Document 6KpkmG16o0kK4NG8epz857YR

Life Sciences Vol. 13, pp. 67-73, 1973. Printed in Great Britain Pergamon Press EFFECT OF FHENOBARBITAL AND 3-METHYLCHOLANTHRENE PRETREATMENT ON THE HEPATOTOXICITY of 1,1,1-TRT"HLOROETHANE AND 1,1,2-TRICHLOROETHANE Gar; P. Carlson Department of Pharmacology and Toxicology, College of Pharmacy University of Rhode Island, Kingston, Rhode Island 02881 (Received 12 February 1973; in final form 7 May 1973) Summary Pretreatment of rats with phenobarbltal potentiated the hepatotoxicity of both 1,1,1- and 1,1,2-trichloroethane given by Inhalation. The toxicity of the 1,1,2- Isomer was increased to a greater extent than that of the 1,1,1- isomer. 3-Methylcholanthrene pretreatment did not result in increased hepatotoxicity. Introduction The two Isomers of trlchloroethane, 1,1,1- (which is methylchloroform) and 1,1,2-, are important as substitutes for carbon tetrachloride. They are used as solvents, degreasers and intermediates in chemical synthesis. The for mer is now often encountered in dry cleaning preparations, and the latter is - vary important industrially (1). The 1,1,1- isomer is markedly less toxic than the 1,1,2- isomer Garner and Mcl.ean (3) demonstrated that pretreatment with the enzyme in ducing agent phenobarbltal potentiated the hepatotoxicity of CCl^ when given po. Its toxicity was also potentiated when inhaled (4), 3-Methylcholanthrene, which is also an enzyme inducer but differs in its mechanism and spectrum of enzymes induced, has been shown to protect against CCl^ hepatotoxicity (5, 6). It was of interest, therefore, to ascertain what effect, if any, pretreatment vlth these agents would have on the hepatotoxicity of 1,1,1- and 1,1,2-trl- chloroethane. $L 036759 67 68 Hepatotoxicity of Trichloroethanes Vol. 13, No, 1 Materlaig and Method* Adult sale albino rata (Charles River Breeding Laboratories, Wilmington, Maaaachuaette) were injected lntraperltoneally with either phenobarbltal (PB) at a dose of SO mg/kg/day for 4 days or 3-methylcholanthrene (MC) at a dose of 40 mg/fcg/day for 2 days or saline or corn oil vehicle as respective controls. At 24 hr after the last dose of FB or 48 hr rfcer the last dose of MC, the ani mals were exposed to either 1,1,1-trlchloroethane (1,1,1-TCE) or 1,1,2-trlchloroethane (1,1,2-TCE) vapors In a dynamic Inhalation chamber previously de scribed (7) for a period of 2 hr. Chamber concentrations were determined us ing a Packard gas chromatograph equipped with flame Ionization detector. Twenty-two hr after the termination of exposure, the animals were lightly anesthetized vlth ether and tall vein blood samples were obtained. Liver and body veights were recorded. Hepatotoxicity was assessed by measuring serum glu tamic oxalacetlc (SCOT) and glutamic pyruvic (SGFT) transaminases by the method of Reltman and Prankel (8) and liver glucose-6-phosphatase according to Harper (9) using maleate buffer, pH 6.25. The value expressed Is the mean t standard error for a group of 5 animals unless otherwise noted, A two-vay analysis of variance technique was used to test the differences between pretreatments. Inhalation of air versus TCE, and the Interaction between the two. The F values arc presented for the inter actions. The level of significance was set at 0.05. Where animals received the same pretreatment, means were compared using the least significant differ ence (led) teat (10). Results The data in Table 1 indicate that there is an effect of phenobarbltal pre treatment on both 1,1,2-TCE and 1,1,1-TCE hepatotoxicity. In the case of the 1,1,2-TCE, no hepatotoxicity was noted in the saline pretreated animals under the conditions of exposure employed (890 ppm for 2 hr) using the parameters In dicated. There was a decrease In glucose-6-phosphatase and 20-fold elevations In the transaminases In the PB pretreated animals. These values are Indicative SL 036760 Vol. 13, No. 1 Hepatotoxicity of Trichloroethanes 69 of a fairly severe degree of hepatotoxicity. TABLE 1 Effect of Phenobarbitel Pretreatment on 1,1,2-Trlchloroethana and 1,1,1-Trichloroathane Hepatotoxicity Treatment Liver Ht Glucose-6- # 100 g B. Ht. Phosphatase SCPTb SG0Tb Saline-Air Saline-1,1,2-TCE* PB-Alr PB-l,l,2-TCE*'f Fd 4.3 4 0.09 4.7 0.17 S.l 0.19 5.5 0.14* 1.65 10.3 4 0.40 10.3 4 0.69 9.7 4 0.36 8.2 4 0.36h 2.42 19 4 1.5 73 4 3.3 20 4 1.7 91 4 7.3 19 1.8 75 4 4.9 465 4 156.5h 1345 4 187.6h 11.89* 65.16* Saline-Air Saline-1,1,1-TCE* PB-Alr PB-1,1,1-TCE* F4 4.1 0.17 4.3 0.20 4.5 4 0.08 5.2 4 0.09h 2.31 13.8 4 0.85 13.2 0.99 15.8 4 0.56 11.3 4 0.98h 5.041 13 4 1.0 17 4 2.2 16 4 1.2 95 4 7.9h 79.91* 44 4 5.3 59 4 4.6 48 4 2.9 178 4 24.8h 19.36* ymoles PO./g/mln v* Reltman-Frankel Units c890 ppm for 2 hr ^computed for Interaction between pretreatment and Inhalation *11,600 ppm for 2 hr f mean of 4 animals *mean of 3 animals *V<o .05 compared to animals with same pretreatment *F<0.05 Exposure to 1,1,1-TCE gave results qualitatively similar to the 1,1,2-TCE exposure. The rats were able to tolerate higher concentrations without mortal ity. The group pretreated with PB and exposed to 1,1,1-TCE had a higher liver to body weight ratio, decreased glucose-6-phosphatase activity, a 6-fold eleva tion In SGPT and a nearly 4-fold increase in SGOT compered to the air exposed ............................. PPUWl SL 036761 70 Hepatotoxicity of Trichloroethanes Vol. 13, No. 1 group. Whan the inducing agent vaa MC (Table 2), there were few indleatlone of either potentiation or protection. Only very ulnor change* were aeen in the parameters measured as a result of the 1,1,2-TCE pretreatment. In the case of the liver to body weight ratio, there was a significant interaction resulting in enlarged livers in the MC pretraated animals inhaling 1,1,2-TCE. This group TABLE 2 Effect of 3-Methylcholanthrene Pretreatment on 1,1,2-Trichloroethane and 1,1,1-TrlchloToethane Hepatotoxicity Treatment Liver Wt. Glucose-6- 100 g B. Wt. Phosphatase SGPTb SGOTb Corn oil-Air Corn oil-1,1.2-TCEc,f MC-Air MC-1,1,2-TCEC Fd 3.5 0.11 3.4 0.12 4.5 0.08 5.5 0.16h 18.891 12.6 0.49 12.3 1 1.25 12.7 i 0.57 9.8 0.71h 2,98 14 1.0 33 12.0h 9 1 0.9 24 5.6h 0.39 50 i 2.9 93 24.5 54 3.7 107 34.5 0.06 Corn oil-Air Corn oil-1,1,1-TCE* MC-Air MC-1,1,1-TCE* P* 4.4 0.19 4.4 t 0.15 6.0 0.32 5.5 0.31 1.28 17.2 1.36 14.8 1 0.46 13.1 t 1.18 15.8 0.66 6.86* 17 1.2 19 3.1 18 1.9* 15 0.9 2.00 54 3.9 56 * 4.8 73 3.1* 60 * 5.5 2.70 *pmoles P0,/g/min bReitman-F*ranfcel Units C2080 ppm for 2 hr dcomputed for interaction between pretreatment and Inhalation *13,070 ppm for 2 hr fone animal died overnight; blood samples were unobtainable from 2 other* mean of 4 animals bp<0 .05 compared to animals with same pretreatment *?<0.05 SL 036762 Vol. 13, No. 1 Hepatotoxiclty of Trichloroethanes 71 also showed a slight decrease In glucose-6-phosphatase activity although the F value Indicates that this was not a significant Interaction. The changes in the scrum transaminases were minimal and were similar with both pretreatments. Higher concentrations of 1,1,2-TCE than the 2080 ppm used could not be employed because of a low survival rate among the animals with increased concentration. Even at the concentration used, one com oil animal died overnight and two others vere so moribund that tall vein blood samples were unobtainable. Following 1,1,1-TCE exposure, there were no changes in either group which could be associated with the inhalation despite the high concentration (13,070 ppm) used. Although the changes in glucose-6-phosphatase activity due to inha lation were not statistically significant, the F value is significant since the changes occurred in opposite directions. In the case of 1,1,1-TCE exposure, therefore, it is not possible to show protection because of the lack of ability of the 1,1,1-TCE to cause significant hepatotoxlcity. Discussion The lack of ability of 1,1,1- and 1,1,2-TCE inhalation to cause hepato toxiclty in the rats is not surprising. Flatt and Cockrlll (11) administered the compounds po for 7 days and found no increase in liver to body weight ratios or decrease in glucose-6-phosphatase. Klaassen and Plaa (12) administered the compounds ip and observed no decrease in glucose-6-phosphatase and no increase in lipid peroxidation. Because of the lack of liver damage in these present experiments, it could not be determined if MC had a protective effect. Mortali ty prohibited the use of higher concentrations. It can be concluded, however, that MC did not potentiate the hepatotoxiclty of the trichloroethanes. FB pretreatment did result in an enhancement of the hepatotoxlcity of both compounds. In actuality, it might better be expressed that hepatotoxi clty was manifested that otherwise would not have appeared. A much greater enhancement was seen with 1,1,2-TCE than with 1,1,1-TCE. It is difficult to speculate how the potentiation occurs since the exact mechanism by which the chlorinated hydrocarbon solvents produce their hepato- m SL 036763 72 Hepatotoxiclty of Trichl roethanes Vol. 13, No. 1 toxicity la unknown. Reeknagel (13) suggests that the metabolism of CCl^ re sults In trlchloromethane free radicals which cause peroxidation of structural lipids and thus necrosis of hepatocytes. Garner and McLean (3) demonstrated that PB pretreatvent results in an Increased metabolism of CCl^. Following the above hypothesis, this would be expected to result In the generation of nore free radicals and consequently more lipid peroxidation and cell destruction. Ton Dyke and Winenan (14) found that 1,1,1-TCE was dechlorlnated In only trace amounts but that 1,1,2-TCE was a good substrate for dechlorination and the re action could be induced by pretreatnent with PB but not with MC. However, Klaassen snd Plan (11) vers unable to detect any Increase In lipid peroxidation in rats treated with 1,1,1-TCE or 1,1,2-TCE. Thus, It is important from both a practical and a theoretical viewpoint that PB will potentiate the toxicity of the trichloroethanea, but the molecular basis of this potentiation, and Indeed the mechanism of hepatotoxlclty of the agents themselves, remains unknown. Acknowledgement The author wishes to acknowledge the technical assistance of Mrs. Barbara Schultz, Mrs. Arlene Johnson and Mrs, Nadylls Wood. I am also Indebted to Dr, Choudary Hanumara for his aid in the statistical analysis of the data. References 1. L. T. FAXRHAIX, Industrial Toxicology, p. 347. Bsfner Publishing Company, New fork (1969). 2. E. BROWNING, Toxicity and Metabolism of Industrial Solvents, p. 253. Elsevier Publishing Company, Amsterdam (1965). 3. R. C. GARNER and A. E. M. McLEAN, Biochem. Pharmacol. 18, 645-650 (1969). 4. H. LAL, S. K. PURI and G. C. FULLER, Toxicol. Appl. Pharmacol. 16. 35-39 (1970). 5. K. A. SUAREZ, G. P. CARLSON, G. C. FULLER and N. FAUSTO, Toxicol. Appl. Pharmacol. 23, 171-177 (1972). 6. B. STRIPP, M. E. HAMRICK and J. R. GILLETTE, Biochem. Pharmacol. 21, 745747 (1972). 7. H. LAL, S. K. PURI and G. C. FULLER, Pharmacol. Res. Conaaup. 2, 143-147 (1970). i \ \ " SL 036764 Vol. 13, No. 1 Hepatotoxicity of Trichloroethanes 73 8. S. REIMAN and S. K. FRA5KEL, Amer, J. Clin. Pathol. 28, 56-63 (1957). 9. A. E. HARPER, Methods of Enzymatic Analysis (H. 0. Bergmeyer, ed.), p.778. Academic Frees, New York (1963). 10. R. G. D. STEEL end J. H. TORRIE, Principles and Procedures of Statistics, . p. 106. McGraw-Hill Book Company, New York (1960). U. D. S. PLATT and B. L. COCKRILL, Blochem. Pharmacol. IS, 445-457 (1969). 12. C. D. KLAASSEN and G. L. PLAA, Blochem, Pharmacol. 18. 2019-2027 (1969). 13. R. 0. RECKNAGEL, Pharmacol. Rev. 19. 145-208 (1967). 14. R. A. VAN DYKE and C. G. WINEMAN, Blochem. Pharmacol, 20, 463-470 (1971). SL 36765