Document QgeL6Rp36EoaEx77jJB40nQNE

't ofsino-aortic denervation .ory responses to cyanide, 68 , PlANTADOSI, C. A., AND ional cerebral blood flow in ind periluorocarbon tiansBiol. 200,59-65. AND Piantadosi, C. A. Ton-for-blood exchange on v in the rat. Amer. J. Phys- 5., and Kameyama, M. titular distribution of cyain the central nervous sys-545. . (1983). A venous outflow rapid changes ofthe cerei consumption in the rat. ASAW'A, S., Narlio, y,, Y. (1982). Elficacy of a L 20%) on cerebral isch- ia. A. L. (1984). Cerebral by cyanide in bloodless v. L., and Jobsis, F. F. chrome a,aj oxidationnn in vivo. J. din. Invest. ~. and Jobsis-Vander:es in brain cytochrome ' and cyanide in vivo. J. 14. L., Saltzman, H. A., F. F. (1985). Carbon tions in the brain ofthe Ippl. Physiol. 58, 665- JURTNER, G. H,, AND Taction ofCO and cyand metabolism. Arch. N, M. A. (1967). The Methods for studying liac output and organ 84. \nd Vega, C. (1963). produced by low dos' 204(2), 309-313. L,, Morgan, r. l,, (thiosulphate on cvaToxicol. Appl. Phar- toxicology and applied pharmacology 94,45-54 (1988) Induction of Strand Breaks in DNA by Trichloroethylene and Metabolites in Rat and Mouse Liver in Vivo Mark A. Nelson and R. J. Bull Pharmacology/Toxicology Graduate Program, College ofPharmacy, Washington State University, Pullman, Washington 99164-6510 Received August 17.1987: accepted January 26,1988 Induction of Strand Breaks in DNA by Trichloroethylene and Metabolites in Rat and Mouse Liver in Vivo. NELSON, M. A., AND BULL, R. J. (1988). Toxicol. Appl. Pharmacol. 94, 45-54. The ability of trichloroethylene (TCE) and selected metabolites to induce single-strand breaks in hepatic DNA of male B6C3F1 mice and Sprague-Dawley rats in vivo was evaluated using an alkaline unwinding assay. Doses of TCE of 22-30 mmol/kg were required to produce strand breaks in DNA in rats, whereas a dose of 11.4 mmol/kg was sufficient to increase the rate of alkaline unwinding in mice. To assess the importance of TCE metabolism to this response, rats were subjected to pretreatments of ethanol, phenobarbital, TCE, or the appropriate vehicle for 4 days prior to challenge doses ofTCE. Phenobarbital and TCE, but not ethanol pretreatments, reduced the dose of TCE required to produce significant increases in single-strand breaks. In another series of experiments, mice and rats were treated with metabolites of TCE. Trichloroacetate. dichloroacetate, and chloral hydrate induced strand breaks in hepatic DNA in a dosedependent manner in both species. Strand breaks in DNA were observed at doses that produced no observable hepatotoxic effects as measured by serum aspartate aminotransferase and alanine aminotransferase levels. The slopes ofthe dose-response curves and the order ofpotency ofthese metabolites differed significantly between rats and mice, suggesting that different mechanisms of single-strand break induction may be involved in the two species. These data provide a potential explanation for the different sensitivity of mice and rats to the hepatocarcinogenic effects of TCE. 1988 Academic Press. Inc. Trichloroethylene (TCE) is used extensively as an industrial solvent and as a household cleaning fluid (Waters et ai., 1977). It is com monly found in waste disposal sites and is a frequent contaminant of both surface and ground waters (Westrick et ai, 1982). TCE produces an increase in the incidence of liver neoplasms in B6C3F1 mice. This mouse strain has a high background inci dence of hepatic tumors, particularly in males. As a consequence, the mechanisms by which such tumors arise are a source of con troversy. TCE also produces a low (4-8%) but significant increase in renal (but not hepatic) neoplasms in Fischer-344 rats (NCI, 1976; NTP, 1983). A number of explanations have been offered for the difference in tumorigenic re sponses between the two species. First, the rate of TCE metabolism is much greater in the mouse than in the rat. This is particularly noticeable at high doses (>7,6 mmol/kg) where the proportion of unchanged TCE ex creted markedly increases in the rat but less so in the mouse (Prout et al,, 1985). Second, TCE is also more effective in inducing liver peroxisomes in mice as compared to rats (Elcombe, 1985), a property that has been asso ciated with hepatic carcinogenesis (Reddy and Lalwani, 1983). Trichloroacetic acid (TCA) is postulated as the metabolite of TCE responsible for peroxisome induction (El- 45 0041-008X/88 $3.00 Copynght 1988 by Academic Press, Inc. All rights of reproduction in any form reserved. SL 034535 46 NELSON AND BULL combe, 1985; Goldsworthy and Popp, 1987). Third, the induction of overt liver damage by TCE has been reported to be greater in mice than in rats (Schumann et ai, 1980). This ac tivity could result in regenerative hyperplasia that could stimulate the outgrowth ofsponta neously initiated cells into neoplasms. TCE covalently binds to DNA when incu bated in the presence of hepatic microsomes or the S9 fraction in vitro (Baneijee and Van Duuren, 1978; Cunningham et al., 1981; DiRenzo et ai, 1982). However, the level of binding of 14C-labeled TCE to hepatic DNA in vivo is so low that binding to protein con taminants in the isolated DNA cannot be ex cluded (Parchman and Magee, 1982; Berg man, 1983). This low level of covalent inter action of TCE with DNA in vivo has lead to the suggestion that TCE is an epigenetic car cinogen (Stott et ai, 1982). The induction of single-strand breakage (SSB) in DNA has been associated with both initiation and promotion events in chemi cally induced carcinogenesis (Walles and Erixson, 1984; Hartley et ai, 1985; Rushmore et ai, 1986). SSBs are known to arise from increases in intracellular calcium (Hartley et ai, 1985), from increased levels of cellular H202 (Cantoni et ai, 1986), through modifi cation ofpoly(ADP)-ribosylation ofDNA-associated proteins (Fujiwara, 1987), topoisomerase-II activity (Markovits et ai, 1987), and through the activity of a variety of endo nucleases (Cleaver and Morgan, 1985). Therefore, measurement of SSB can detect effects upon DNA by mechanisms that do not require direct interaction ofthe compound or its metabolites with DNA. For these reasons, we compared the ability of TCE and its me tabolites to induce SSB in hepatic DNA of mice and rats in vivo. METHODS Chemicals. TCE (epichlorohydrin free) was obtained from Fisher Scientific. Chloral hydrate (CH), dichloroacetate (DCA), trichloroethanol (TCEOH), and trichloroacetate (TCA) were purchased from Sigma Chemical Co. (St. Louis, MO). Purity of administered chemicals was found to be 99+% using gas-liquid chromatography methods described by Prout et al. (1985). Animats and exposures. Male Sprague-Dawley rats weighing 300-400 g (from Washington State University Laboratory Animal Resource Center) and male B6C3F1 mice (from Simonsen Labs, CA) weighing 25-30 g were used. The animals were housed in temperature-con trolled rooms with a 12-hr. light/dark cycle. Food (Pur ina Laboratory Rodent Chow, Ralston-Purina Co., St. Louis, MO.) and water were provided ad libitum. Each experiment involved four groups containing three ani mals each, with one group always as a vehicle control. Animals were fasted overnight before dosing. The occa sional animals that died or suffered respiratory distress due to intubation errors were excluded from the experi ments. In the first set ofexperiments, single oral doses ofTCE or its metabolites were administered in a total volume of 1 ml of 1% aqueous of Tween 80/kg body wt. Control animals received an equivalent volume of vehicle alone. Four hours after treatment, the animals were killed and 10% liversuspensionsprepared, andSSBwasdetermined as described below. The timing ofthe animal deaths was based upon the time course of DCA accumulation in the liver following oral intubation (Evans, 1982). In a second series of experiments, rats were subjected to daily treatments ofethanol (0 or 3.0 g/kg, po), phenobaibital (0 or 50 mg/kg, ip), TCE (0 or 3.8 or 11.4 mmol/ kg, po), or the appropriate vehicle for 4 days prior to the administration of challenge doses of TCE ranging from 3.8 to 30 mmol/kg. Animals were killed 4 hr after admin istration ofthe TCE challenge dose and SSB in DNA was determined. To be certain that alkaline unwinding was responsible for the different fractions of single-stranded DNA seen at a given time, the rate of unwinding was followed at intervals of 30,60, and 120 min of incubation. The ani mals received a dose of 3.9 mmol/kg DCA and controls were given an appropriate volume ofvehicle. At the indi cated time intervals DNA unwinding was stopped and the amount converted to single-stranded DNA deter mined. Alkaline unwinding assay. To measure SSB in DNA, the alkaline unwinding assay of Morris and Shertzer (1985) was used. This assay measures the rate of transi tion of double-stranded DNA to single-stranded DNA during alkaline denaturation. DNA strand breaks serve as points at which unwinding is initiated (Rydberg, 1975). Termination of animals and preparation of liver sam ples were conducted in subdued, indirect incandescent lighting to avoid the introduction ofstrand breaks by uv irradiation. DNA was assayed using Setaro and Morley's (1976) modification of Kissane and Robins' (1958) diaminobenzoic acid flourometric assay. The fraction of DNA unwound was calculated as SL 03A536 jmimstered chemicals was is-liquid chromatography r a/.(l985). tale Sprague-Dawley rats ashington State University Center) and male B6C3F1 'A) weighing 25-30 g were used in temperature-con* dit/dark cycle. Food (Purv, Ralston-Purina Co., St. provided ad libitum. Each >ups containing three aniways as a vehicle control, t before dosing. The occaiffered respiratory distress excluded from the experi- s, single oral doses ofTCE nered in a total volume of i 80/kg body wt. Control t volume ofvehicle alone. ; animals were killed and . and SSB was determined . ofthe animal deaths was yZA accumulation in the ^Evans, IS 82), fcnts, rats were subjected > or 3,0 eJkg, po), phenoE (0 or 3.8 or 11.4 mmol/ cle for 4 days prior to the ses of TCE ranging from re killed 4 hrafter adminose and SSB in DNA was winding was responsible igJe-stranded DNA seen winding was followed at t of incubation. The aniol/kg DCA and controls ie ofvehicle. At the indi ting was stopped and e-stranded DNA deter- measure SSB in DNA, 'f Morris and Shertzer sures the rate of transi> single-stranded DNA ^A strand breaks serve is initiated (Rydberg, eparation ofliversam indirect incandescent of strand breaks by uv ng Setaro and Morlevs md Robins' (1958) di assay. The fraction of DNA STRAND BREAKS BY TRICHLOROETHYLENE 47 Total DNA - PS DNA), (Total DNA - PS DNA)p (Total DNA) (Total DNA) .here the subscript indicates the amount of doubletranded DNA at time "0" and ofincubation in alkaine solution. Serum enzyme determinations. Animals, treated in a arailel fashion to those used in the experiments decribed above, were killed 24 hr after a challenge dose of CE or its metabolites to determine the extent ofhepatic njury, indicated by increases in aspartate aminotransfer.se (AST) and alanine aminotransferase (ALT) enzyme _`vels. The animals were killed at 24 hr, rather than 4 hr, o provide full opportunity for expression of the enymes's increase (Pappas, 1986). Serum was separated by centrifugation from blood Irawn from the inferior vena cava and assayed for serum ALT and AST levels using diagnostic kits purchased from Sigma. ALT and AST serum determinations are ex pressed in international units/liter. One international unit (U) of enzyme activity is defined as the amount of enzyme that produces 1 umol of NAD/min (Sigma pro cedure No. 59-UV, 1987). Statistical analysis. All values are expressed as means SE. To simplify construction ofdose-response curves, data from the multiple control groups employed were combined and expressed as a single mean SE. How ever, when testing the various treatments for significance the statistical analyses were performed using the concur rent control. Log dose-response curves were constructed and regression analysis was used to determine the slopes. Student's t test was used to test whether differences in slopes were statistically significant (Goldstein, 1964). Analysis of variance was used to determine significant differences among treatment groups (a level of p 0.05 being considered significant), with Duncan's multiple range test used for mean separation. RESULTS The induction of strand breaks in rat and mouse hepatic DNA by TCE in vivo. For sim plicity, increases in the rate of DNA unwind ing under alkaline conditions will be referred to as increases in single-strand breaks (SSBs) in DNA, SSBs have been demonstrated to be the major factor involved in increased rates of alkaline unwinding (Rydberg, 1975). TCE induces SSB in hepatic DNA of both rats and mice in vivo (Table 1). However, the characteristics of the dose-response curves vary between the two species. The slopes of the dose-response curves (change in fraction TABLE 1 The Induction of Strand Breaks in Rat and Mouse Hepatic DNA by TCE in Vivtf Dose N Species Fraction DNA unwound 0 3.9 11.4 22.9 30.4 7 SD-rats 5 SD-rats 5 SD-rats 7 SD-rats 7 SD-rats 0.22 0.03* 0.25 0.06 0.22 0.03 0.34 0.05c 0.47 0.05' 0 5 B6C3Fl-mice 0.13 0.04 0.76 4 B6C3Fl-mice 0.20 0.03 5.8 6 B6C3Fl-mice 0.22 0.03 11.4 6 B6C3FI-mice 0.29 0.03' 22.9 6 B6C3Fl-mice 0.32 0.04c " Male Sprague-Dawley rats and male B6C3F1 mice were given single oral doses of TCE suspended in 1% Tween 80 in distilled water. Control animals received an equal volume of vehicle alone. * Values are means SE ofN animals. ` Significantly different from vehicle control, p < 0.05, by ANOVA and Duncan's multiple range test. unwound/log dose) were significantly differ ent (p = 0.05). Slopes of 0.22 0.07 (95% confidence interval) and 0.08 0.03 were found in the rat and mouse, respectively. In rats, single oral doses of 22.9 and 30 mmol/ kg TCE were required to significantly in crease SSB relative to controls in the rats. In mice, TCE produced increased rates of DNA unwinding following the administration of a dose of 11.4 mmol/kg (1.5 g/kg). Therefore, mice are more sensitive to the induction of SSB in hepatic DNA than rats. Doses of TCE above 22.9 mmol/kg (i.e., 30.0 mmol/kg) were lethal to mice, preventing valid experi mentation in this range and accounting for the smaller maximum response observed rel ative to rats. The effects ofphenobarbital, ethanol, and TCE pretreatments on the induction ofsinglestrand breaks in hepatic DNA of the rat by TCE. Phenobarbital pretreatment is known to increase the metabolism of TCE (Leibman and McAllister, 1967; Miller and Guenger- SL 034537 48 NELSON AND BULL Challenge Dose TCE (mmol/Kg) Fig. 1. Modification ofthe induction of SSB in rat he patic DNA by TCE with phenobarbital pretreatment. Phenobarbital was administered ip at a dose of50 mg/kg for 4 consecutive days. On the fifth day animals received the indicated doses ofTCE and were killed 4 hr later. For simplicity, control animals receiving either phenobarbi tal or vehicle pretreatments were combined ( = 20, 17, respectively). Each experimental point represents the mean ofat least five animals SE. `Different from vehicle-pretreated animals administered the same dose of TCE (11.4 mmol/kg) and concurrent controls, p 0.05, by ANOVA and Duncan's multiple range test. ich, 1983). To investigate the role that metab olism of TCE might play in the induction of SSB, rats were pretreated with 50 mg/kg phe nobarbital (ip) for 4 days. On the fifth day, the challenge doses of TCE were adminis tered. Under these conditions, the dose of TCE required to produce significant in creases in SSB was lowered to 11.4 mmol/kg (1-5 g/kg) (Fig. 1). Ethanol pretreatment was used as a second means of modifying the cytochrome P-450 activities of the liver. Ethanol induces P450c, a form much less effective in metabo lizing TCE than /MSO,*, (Koop et al., 1982; Ryan et al., 1984). No significant difference in the mean rate of alkaline unwinding was observed between ethanol-treated and con trol animals (Fig. 2). The biotransformation of TCE to CH by rat liver microsomal preparations has been shown to increase when rats were pretreated with TCE (Leibman and McAllister, 1967). Therefore, we investigated the influence of prior doses of TCE on the induction of SSB. Previous experiments demonstrated that sin gle doses of 3.8 (0.5 g/kg) and 11.4 mmol/kg (1.5 g/kg) TCE failed to produce SSB, thus, these doses were chosen for the pretreatment. In animals pretreated with either 3.8 or 11.4 mmol/kg TCE, a challenge dose of 11.4 mmol/kg of TCE increased the number of SSB in DNA (Fig. 3). If one assumes that the fourth dose of 11.4 mmol/kg TCE was as effective as the fifth, it would appear from the control data that the SSB produced by the fourth pretreatment dose were repaired by the time of measurement (i.e., 28 hr after the last pretreatment dose). This has been con firmed in subsequent experiments (data not shown). The induction of strand breaks in rat he patic DNA by metabolites of TCE. It was found that several of the stable metabolites of TCE were able to cause SSB in rat hepatic DNA at much lower doses than TCE (Fig. 4). DCA was clearly the most potent metabolite, with doses as low as 0.23 mmol/kg producing significant increases in the rate ofalkaline un- ] Challenge Dose TCE (mmol/kg) FlO. 2. The lack of effect of ethanol pretreatment on the induction of SSB in rat hepatic DNA. Ethanol was administered by gavage at a dose of3 g/kg for 4 consecu tive days. On the fifth day animals were given the indi cated doses ofTCE and killed 4 hr later. Each experimen tal point represents the mean of at least five animals SE. No differences were found between ethanol- and vehicle-pretreated animals, p - 0.05, by ANOVA and Duncan's multiple range test. V t M SL 034538 the induction of SSB. demonstrated that sin<g) and 11.4 mmol/kg to produce SSB, thus, t for the pretreatment, vith either 3.8 or 11.4 lllenge dose of 11.4 eased the number of one assumes that the imol/kg TCE was as ould appear from the 5SB produced by the >se were repaired by it (i.e., 28 hr after the . This has been can 'tperiments (data not nd breaks in rat heites of TCE. It was e stable metabolites se SSB in rat hepatic es than TCE (Fig. 4). |t potent metabolite, Tnmol/kg producing e rate ofalkaline un- i t 30 40 :E (mmol/kg) lanol pretreatment on tic DNA. Ethanol was if 3 g/kg for 4 consecuIs were given the mdilater. Each experimen' at least five animals between ethanol- and >,05, by ANOVA and DNA strand breaks by trichloroethylene 49 Pretreatment Dose TCE (mmol/kg) Fig. 3. The induction of SSB in rat hepatic DNA by "E and alteration of this response by TCE pretreatents. Pretreatment with doses of TCE were given by ivage for 4 consecutive days. On the fifth day the aniials were given a dose ofTCE (11.4 mmol/kg) by gavage nd killed 4 hr later. For simplicity, Tween/Tween (n = 18) and TCE/Tween (n - 18) controls have been cornlined. Values for Tween/TCE and TCE/TCE treatments are the means from at least five animals SE. `Different from concurrent controls; Tween/Tween, Tween/TCE (11.4 mmol/kg), and TCE (3.8 mmol/kg)/Tween group, p s 0.05, by ANOVA and Duncan's multiple range test. "Different from concurrent controls; Tween/Tween, Tween/TCE (11.4 mmol/kg) and TCE (11.4 mmol/kg)/ Tween group, p 0.05, by ANOVA and Duncan's multi ple range test. ated with the induction of SSB (Fig. 5). Dichloroacetate treatment accelerated the alka line unwinding ofDNA, a pattern of response similar to that observed by Morris and Shertzer (1985) with /V-nitrosodimethylamine. The induction of strand breaks in mouse liver DNA by TCE metabolites. As with rats, a dose-dependent increase in SSB was observed with DCA, TCA, and CH (Fig, 6). Unlike the rat, the most potent metabolite in inducing SSB in mouse liver DNA was TCA. A dose of only 0.006 mmol/kg was required to produce a significant increase in the rate of alkaline unwinding. A similar dose of DCA also in creased the rate of alkaline unwinding, but the effect was less than that observed with TCA. CH was considerably less potent than the other metabolites, requiring a dose of 0.6 mmol/kg before a significant increase in the rate of alkaline unwinding was observed. The slopes of the log dose-response curves for DCA, TCA, and CH in the mouse were winding. The lowest dose of TCA that pro duced significant SSB in hepatic DNA was 0.6 mmol/kg. The dose-response curve of chloral hydrate closely approximated that of TCA, but the lowest dose to cause significant increases in SSB was 1.8 mmol/kg. Trichloroethanol did not induce significant SSB at any dose tested. The dose-response curves obtained from rats were uniformly steep. As mentioned ear lier, a slope of 0.22 was observed for TCE. The corresponding slopes and 95% confi dence intervals for DCA, TCA, and CH were 0.29 0.02, 0.31 0.06, and 0.37 0.06, respectively. The time course of DNA unwinding under alkaline conditions was consistent with the assumption that the proportion of singlestranded DNA after 2 hr incubation is associ ^ z c ~ 2 FIG. 4. The induction ofSSB in rat hepatic DNA after exposure to DCA, TCA, CH, and TCEOH. The indi cated doses ofthese metabolites were given by gavage and 4 hr later the animals were killed. For simplicity, control data were combined ( = 27). Each experimental point represents the mean from at least five animals SE. `Different from concurrent vehicle control, p 0,05, by ANOVA and Duncan's multiple range test. SL 034539 50 NELSON AND BULL these compounds, did not have increased ALT or AST levels relative to vehicle control treated animals (Table 5). DISCUSSION Fig. 5. The effect of DCA on the rate of alkaline un winding of rat liver DNA. Values given are the means from at least four animals SE. Rates of unwinding be tween DCA and vehicle control arc significantly different (p 0.05) by regression analysis and Student's t test. The present study demonstrates that TCE is capable ofinducing SSB in hepatic DNA of both mice and rats in vivo. In both species, the induction of SSB by TCE required massive doses. The doses required for SSB induction in mice correspond to those used to induce liver tumors in chronic bioassays (NCI, 1976; NTP, 1983). The doses required to induce SSB in the hepatic DNA of rats were higher than the maximum tolerated dose in chronic studies (NCI, 1976). Walles (1986) also dem onstrated that there was a linear increase in the level of SSB in mouse liver DNA follow ing ip administration of TCE, reporting that the strand breaks are repaired within 24 hr. not as steep as they were for these compounds in the rat. In the mouse, the slopes and 95% confidence intervals for DCA, TCA, and CH were 0.11 0.02, 0.13 0.01, and 0.09 0.04, respectively. These slopes are signifi cantly different from the slopes in the rat (p = 0.05). Serum AST and ALT determinations. Se rum ALT and AST levels were monitored at 24 hr to determine whether hepatic injury oc curred with the doses used in the various SSB experiments. In the rat, TCE increased the se rum ALT and AST levels only after phenobarbital pretreatment and at the high dose of 30 mmol/kg at (Table 2). Neither ethanol nor TCE pretreatment resulted in any increases in these serum enzymes following challenge doses of TCE (Table 3). CH, DCA, and TCA also failed to increase serum ALT and AST levels (Table 4). Similar evaluation ofserum ALT and AST levels were conducted in mice using doses of TCE, CH, DCA, and TCA that produced a significantly increased rate of alkaline DNA unwinding. Serum samples, collected from mice 24 hr after the acute administration of Fig. 6. The induction of SSB in mouse hepatic DNA after exposure to DCA, TCA, and CH. The indicated doses ofthese metabolites were given by gavage and 4 hr later the animals were killed. For simplicity, control data were combined {n = 28). Each experimental point repre sents the mean from at least five animals SE. 'Different from concurrent vehicle control, p = 0,05, by ANOVA and Duncan's multiple range test. SL 0-34540 * id not have increased Native to vehicle control -*5). JSSION iemonstrates that TCE SSB in hepatic DNA of ivo. In both species, the TCE required massive ired for SSB induction ) those used to induce : bioassays (NCI, 1976; es required to induce 4A of rats were higher crated dose in chronic alles (1986) also demas a linear increase in use liver DNA follow)f TCE, reporting that epaired within 24 hr. 8 in mouse hepatic DNA . and CH. The indicated given by gavage and 4 hr >r simplicity, control data _'xpenmental point repre animals SE. `Different >1. p * 0.05, by ANOVA st. DNA STRAND BREAKS BY TRICHLOROETHYLENE 51 TABLE 2 Effects of TCE on Aspartate Aminotransfer. \se (AST) and Alanine Aminotransferase (ALT) AcnviTiES in Serum of Rats following Pretreat ment wtth Phenobarbital or TCE Pretreatment/ Challenge Dose AST ALT N (U/liter) (U/liter) Saline/Tween 5 30 6* 15 6* Phenobarbital/Tween 5 33 2 14 1 Tween/Tween 4 22 2 3 1 Saline/TCE 11.4 mmol/kg 5 36 9 12 + 2 30.5 mmol/kg 5 24 3 9 1 Phenobarbital/TCE 11.4 mmol/kg 5 30 4 13 2 30.5 mmol/kg 4 136 3' 47 16' TCE/TCE 11.4 mmol/kg 5 27 2 7 1 " Male Sprague-Dawley rats were given ip injections of 50 mg/kg (in saline) or 11.4 mmol/kg TCE (in 1% aqueous Tween 80)/day for 4 days. Control animals re ceived an equivalent volume of vehicle alone. Challenge doses of TCE were administered 24 hr after the last pre treatment dose. AST and ALT levels were determined 24 hr after the challenge dose of TCE. b Values are expressed as means SE ofN animals. 'Significantly different from Saline/Tween and Phenobarbital/Tween groups, p 0.05, by ANOVA and Duncan's multiple range test. This is consistent with observations made in the rat in the present study. The increased number of SSB produced by TCE following phenobarbital pretreatment suggests that metabolic activation is neces sary for DNA damage to occur. Metabolism of TCE has been shown to be saturated at doses of this magnitude in the rat (Prout et al., 1985). Apparently, phenobarbital pre treatment increased the enzymatic activity of the rat mixed-function oxidase system such that a comparatively larger fraction of the 11.4 mmol/kg dose was metabolized. Metab olism studies from our laboratory show that peak concentrations of TCE in blood only slightly increase with doses of TCE in this range. This limits the concentrations of me tabolites measured in blood (Larson and Bull, 1988), which could account for the fail ure of higher doses of TCE (30 mmol/kg) to further increase the response in animals pre treated with phenobarbital. Unlike pretreatments with phenobarbital or TCE, ethanol did not increase the ability of TCE to cause SSB in DNA. The relative ineffectiveness of ethanol is probably related to the fact that it induces different isozymes of cytochrome P-450 than does phenobarbital. Ethanol administration is known to induce a unique cytochrome P-450 isozyme with spe cific catalytic properties (Ko et al., 1987; Ryan et al., 1984; Koop et al., 1982). Miller and Guengerich (1983) have shown that cyto chrome P-450pb-b is particularly efficient in converting TCE to CH, which in turn is con verted to TCA and TCEOH. Our data suggest that TCE pretreatment also activates this pathway. Several studies have reported that covalent binding of TCE to exogenous DNA takes place in the presence of liver microsomes TABLE 3 Effects of TCE on Aspartate Aminotransfer ase (AST) and Alanine Aminotransferase (ALT) Activities in Serum of Rats following Pretreat ment with Ethanol11 Pretreatment/ challenge Dose AST ALT N (U/liter) (U/liter) Water/Tween 5 30 2*' 8 1*' EtOH/Tween 5 26 6 7+1 EtOH/TCE 11.4 mmol/kg 5 23 + 2 8 1 22.7 mmol/kg 5 22 1 9 1 Water/TCE 11.4 mmol/kg 5 24 2 10 1 22.7 mmol/kg 5 22 1 9+ 1 ` Male Sprague-Dawley rats received oral doses ofeth anol 3.0 g/kg (in wateij/day for 4 days. Challenge doses of TCE were administered in a 1% Tween 80 in distilled water 24 hr after the last pretreatment dose. Control ani mals received an equivalent volume of vehicle alone. AST and ALT levels were determined 24 hr after the challenge dose ofTCE. b Values expressed as means SE ofIV animals. 'No significant differences found, p < 0.05, by ANOVA and Duncan's multiple range test. 034541 52 NELSON AND BULL TABLE 4 Effects of DCA, TCA, TCEOH, and CH on Aspar tate Aminotransferase (AST) and Alanine Ami notransferase (ALT) Activities in the Serum of Rats" Challenge dose AST ALT (mmol/kg) V (U/liter) (U/liter) Tween DCA (3.9) TCA (3.1) TCEOH (3.3) CH (3.4) 5 16 2*,c 5 ltc 4 16 + 1 4+ 1 4 24 7 62 4 18 + 2 3+1 4 17 + 0 2 1 " Male Sprague-Dawley rats were administered agents by gavage in 1% Tween 80 in distilled water solution. Control animals received an equivalent volume of vehi cle alone. AST and ALT levels were determined 24 hr after the agents were administered. b Values expressed as means SE ofA'animals. 'No significant differences found, p 0.05, by ANOVA and Duncan's multiple range test. (Cunningham et al., 1981; DiRenzo et al,, 1982; Bergman, 1983). The low level of DNA binding in vivo suggests that the DNA bind ing observed in the in vitro studies is not di rectly related to the induction of SSB re ported here. It is presumed that such binding would involve the epoxide or acid chloride intermediates of TCE metabolism, whereas the present data indicate that the more stable metabolites can produce the effect at low doses. The mechanism by which SSBs are in duced by these agents requires further study. The most interesting result of this study was the substantial differences in sensitivity of mice and rats to TCE and its metabolites. Significantly different responses were ob served at much lower doses in mice than in rats. This was due to the large differences in the slopes of the dose-response curves for the two species. The differences in slopes of the dose-re sponse curves constructed for the two species also have mechanistic implications. The steep dose-response curves for the rat are consistent with a process requiring more steps than would be involved in producing the shallower slopes observed for mice (Goldstein et al., 1974). We do not believe this difference can be explained on pharma cokinetic grounds, since metabolism studies show blood concentrations of the metabolites are limited at high doses of TCE (Larson and Bull, 1988). One explanation that may be re sponsible for the differing slopes could be in efficient mechanism(s) for recognition and repair of strand breaks. However, there have been no studies that identify such aberrations in the B6C3F1 mouse. While the mecha nisms responsible for the response are not clear at present, the observation is interesting, considering the difference in the sensitivity of these species to the hepatocarcinogenic effects of TCE. While there was a tendency for mice to be more sensitive to the metabolites of TCE tested, the dose of TCA required to increase the rate of alkaline unwinding in mice was 1 / 100 of that required in the rat. These results parallel the sensitivity of rats and mice to the induction of hepatic peroxisomes by TCA (Elcombe, 1985; Goldsworthy and Popp, 1987). TABLE 5 Effects of CH, DCA, TCA, and TCE on Aspar tate Aminotransferase (AST) and Alanine ami notransferase (ALT) Activities in the Serum of Mice" Challenge dose AST ALT (mmol/kg) N (U/liter) (U/liter) Tween CH (3.4) DCA (3.9) TCA (3.1) TCE (11.4) 5 23 6tc 16 + 4*' 5 18 1 16 + 4 4 20 3 15 + 2 5 24 2 11 + I 5 24 3 24 + 6 " Male B6C3F1 mice were administered agents by ga vage in 1% Tween 80 in distilled water. Control animals were given equivalent volumes of vehicle. AST and ALT levels were determined 24 hr after the agents were admin istered. * Values expressed as means SE ofA animals. c No significant differences found, p 0.05, by ANOVA and Duncan's multiple range test. SL 034542 4# observed for mice l). We do not believe explained on pharma:e metabolism studies ons ofthe metabolites s ofTCE (Larson and nation that may be ra ng slopes could be infor recognition and However, there have ttify such aberrations . While the mechahe response are not rvation is interesting, e in the sensitivity of hepatocarcinogenic iency for mice to be netaboliies of TCE required to increase idingin mice was 1/ ne rat. These results "hts and mice to the foxisomes by TCA worthy and Popp, and TCE on aspar. 0 and Alanine Ami ties in THE Serum of ALT (U/liter) nistered agents by ga*Uer- Control animals ehicle. AST and ALT ie agents were admin- - ofNanimals, ind. p ^ 0.05. by nge test. DNA STRAND BREAKS BY TRICHLOROETHYLENE 53 Preliminary data (Nelson and Bull, 1988) dicate that the induction of SSB with these emicals in mice occurs long before there is ;y evidence of peroxisome proliferation, lerefore, the increased d oxidation of lipids peroxisomes could not account for the B observed in the present study. Neverthes, it is interesting to see that these two reonses parallel one another in their responeness to TCA, suggesting a less obvious ikage between the two responses. In summary, we have demonstrated that CE is capable of inducing SSB in hepatic NA of mice and rats in vivo. Pretreatment ith phenobarbital or low doses of TCE, but ot with ethanol, decreases the dose of TCE .quired to induce SSB. This suggests the inolvement of a metabolite(s) of TCE, pro duced by pathways inducible by phenobarbial or TCE. The induction of SSB occurred at ouch lower doses of the stable metabolites of TCE (DCA, TCA, and CH), suggesting that one or more of these compounds are respon sible. Variation in the relative potency of TCA in mice and rats parallels the species specificity of the hepatocarcinogenic effects of TCE. More importantly, the dose-re sponse relationships observed suggest differ ences in the mechanisms by which strand breaks are induced in the hepatic DNA of the two species. The induction of SSB appears to be independent of the hepatotoxicity of these chemicals, since increased damage occurred in the absence of significant elevations in se rum AST and ALT levels. ACKNOWLEDGMENTS We thank Ms. Alexis Lansing, Ms. Lilan Basse, and Ms. Idalia Sanchez for excellent technical assistance; and Mrs. Jessica Nelson for editing the manuscript. 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