0.00, respectively), showing the strong enhancing effect of TCE. These results dem onstrate that low doses ofthe hepatotoxicant, CCL,, can still induce severe liver injury when administered as a mixture with high doses of a very weak hepatotoxicant, TCE. Acetone-pretreated rats challenged with CCI4 (0.25 ml/kg) exhibited severe liver in jury, whereas animals challenged with TCE [~~| CCI4 or TCE alon* ^ TCE-CCI4 mixture Fig. 2. Plasma ALT activities measured in acetone-pretreated rats challenged with TCE-CCl, mixtures of different proportions. Rats were pretreated orally with acetone (0.75 ml/kg) and were challenged (ip) 18 hr later with CCU. TCE. or a TCE-CCU mixture (see Fig. 1 for dosages). Plasma samples were obtained 24 hr after the challenge. Results represent the mean SE for six animals/group. SL 035052 i 436 CHARBONNEAU ET AL. cci4 or TCE alone TCE-CCI4 mixture CCI4 0.25 TCE 0.38-CCU 0-18 TCE O.75-CCI4 0.13 TCE I.II-CCI4 0.06 TCE 1.5 0.5 1.0 1.5 2.0 2.5 PLASMA BILIRUBIN CONCENTRATION ( mg/dl ) .Fig. 3. Plasma bilirubin concentrations measured in acetone-pretreated rats challenged with TCE-CCU mixtures of different proportions. Rats were pretreated orally with acetone (0.75 ml/kg) and were chal lenged (ip) 18 hr later with CCU, TCE, or a TCE-CCU mixture (see Fig. 1 for dosages). Plasma samples were obtained 24 hr after the challenge. Results represent the mean SE for six animals/group. i (1.5 ml/kg) showed no hepatotoxicity (Fig. 2 and Table 3). Plasma ALT activities mea sured in acetone-pretreated rats challenged with (doses in ml/kg) [TCE (0.38)-CCl4 (0.19)], [TCE (0.75)-CCL (0.13)], or [TCE (1.11)--CCI4 (0.06)] were similar to each other, and were increased twofold compared to CC14 (0.25 ml/kg)-treated rats (Fig. 2). His tological evaluation ofliver injury in acetonepretreated rats revealed a more severe re sponse in rats challenged with only CC14 (0.25 ml/kg) than those treated with any of the three mixtures studied (Table 3). The results suggest that none of the three mixtures stud ied was subjected to a greater potentiating elfect of the haloalkane-induced liver injury, although the severe response obtained with CC14 (0.25 ml/kg) alone (45% of the lobule being composed of necrotic hepatocytes) re stricted the possibility of observing further marked increases. The cholestatic component of the liver le sion, indirectly evaluated by the increase of plasma total bilirubin concentration, was un- TABLE 3 Histological Evaluation of the Effects of TCE and CC14 Dosage on the Severity of Haloalkane Mixture-Induced Liver Injury in Male Rats Pretreated with Corn Oil or Acetone Dosage ofTCE (ml/kg) Dosage ofCCU (ml/kg) Com oil-pretreated rats Nec Bal Nor Nec/Nor Acetone-pretreated rats Nec Bal Nor Nec/Nor 0 0.38 0.75 1.11 1.5 0.25 0.19 0.13 0.06 0 68 179 182 162 70 184 120 130 162 126 108 178 0 0 396 0.37 0.88 0.74 0.71 0 196 103 135 200 29 171 193 0 207 238 0 184 0 0 432 1.45 1.17 0.93 1.29 0 Note. Rats were pretreated orally with com oil (10 ml/kg) or acetone (0.75 ml/kg) and were challenged 18 hr later with CCU, TCE, or a TCE-CCU mixture. Liver samples were obtained 24 hr after the challenge. Results are expressed in distance (4m) occupied by cell type from the portal triad to the central vein: values represent the mean of 10 observations/animal for six rats/group. Nec, necrotic hepatocytes; Bal, ballooning ofhepatocytes; Nor, normal hepa tocytes. jally severe z. 3). Plas asured in ged with (; U (0.19)], CEO-llM . 2.9, and ' tained for r. /kg). Acett Lh TCE (1. :ase above t ;S (0.31 0.1 1US, the lov e mixture-ii It was pre iuced hepati nultan^us; ated by pretri neau et al., 1 periments v. MED of acei induced live haloalkane c CC14 instead that the ME 0.25 to 0.05 ml/kg) was: lution (Tal amounts of tiate the 1 than those 1 The relev cupational: viously rep< that in rats CCU liver ii sure by in equal to th ministratic (MED); thv tion exposi late very w minjtffeu ME^w a SL 035053 TCE alone 14 mixture % i i \ 2.5 /dl ) ged with TCE-CCU /kg) and were chal-s). Plasma samples Is/group. "able 3). The results hree mixtures studgreater potentiating nduced liver injury, onse obtained with (45% of the lobule >tic hepatocytes) ref observing further nent of the liver le1 by the increase of icentration, was un- 1TY of Haloalkane or acetone -pretreated rats Nor 135 171 207 184 432 Nec/Nor 1.45 1.17 0.93 1.29 0 c challenged 18 hr later :e. Results are expressed ;>resent the mean of 10 ytes; Nor, normal hepa- ACETONE-POTENTIATED TCE-CCL, TOXICITY 437 qually severe for the three mixtures studied ~ig. 3). Plasma bilirubin concentrations teasured in acetone-pretreated rats chalnged with (doses in ml/kg) [TCE (0.38)Cl4 (0.19)], [TCE (0.75)-CCl4 (0.13)], or ,`CE (1.11)--CCL* (0.06)] were, respectively, 1, 2.9, and 3.5 times higher than the value otained for rats treated with only CC14 (0.25 1/kg). Acetone-pretreated rats challenged ith TCE (1.5 ml/kg) alone showed no in ease above the control plasma bilirubin val es (0.31 0.03 vs 0.29 0.01, respectively), hus, the lower the CCI4 dosage, the higher le mixture-induced cholestatic response. DISCUSSION It was previously reported that CCL-inluced hepatotoxicity is enhanced by the sinultaneous administration ofTCE (Pessayre 't al., 1982), and that this response is potentiited by pretreatment with acetone (Charbonneau et al., 1986b). In the present study, ex periments were performed to verify if the MED of acetone required to potentiate CCLinduced liver injury is decreased when the haloalkane challenge is a mixture ofTCE and CCL, instead of CCI4 alone. The results show that the MED of acetone was decreased from 0.25 to 0.05 ml/kg, or lower, when TCE (0.25 ml/kg) was added to the CCU (0.1 ml/kg) so lution (Tables 1 and 2). Thus, smaller amounts of the ketone are required to poten tiate the TCE-CCL* hepatotoxic response than those needed to potentiate CCI4 toxicity. The relevance ofthis observation to the oc cupational setting may be important. We pre viously reported (Charbonneau et al., 1986a) that in rats the severity of ketone-potentiated CC14 liver injury resulting from acetone expo sure by inhalation (2500 ppm for 4 hr) is equal to that obtained after a single oral ad ministration of acetone of 0.25 ml/kg (MED); the injury arising from larger inhala tion exposures (multiples of the MED) corre late very well with the corresponding oral ad ministration dosages of acetone. Since the MED of acetone was shown to be five times lower when TCE was added to the CCL solu tion in the present study, one can infer that the corresponding acetone MED for expo sures by inhalation would be 500 ppm (4 hr exposure). Recently, Landry and Brodeur (unpublished data) observed that exposure of rats to acetone by inhalation (500 ppm for 4 hr) resulted in potentiated liver injury, when they were challenged with CCL (2500 ppm for 6 hr) 18 hr later. Extrapolation of these data to humans in the occupational setting suggests that such an ambient concentration ofacetone would be below the recommended threshold limit value of 750 ppm (8 hr/day, 5 days/week) for industrial exposure. Thus, the acetone dose involved in the potentiation phenomenon falls within that which might arise from exposure in the workplace. Small doses of acetone and TCE, two widely used solvents, simultaneously admin istered with CCL can lead to liver injury. It is probably of greater importance, however, to verify if the administration of larger doses of these solvents combined with the administra tion of a smaller dose of the hepatotoxicant results in liver injury. The data show that, compared to rats challenged with CCL alone, a smaller dose of CCL produced severe liver injury when administered in a mixture with proportionately increasing doses ofTCE (Fig. 1). In all cases, pretreatment with the larger dose of acetone prior to the challenge led to a more severe liver injury (Fig. 2). Normally, CCL liver injury, resulting in ex tensive necrosis, is not associated with marked increases in plasma bilirubin reten tion (Traiger and Plaa, 1971). However, a small dose of CC14 potentiated by isopropa nol, which is biotransformed to acetone (Traiger and Plaa, 1982), produces markedly elevated plasma bilirubin concentrations (Traiger and Plaa, 1971); de Lamirande and Plaa (1981) demonstrated that isopropanol enhances the cholestatic as well as the necrogenic properties of CCL. Thus, with CCL, el evated plasma bilirubin concentrations are an indirect indication ofthe cholestatic prop erties of this haloalkane. Interestingly, results shown in Fig, 3 indicate that elevated plasma SL 035054 438 CHARBONNEAU ET AL. I bilirubin levels were observed when the po tentiation was more severe: [TCE (1.11)-- CCU (0.06)] > [TCE (0.75)-CCL, (0.13)] > [TCE (0.38MXU (0.19)]. These observa tions suggest that the liver injury resulting from the potentiation are associated with a marked cholestatic component as well as ex tensive necrosis. Exposures to haloalkane mixtures may be frequent in the occupational setting. Further more, acetone is quite widely used industri ally. The interactive phenomenon described in the present study is quite evident, but its application to the workplace is yet to be es tablished. It is already evident, however, that the rule of adjusting TLVs for mixtures of chemicals on the assumption that their effects are additive (ACGIH, 1986) is not valid for all combinations of such chemicals. Further research is needed to determine if the obser vations reported herein can be observed with other haloalkanes. Finally, if they can be ob served in laboratory animals, one needs to es tablish the likelihood that the interactions might occur with chemical exposure patterns that exist in occupational environments. REFERENCES ACGIH (1986). Appendix C: Threshold limit values for mixtures. In Threshold Limit Values and Biological Exposure Indicesfor 1986-1987, pp. 45-47. American Conference of Governmental Industrial Hygienists, Cincinnati. CHARBONNEAU, M., BRODEUR, J., DU SOUICH, P.. AND Plaa, G. L. (1986a). Correlations between acetonepotentiated CCU-induced liver injury and plasma con centrations after inhalation or oral administration. Toxicol. Appl. Pharmacol. 84,286-294. CHARBONNEAU, M., OLESKEVICH, S., BRODEUR, J., and Plaa, G. L. (1986b). Acetone potentiation of rat liver injury induced by trichloroethylene-carbon tetra chloride mixtures. Fundam. Appl. Toxicol. 6, 654661. De Lamirande, E., and Plaa, G. L. (1981). 1,3-Butanediol pretreatment ofthe cholestasis induced in rats by manganese-bilirubin combination, taurolithocholic acid or a-naphthylisothiocyanate. Toxicol, Appl. Pharmacol. 59,467-475. Hewitt, W. R,, Miyajima, H., Cote, M. G., and Plaa, G. L. (1980). Modification ofhaloalkane-induced hepatotoxicity by exogenous ketones and metabolic keto sis. Fed. Proc. 39,3118-3123. Iuima, M,, Cote, M. G., and Plaa, G. L. (1983). Semi- quantitative morphologic assessment of chlordeconepotentiated chloroform hepatotoxicity. Toxicol. Leu. 17,307-314. Jendrassik, L., and Grof, P. (1983). Vereinfachte photometrische Methoden zur Bestimmung des Blutbilirubins. Biochem. Z. 297,81-89. Nosslin, B. (1960). The direct diazo reaction ofbile pig ments in serum. Scand. J. Clin. Lab. Invest. (Suppl. 49) 12, 1-176. Pessayre, D., Cobert, B., Descatoire, V., Degott, C., Babany, G,, Funck-Brentano, C,, Delaforge, M,, and Larrey, D. (1982). Hepatotoxicity of tri chloroethylene-carbon tetrachloride mixtures in rats. Gastroenterology 83,761 -772. Pilon, D., Brodeur, J., and Plaa, G. L. (1986). 1,3Butanediol-induced increases in ketone bodies and po tentiation ofCCU hepatotoxicity. Toxicology 40,165- 180. Plaa, G. L., and Hewitt, W. R. (1982). Potentiation of liver and kidney injury by ketones and ketogettic substances. In Advances in Pharmacology & Thera peutics II (H. Yoshida, Y. Hagihara and S. Ebashi, Eds.), Vol. 5, pp. 65-75. Pergamon, New York. Plaa, G. L., Hewitt, W. R,, du Souich, P,, Caille, G., and Lock, S. (1982). Isopropanol and acetone po tentiation of carbon tetrachloride-induced hepatotox icity: single versus repetitive pretreatments in rats. J. Toxicol. Environ. Health 9,235-250. Reitman, S., and Frankel, S. (1957). A colorimetric method for the determination of serum oxaloacetic and glutamic transaminase. Amer. J. Pathol. 28, 5663. Traiger, G. J., and Plaa, G. L. (1971). Differences in the potentiation ofcarbon tetrachloride in rats by etha nol and isopropanol pretreatment. Toxicol. Appl. Pharmacol. 20, 105-112. Traiger, G. J., and Plaa, G. L. (1972). Relationship ofalcohol metabolism to the potentiation ofCCU hep atotoxicity induced by aliphatic alcohols. J. Pharma col. Exp. Ther. 183,481-488. SL 035055 LAMENTAL OCHELLE exti (3 - (3,4-e lane (ECI dustrial si adhesive mixtures epoxy coi ECEMS oral LD5 1 To wb Bush^Lun ExApa