Document RvEQ21j6KJvQm0eYNDOjr8Mz

COMPARISON OF THE BIOCHEMICAL ALTERATIONS ELICITED IN LIVERS FROM RATS TREATED WITH CARBON TETRACHLORIDE, CHLOROFORM, 1,1,2-TRICHLOROETHANE AND 1,1,1-TRICHLOROETHANE* Curtis D. Klaassenx and Gabriel L. Plaa; Oakdale Toxicology Center, Department of Pharmacology, College of Medicine, University of Iowa, Iowa City, Iowa 52240, U.S.A. (Received 12 November 1953; accepted 21 February 1969) Abstract--Chlorinated hydrocarbons differ in their capacity to produce liver damage. . The comparative effects of carbon tetrachloride (CCD, chloroform, 1.1,1-trichloro- ethane and 1,1,2-trichlorcethane were studied in rats, CCh produced the highest increase in hepatic triglycerides and also produced elevated levels at the lowest dose (0 03 ml/kg). Chloroform was intermediate in producing elevated liver triglyceride levels; with 1,1,2-trichloroethane, enhanced triglyceride levels'were demonstrated only at near-lethal dosages. No enhanced hepatic triglyceride level was demonstrated with 1,1,1-trichloroethane. Significant dose-related decreases rn glucose 6-phosphatase activity were demonstrated with doses of 0-3 ml/kg of CCU or greater. With the other chlorinated hydrocarbons, no decrease in hepatic glucose 6-phosphatase activity was detected. When the hydrocarbons were added directly to liver homogenates, only those incubations containing CCU exhibited increased lipid peroxidation (enhanced thiobarbituric acid reactants). In vivo, evidence of lipid peroxidation (diene conjugates) in the liver was obtained 15 min after CC1: treatment; this effect reached its peak at 30 min. Enhanced diene conjugates were detected with doses of 0*J^il/kg of CCU or greater. However, with the other three hydrocarbons, no increase in diene conjugates was detected. Thus, this study shows that while the temporal relationships of CCU-indcced hepatotoxicity are compatible with the lipid peroxidation hypothesis, the dosage relationship is weaker. Also the lack of qualitative similarity in the results obtained with chloroform is disturbing. A large number of studies have been performed to determine the mechanism of carbon tetrachloride hepatotoxicity. The hypotheses concerning the mechanism of the fatty changes seen after carbon tetrachloride have been recently reviewed by Recknagel.1 A current hypothesis is that the primary event in the hepatotoxic response is the homolytic cleavage of carbon tetrachloride to trichloromethyl free-radical and monatomic chlorine. This cleavage presumably takes place in or near the endoplasmic reticulum of the hepatic parenchymal cell, and the free-radicals are believed.to attack the lipoidal elements of the endoplasmic reticulum, thus initiating the autocatalytic, peroxidative breakdown of these membranes. The liver celts are then unable to secrete triglycerides into the plasma, and the fatty liver develops. * This work was supported by funds from USPHS Research Grant GM-12675. t USPHS Predoctoral Fellow (5-F!-G_M-30,995). Present address; Department of Pharmacology, University of Kansas Medical School, Kansas City, Kansas. t Present address: Department of Pharmacology, Faculty of Medicine, University of Montreal, Montreal, Canada. 2019 SL 036890 2020 Curtis D. Klaassen and Gabriel L. Plaa Chlorinated hydrocarbons other than carbon tetrachloride also produce liver dysfunction. Klaassen and Plaa2-3 reported that in both mice and dogs carbon tetrachloride caused severe hepatic dysfunction, while moderate to severe changes were seen with chloroform; moderate alterations were seen with 1,1,2-trichloroethane, and only mild dysfunction was observed with trichloroethylene, tetrachloroethylene, dichloromethane and 1,1,1-trichIoroethane, when administered at near-lethal doses. The purpose of the present investigation was to study the temporal and dosage relationship in regard to the biochemical alterations seen after carbon tetrachloride and also to determine if similar changes could be exhibited after chloroform, 1,1,2trichloroethane and 1,1,1-trichIoroethane. METHODS Male, Sprague-Dawley rats (300-400 g) were used throughout. Analytical grades of the following hydrocarbons were employed: carbon tetrachloride; chloroform; 1,1,2-trichloroethane; and 1,1,1-trichIoroethane. All agents were administered intraperitoneally and were made up in corn oil solutions to deliver the proper dosage in a final volume of 1 ml/100 g of body weight. Lethality. For the 24 hr LD50 determinations, four or five groups of rats, five to ten per group, were injected with a graded series of doses of the hydrocarbons and the number of deaths was recorded at the end of 24 hr. The median lethal dose (ld50) was then calculated for each hydrocarbon by the method of Litchfield and Wilcoxon.4 Liver triglycerides. Hepatic triglycerides were measured by a modified method of Van Handel and Zilversmit.5 Glucose 6-phosphatase. Activity of this enzyme was determined in rat liver homogen ized in 01 M maleic acid (pH 6-25). Final conditions for the enzyme assay were as follows: 0-5 ml of 0-2 M disodium glucose 6-phosphate, pH 6-25; 1-5 ml of 0T M maleic acid buffer, pH 6-25; 0-5 ml liver homogenate, 20 mg/ml; 40 min of incubation at 37. The reaction was terminated with the addition of 10 ml of 10% trichloroacetic acid. Inorganic phosphorus was determined in 2-ml aliquots of the supernatant by the method of Fiske and Subbarow, using reagents obtained from the Sigma Chemical Company. Lipidperoxidation in vitro. The peroxidation of rat liver homogenates after addition of various amounts of chlorinated hydrocarbons was determined by using the thiobarbituric acid (TBA) method of Comparti et alJ<3 Final conditions for the enzyme assay were as follows: 2 ml of 0-15 M potassium phosphate buffer, pH 7-4; 1 ml of 0-15 M potassium chloride; 1 ml of 30% liver homogenate in ice cold 0-15 M KCI; varying volumes of the hydrocarbons; the flasks stoppered, incubated, and shaken for 45 min at 37. The reaction was stopped by the addition of 4 ml trichloroacetic acid. After centrifugation, the amount of peroxide formed was estimated in 2-0 ml of supernatant by adding 2*0 ml of 0*67% TBA and boiling for 10 min. Optical densities were measured at 543 mp. Lipid peroxidation in vivo. The quantitative estimation of the lipid peroxidation in vivo was determined by measuring diene conjugate ultraviolet absorption of lipid extracts of the microsomal fraction of rat livers, as described by Reynolds (personal communication). Eight g of liver was homogenized with a Potter-Elvehjem homogenizer in 16 ml of ice-cold 0*3 M sucrose which was 3 mM with respect to EDTA. The homogenate was centrifuged at 9000 g for 20 min and 13 ml of the supernatant was 036891 SL UP.TL Jc also produce liver nice and dogs carbon rate to severe changes h 1,1,2-trichloroethane, le, tetrachloroethylene, I at near-lethal doses. : temporal and dosage ;r carbon tetrachloride fter chloroform, 1,1,2- tout. Analytical grades achloride; chloroform; ere administered intrathe proper dosage in a oups of rats, five to ten : hydrocarbons and the xlian lethal dose (LD50) itchfield and Wilcoxon.4 a modified method of ed in rat liver homogen- II enzyme assay were as 6-25; 1-5 ml of 0-1 M 1; 40 min of incubation 1 of 10% trichloroacetic >f the supernatant by the >m the Sigma Chemical nogenates after addition lined by using the thio- nl of 0-15 M potassium ide; 1 ml of 30% liver the'hydrocarbons; the reaction was stopped by the amount of peroxide , ml of 0-67% TBA and f the lipid peroxidation iolet absorption of lipid 1 by Reynolds (personal Uer-Elvehjem homogenii respect to EDTA. The 1 of the supernatant was i i! i | Chlorinated hydrocarbons and biochemical alterations 2021 f then centrifuged at 94,000 g for 60 min. The supernatant was decanted and the } microsomal fraction was resuspended in 2 ml of the sucrose-EDTA solution. An [ aliquot of the microsomal suspension (0-5 ml) was .added to 9-5 ml of a chloroform; methanol (2:1) mixture. After centrifugation, the supernatant was decanted into a graduated centrifuge tube and brought to a volume of 10 ml by the addition of ! chloroform-methanol mixture. Distilled water (2-0 ml) was added and the two phases f were thoroughly mixed and then centrifuged. The upper phase was aspirated off and discarded. The interface was washed twice with 0-5 ml of fresh upper phase. Stock ' upper and lower phases were made each day by adding 25 ml water to 95 ml of the chloroform-methanol mixture and shaking. The upper phase was used for washing the interface, and the lower phase was used as a blank for spectrophotometry. Methanol (200 /d) was added to each tube and the absorbance of an aliquot was read ; at 243 m/i in a Gilford spectrophotometer. j Statistics. Means were compared by an analysis of variance. When a significant | difference (P < 0 05) was found, the means were then compared by the Student ` f-test.9 RESULTS Lethality. The 24 hr LD50 values and their per cent confidence limits were as follows; 1,1,2-trichloroethane, 0-65(0-60 to0-70) ml/kg; chloroform, 1 -3 (l-l to 1 -4) ml/kg;carbon tetrachloride, 1-9 (1-7 to 2-1) ml/kg; aiid 1,1,1-trichloroethane, 3-8 (3-3 to 4-2) ml/kg. Similar LD50 values have been previously reported for these hydrocarbons in mice and dogs.2-3 These LD50 values were used in the later studies to calculate the 0*75 X LD50 doses used in the time-response studies: carbon tetrachloride, 1-4 ml/kg; chloroform, 0-94 ml/kg; 1,1,2-trichloroethane, 0-49 ml/kg; and 1,1,1-trichloroethane, 2-8 ml/kg. Fig. 1. Temporal accumulation of hepatic triglycerides (per g wet wt. of liver) after a high dose of each hydrocarbon (0-75 x ldso)-Each point represents the mean S.E. of three rats. BP SP SL 036892 2022 Curtis D. Klaassen and Gabriel L. Plaa Liver triglycerides. Figure l demonstrates the 36 hr time response for the accumula tion of hepatic triglycerides after treatment with the four hydrocarbons (0.75 x LD50). The highest hepatic triglyceride levels were demonstrated after carbon tetra chloride treatment; enhanced levels were demonstrated 1-5 hr after its administration, which was the earliest time tested. The triglyceride level increased until the 12 hr interval and remained quite constant thereafter. Chloroform and 1,1,2-trichloro* ethane produced intermediate elevations in hepatic triglycerides and tended to increase throughout the 36-hr interval. With 1, 1, l-trichloroethane, no increase in hepatic triglycerides was demonstrated at any time interval. Figure 2 depicts the dose-response relatinship for the accumulation of triglycer ides 24 hr after the administration of the chlorinated hydrocarbons. Carbon tetra chloride produced the highest hepatic levels of triglycerides and also produced Fio. 2. Dose-response relationship for the accumulation of hepatic triglycerides (per g wet wt. of liver) 24 hr after administration of the hydrocarbon. Mean S.E. of three rats. Five doses were used for carbon tetrachloride, four for chloroform, and two for 1,1,2-trichloroethane. elevated levels at the lowest dose. Chloroform was intermediate in producing en hanced liver triglyceride levels. With 1,1,2-trichloroethane enhanced triglyceride levels were demonstrated only at near-lethal doses. Glucose 6-phosphatase. Figure 3 depicts the glucose 6-phosphatase activity of the liver of rats at various times after treatment with carbon etrachloride (0-75 x LDso). Three hr after treatment, the enzyme activity was still normal, but by 6 hr it was depressed to below 50 per cent of control activity. The activity remained low for the rest of the time intervals tested. With the other chlorinated hydrocarbons, no decrease in glucose 6-phosphatase was exhibited at any of the time intervals. These values ranged from 9*9 0*7 to 12-7 1-2, 9*1 2-5 to 14*8 1*9, and 10*6 1-1 to 13*8 1*5 mg Pi/g, for chloroform, 1,1,2-trichloroethane and 1,1,l-trichloro ethane respectively. Figure 4 demonstrates the glucose 6-phosphatase activity 12 hr after varying doses of CCI4. There was a significant decrease in activity from control levels in the rats treated with 1*0 and 0-3 ml/kg, but not with 0-1 and 0*03 ml/kg. SL 036893 *-.ip it * 1 ;ponse for the accumula hydrocarbons (0.75 X ated after carbon tetra after its administration, icreased until the 12 hr rm and 1,1,2-trichloroes and tended to increase no increase in hepatic emulation of triglycerocarbons. Carbon tetrades and also produced Tttraclilorid* Chlorinated hydrocarbons and biochemical alterations 2023 Fig. 3. Time-response relationship for glucose 6-phosphatase activity (per g wet wt. of liver) after a dose of CCU (0-75 x LDso). Mean S.E. of three rats. (glycerides (per g wet wt. of hree rats. Five doses were used ,2-trichIoroethane. tediate in producing enie enhanced triglyceride osphatase activity of the rachloride (0-75 X LDso). mal, but by 6 hr it was ctivity remained low for inated hydrocarbons, no the time intervals. These -S 1-9, and 10-6 1-1 iane and 1,1,1-trichloro- 12 hr after varying doses control levels in the rats kg. Fig. 4. Dose-response relationship for glucose 6-phosphatase activity (per g wet wt. of liver) 12 hr after CCI4. Mean S.E. of three rats. Table 1. Effect of various concentrations of the chlorinated hydrocarbons on lipoperoxidation in vitro Carbon tetrachloride Chloroform 1 0-210 4- 0024U 0-148 0-024 2 0-236 0-020) 0160 0-024 5 0-268 0-041) 0-156 0017 10 0-290 0 051) 0-128 0-16 25 0239 0-072) 0 093 0-005 50 0-111 0 009 0070 -1- 0-0055 100 0 111 i 0-011 0-079 00095 200 0-112 0 011 0 063 0 0095 1,1,2-Trichloroethane 1,1,1-TrichIoroethane 0-151 0023 0-111 0024 0 095 dr 0 009 0-072 00055 0-038 0-0065 0-071 i 0 0085 0-080 00075 0-064 0-0085 0-132 -U 0021 0131 _L 0-019 0-145 0-023 0-177 0031) 0-107 0-013 0-078 00075 0-068 0-012 0-059 00105 * Volume of hydrocarbon added to the incubation medium. t Mean S.E. of the optical density of the TBA reactants of six treated rats. The mean S.E. of 24 control rat livers was 0-123 0-008. $ Significantly greater (P < 0-05) concentration of TBA reactants than in control rat liver. 5 Significantly lower (P < 0-05) concentration of TBA reactants than in control rat liver. SL 036894 2024 Curtis D. Klaassen and Gabriel L. Plaa Fio. 5. Time-response relationship for appearance of diene conjugates in microsomal lipids after administration of CCU (0-75 x ldjo). Mean S.E. of three rats. % Lipidperoxidation in vitro. Table 1 demonstrates the ability of the four chlorinated hydrocarbons to enhance peroxidation in vitro, as measured by the thiobarbituric acid method. Increased TBA reactants were detected after 1, 2, 5, 10 and 25 pi carbon tetrachloride, but not with 50, 100 and 200 p\. With chloroform and 1,1,2-trichloroethane, no enhanced lipid peroxidation was detected with any of the doses used. However, decreased lipid peroxidation was observed with the higher doses of these two hydrocarbons. With 1,1,1-trichloroethane, enhanced lipid peroxidation was detected with one dose of the hydrocarbon (10/d) and decreased with the higher doses (50-200 /d). Lipid peroxidation in vivo. Figure 5 depicts the amount of lipid peroxidation detected at various time intervals in livers from rats treated with carbon tetrachloride (0-75 x LD50) as measured by the ultraviolet absorption of microsomal lipid extracts. Figure 5 demonstrates that the appearance of enhanced quantities of the products of lipid peroxidation was quite rapid, with peak quantities detected at 30 min. The amount then decreased to control values by 12 hr. With the other three hydrocarbons, no increased amounts of lipid peroxides were detectable at any of the time intervals. The optical density values ranged from QT51 0 025 to 0-236 0 053, 0 138 0-016 to 0170 0 007, and 0-124 0-023 to 0-208 0 031 for chloroform, 1,1,2trichloroethane and 1,1,1-trichloroethane respectively. Figure 6 depicts the amount of diene conjugates in the microsomal lipid extracts 30 min after the various doses of carbon tetrachloride. These were significantly higher than control in the rats treated with 0-3 or 1-0 ml/kg, but not with 0-1 ml/kg, DISCUSSION In determining the mechanism of action of any toxic agent, temporal and dosage parameters are important considerations. These two aspects should correlate reason ably well when morphologic effects are compared to the supposed alterations in SL 036895 Chlorinated hydrocarbons and biochemical alterations 2025 l 12 tes in microsomal lipids after of three rats. ty of the four chlorinated by the thiobarbituric acid 5, 10 and 25 ^1 carbon ^orm and 1,1,2-trichloroJany of the doses used, the higher doses of these I lipid peroxidation was ised with the higher doses int of lipid peroxidation with carbon tetrachloride microsomal lipid extracts, quantities of the products s detected at 30 min. The other three hydrocarbons, any of the time intervals. 3 0-236 0-053, 0-138 331 for chloroform, 1,1,2- microsomal lipid extracts >c were significantly higher ;t with 0-1 ml/kg. ^ent, temporal and dosage ts should correlate reasone supposed alterations in Fic. 6. Dose-response relationship for appearance of diene conjugates in microsomal lipids 30 min after administration of CCI4. Mean S.E. of three rats. biochemical function which are believed to lead to the alterations in structure. A lack of correlation can minimize cause and effect considerations, as has been demon strated with the demise of the mitochondria as the initial site of action of carbon tetrachloride.1*10 The results of the present study demonstrate that triglycerides accumulate rapidly during the first 12 hr after treatment with a high dose of carbon tetrachloride. If one assumes that this accumulation is somehow associated with changes in function of the endoplasmic reticulum, one also sees that one index of its integrity, microsomal glucose 6-phosphatase activity, is also affected quite early by the treatment. Recknagel and Lombardi11 reported significant decreases 2 and 4 hr after carbon tetrachloride, whereas we observed significant decreases from 6 to 36 hr later. Reynolds and Yee,12 using a histochemical procedure for measuring glucose 6-phosphatase activity, detected a decrease within 1 hr after treatment. Recknagel and Ghoshal13*14 have shown that hepatic microsomes prepared from rats treated with carbon tetrachloride contain more diene conjugates than normal. These products of lipid peroxidation were found to be elevated as early as 1-5 hr after treatment with carbon tetrachloride. In the present study, this observation was confirmed, and evidence of increased diene conjugation was found 15 min after treatment. Therefore, temporal changes in^ the endoplasmic reticulum correlate reasonably well with the reported morphologic changes observed by both light and electron microscopy10 and are compatible with the lipoperoxidation hypothesis of Recknagel. When one turns to the dose-response relationships, a correlation also seems to exist, although perhaps not as well as that seen with time. With glucose 6-phosphatase, we observed a significant decrease only at carbon tetrachloride doses of 0-3 ml/kg or greater. However, Reynolds and Yee,12 using a histochemical technique, have detected decreases in the level of this enzyme after treatment with lower doses of carbon tetrachloride. When microsomal diene conjugates were measured, again we found that doses of 0-3 ml/kg or greater of carbon tetrachloride were required to show significant increases. Yet, the triglyceride data show that hepatic triglyceride accumulation occurs at doses of carbon tetrachloride below 0-3 ml/kg. In previous work,15 it has been shown that sulfobromophthalein retention can occur in rats SL 036896 2026 Curtis D, Klaassen and Gabriel L. Plaa after a carbon tetrachloride dose of 0-1 ml/kg. With light microscopy, morphologic changes have been observed16 in rats treated with 0-13 ml/kg. The correlation begins to falter when one compares the results obtained in the various parameters tested with the different halogenated hydrocarbons employed. Only carbon tetrachloride resulted in increased hepatic triglycerides, decreased microsomal glucose 6-phosphatase activity, and increased microsomal diene con jugates. Chloroform, which causes pathologic changes similar to those produced by carbon tetrachloride,2-3*17 caused an increase in triglycerides, but did nothing to glucose 6-phosphatase activity or diene conjugation. Reynolds and Yee12 also were unable to find alterations in glucose 6-phosphatase activity 1 hr after treatment with chloroform. We found no effect on either parameter 3-36 hr after chloroform treat ment. Our results obtained with the 1,1,1-trichloroethane are not surprising in view of its low order of hepatotoxicity; the lesion produced by 1,1,2-trichloroethane is also milder than that produced by carbon tetrachloride.2'3 However, even when the hydrocarbons were added directly to liver homogenates, only those incubations containing carbon tetrachloride exhibited increased lipid peroxidation (enhanced TBA reactants). It was further shown that this effect only applied when 1-25 y\ was added; with larger amounts, no increase in TBA reactants was observed. Inhibi tion of lipid peroxidation in vitro with high concentrations of carbon tetrachloride has been observed by Comparti et al.7 We observed similar effects with high concentra tions of chloroform and the tric'hloroethanes. Thus, this study shows that, while the temporal relationships of carbon tetra chloride-induced hepatotoxicity are compatible with the lipid peroxidation hypothesis, the dosage relationships are weaker. Finally, the lack of a qualitative similarity in the results obtained with the other halogenated hydrocarbons, particularly chloro form, is disturbing. It is difficult to interpret negative data, such as the lack of effect seen with the lower doses of carbon tetrachloride and with the other hydrocarbons. Perhaps our biochemical techniques are still too insensitive to detect the absolute minimum amount of change necessary to produce a lesion, and what one measures after carbon tetrachloride is a response which is greater than that necessary to elicit the lesion. In this light, the lack of a response observed with chloroform could mean that with this agent only a minimal effect occurs, and that this effect is undetectable by our techniques, although it is sufficient to elicit the lesion. On the other hand, it might be that carbon tetrachloride and chloroform have different mechanisms of action. It has already been demonstrated in mice and dogs.2*3 that while the end results are qualitatively similar for both agents, quantitative comparisons of potency show that carbon tetrachloride exerts its action with doses which are about 10-13 times lower than those required for chloroform. Reynolds and Yee12 reported that the transient influx of calcium seen after carbon tetrachloride treatment is not seen with other chloromethanes. Our present triglyceride results also indicate a difference between these agents in the time required to reach peak effect. Perhaps carbon tetra chloride has two mechanisms of action and chloroform shares only one of these mechanisms. Finally, these results could indicate that the changes seen with glucose 6-phosphatase and lipoperoxidation after carbon tetrachloride are only secondary effects and have little to do with the initiating event in the development of the lesion. Thus, it must be admitted that the molecular basis of carbon tetrachloride hepato toxicity is not yet solved and many aspects need to be resolved. SL 036897 f microscopy, morphologic ; results obtained in the hydrocarbons employed. triglycerides, decreased ! microsomal diene conjar to those produced by ides, but did nothing to olds and Yee12 also were I hr after treatment with ir after chloroform treatire not surprising in view y 1,1,2-trichloroethane is However, even when the , only those incubations peroxidation (enhanced ly applied when 1-25 /d mts was observed. Inhibif carbon tetrachloride has ects with high concentra- onships of carbon tetrai peroxidation hypothesis, a qualitative similarity in Bans, particularly chloroTMsuch as the lack of effect l the other hydrocarbons, ve to detect the absolute i, and what one measures an that necessary to elicit th chloroform could mean this effect is undetectable on. On the other hand, it different mechanisms of )gs.2-3 that while the end /e comparisons of potency es which are about 10-13 s and Yee12 reported that >ride treatment is not seen s also indicate a difference feet. Perhaps carbon tetrashares only one of these changes seen with glucose loride are only secondary development of the lesion, rbon tetrachloride hepatoIved. j ; J I : ! ! ` ; ; 1 1 i ! i ; | ! I Chlorinated hydrocarbons and biochemical alterations 2027 Acknowledgement--We wish to acknowledge the excellent technical assistance of Mrs. Carol Butters and Mrs. Jana Nieman. REFERENCES 1. R. O. Recknagel, Pharmac, Rev. 19,145 (1967). 2. C. D. Klaassen and G. L. Plaa, Toxic, appl. Pharmac. 9, 139 (1966). 3. C. D. Klaassen and G. L. Plaa, Toxic, appl. Pharmac. 10,119 (1967). 4. J. L. Litchfield, Jr. and F. Wilcoxon, J. Pharmac. exp. Ther. 96,99 (1949). 5. W. M. Butler, A. M. Maling, M. G. Horning and B. B. Brodie, J. Lipid Res. 2,95 (1961). 6. C. H. Fiske and Y. Subbarow, J. biol. Chem. 66, 375 (1925). 7. M. Comparti, C. Saccocci and M. U. Dianzani, Enzymolgia 29,185 (1965). 8. M. D. Comparti, R. Hartman and H. R. DtLuzio, Lab. Invest. 16, 616 (1967). 9. R. G. Steele and J. G. Torrie, Principles and Procedures of Statistics. McGraw-Hill, New York, (1960). 10. G. L. Plaa and R. E. Larson, Archs, envir. Hlth. 9,536 (1964). 11. R. O. Recknagel and B. Lombardi, J. biol. Chem. 236,564 (1961). 12. E. S. Reynolds and A. G. Yee, Lab. Invest. 16,591 (1967), 13. R, O. Recknagel and A, K. Ghoshal, Explmolec. Path. 5,473 (1966). 14. R. O. Recknagel and A. K. Ghoshal, Lab. Invest. 15,132 (1966). 15. C. D. Klaassen and G. L. Plaa, Toxic, appl. Pharmac. 12,132 (1968). 16. R. E. Larson, G. L. Plaa and L. M. Crews, Toxic, appl. Pharmac. 6,154 (1964).. 17. W. F. VonOettingen, The Halogenated Hydrocarbons ofIndustrial andToxicological Importance. Elsevier, Amsterdam (1964). i i SL 036898