effect of dichloroacetic acid and trichloroacetic acid on dna

TOXJCOLOGICAL SCIENCES 43, 139-144 (1998)

ARTICLE NO. TX982449

Effect of Dichloroacetic Acid and Trichloroacetic Acid on DNAMethylation in Liver and Tumors of Female B6C3F1 Mice

Lianhui Tao, Paula M. Kramer, Rongrong Ge, and Michael A. Pereira

Department of Pathology, Center for Environmental Medicine, Medical College of Ohio, 3055 Arlington Avenue, Toledo, Ohio 43614-5806

Received October 27, 1997; accepted February 23, 1998

Effect of Dichloroacetic Acid and Trichloroacetic Acid on DNAMethylation in Liver and Tumors of Female B6C3F1 Mice. Tao,L., Kramer, P. M., Ge, R., and Pereira, M. A. (1998). Toxicol. Sci.43, 139-144.

Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) arefound in drinking water and are metabolites of trichloroethylene.They are carcinogenic and promote liver tumors in B6C3F1 mice.Hypomethylation of DNA is a proposed nongenotoxic mechanisminvolved in carcinogenesis and tumor promotion. We determined theeffect of DCA and TCA on the level of DNA methylation in mouseliver and tumors. Female B6C3F1 mice IS days of age were admin-istered 25 mg/kg N-methyl-N-nitrosourea and at 6 weeks started toreceive 25 mmol/liter of either DCA or TCA in their drinking wateruntil euthanized 44 weeks later. Other animals not administeredMNU were euthanized after 11 days of exposure to either DCA orTCA. DNA was isolated from liver and tumors, and after hydrolysis5-methylcytosine (SMeC) and the four bases were separated andquantitated by HPLC. In animals exposed to either DCA or TCA for11 days but not 44 weeks, the level of 5MeC in DNA was decreasedin the liver. SMeC was also decreased in liver tumors from animalsexposed to either chloroacetic acid. The level of SMeC in TCA-promoted carcinomas appeared to be less than in adenomas. Termi-nation of exposure to DCA, but not to TCA, resulted in an increasein the level of 5MeC in adenomas to the level found in noninvolvedliver. Thus, hypomethylated DNA was found in DCA and TCApromoted liver tumors and the difference in the response of DNAmethylation to termination of exposure appeared to support thehypothesis of different mechanisms for their carcinogenic activity.C 1998 Society of Toxicology.

The use of chlorine for the disinfection of drinking waterwas an important breakthrough of this century in avoidingwaterborne infections in the general population. The discoveryof chlorinated organic by-products of disinfection in the 1970sand 1980s has raised questions about the health effects ofchronic exposure to chlorine and its by-products in drinkingwater. Dichloroacetic acid (DCA)1 and trichloroacetic acid(TCA) are two of the more common by-products of chlorina-

1 Abbreviations used: DCA, dichloroacetic acid; GST-ir, glutathione S-transferase-Tr; HPLC, high-performance liquid chromatography; LOH, loss ofheterozygosity; 5MeC, 5-methylcytosine; MNU, yV-methyl-/V-nitrosourea;TCA, trichloroacetic acid.

tion of drinking water (Uden and Miller, 1983; Krasner et al,1989). They are formed by the reaction of chlorine with humicacid, fulvic acid, and other natural organic compounds in thesource water (Miller and Uden, 1983; Coleman et al, 1984).The two chloroacetic acids are also major metabolites of tri-chloroethylene, an important industrial and commercial solventand multimedia contaminant (Bruckner et al, 1989; Dekant etal, 1984; IARC, 1979). Thus, human exposure to DCA andTCA results from chlorinated drinking water and possibly frommetabolism of trichloroethylene found in the environment.

Administering DCA and TCA in drinking water has beenshown to induce hepatocellular adenomas and carcinomas inB6C3F1 mice (Herren-Freund et al, 1987; Bull et al., 1990;Pereira, 19%). DCA and TCA in drinking water have also beenshown to promote N-methyl-/v'-nitrosourea (MNU)-initiated fociof altered hepatocytes and tumors (Pereira and Phelps, 1996;Pereira et al., 1997). However, the mechanism by which theyinduce and promote liver tumors is unclear. DCA and TCA havebeen proposed to be nongenotoxic hepatocarcinogens since thereis little evidence from in vivo and in vitro studies to indicate eithergenotoxic or mutagenic activity (Rapson et al., 1980; Meier andBlazak, 1990; Chang et al, 1992; Fox et al., 19%). Mouse livertumors induced by DCA and TCA have been demonstrated not tocontain a unique mutation in either the H- or K-ras oncogenerelative to spontaneous tumors (Anna et al, 1994; Ferreira-Gonzalez et al, 1995). The absence of a unique mutation furthersupports an epigenetic, nonmutagenic mechanism for their carci-nogenic activity. DCA and TCA have been shown to induceproliferation of peroxisomes in the liver of mice, an activityshared with some other hepatocarcinogens and tumor promoters(Elcombe, 1985; Elcombe et al., 1985; Goldsworthy and Popp,1987; Odum et al, 1988).

Although DCA and TCA have many chemical and biologi-cal properties in common, the mechanism and pathogenesis oftheir carcinogenic and tumor promoting activity in mouse liverappear to be different (Herren-Freund et al, 1987; Bull et al,1990; Pereira, 1996; Pereira and Phelps, 1996; Tao et al, 1996;Latendresse and Pereira, 1997; Pereira et al, 1997). In the liverof female B6C3F1 mice, DCA induced and promoted alteredhepatocyte foci and tumors that were predominantly eosino-philic, contained glutathione S-transferase-ir (GST-TT), and didnot demonstrate loss of heterozygosity (LOH) on chromosome

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140 TAO ET AL.

6. In contrast, TCA induced and promoted very few alteredhepatocyte foci. The tumors in animals exposed to TCA werebasophilic, lacked GST-TT, and demonstrated LOH of a portionof chromosome 6. When exposure to DCA or TCA was ter-minated, altered hepatocyte foci and adenomas induced byDCA regressed, while those induced by TCA did not regressbut rather progressed to carcinomas (Bull et al, 1990; Pereiraand Phelps, 1996). Hence, the pathogenesis of the carcinogenicactivity of DCA and TCA is apparently different.

Mammalian DNA naturally contains the methylated base,5-methylcytosine (5MeC), which plays a role in regulation ofgene expression and DNA imprinting (Bird, 1986; Razin andKafri, 1994; Stoger et al, 1993). An overall decrease in thecontent of 5MeC in DNA is a frequent finding in tumors and isconsidered to represent a key factor in expanding clones ofprecancerous cells during neoplastic progression (Gama-Sosaet al, 1983; Jones and Buckley, 1990; Herman et al., 1994;Counts and Goodman, 1994, 1995). The level of 5MeC inDNA is lower in chemical-induced liver tumors than in normalliver tissue (Lapeyre and Becker, 1979; Lapeyre et al, 1981).Hypomethylation is also an early event in carcinogenesis inhumans (Vogelstein et al, 1988). The level of genomic 5MeCdecreased with the progression from normal to benign neo-plasm to metastatic neoplasm in humans (Gama-Sosa et al,1983). Additional support for a role for hypomethylation incarcinogenesis comes from studies involving 5-azacytosine(Razin and Cedar, 1991). The deoxynucleotide of 5-azacy-tosine can be incorporated into DNA and noncompetitivelyinhibits DNA methyltransferase, leading to hypomethylation.5-Azacytosine can act as a tumor promoter in rat liver and canlead to malignant transformation in vitro (Carr et al, 1984;Huggett et al, 1991; Rimoldi et al, 1991).

Hypomethylation of DNA has been hypothesized to be anepigenetic, nongenotoxic mechanism involved in tumor pro-motion by facilitating aberrant gene expression (Counts andGoodman, 1995). Since DCA and TCA are nongenotoxic tu-mor promoters, they should induce DNA hypomethylation intumors. Furthermore, the pathogenesis of their carcinogenicactivity appears to be different including the regression ofadenomas on termination of exposure to DCA but not TCA(Bull et al, 1990; Pereira and Phelps, 1996). Similarly, termi-nation of exposure to the chloroacetic acids might affect DNAmethylation differently. The studies reported here determinedwhether DCA and TCA decreased the level of 5MeC in theDNA of liver and tumors from B6C3F1 mice and whethertermination of exposure resulted in an increase in 5MeC intumors from DCA- but not TCA-treated animals.

MATERIALS AND METHODS

Animal experiments and liver tumors. The source of the liver tumors usedin this study has been published (Pereira el at, 1997). Briefly, VAF (viralantibody free) female B6C3F1 mice were purchased from Charles RiverBreeding Laboratories (Portage, MI). At 15 days of age, the mice wereadministered 25 mg/kg MNU (Sigma Chemical Co., St. Louis, MO) byintraperitoneal injection. At 6 weeks of age, the mice started to receive 25

mmol/liter of either DCA or TCA (neutralized with sodium hydroxide to pH6.5-7.5) in their drinking water until being euthanized after 44 weeks ofexposure. There were also two recovery groups in which exposure wasterminated 1 week prior to euthanization at Week 44 in order to determinewhether DNA methylation would increase prior to regression of the tumors.The effect of short-term exposure to either DCA or TCA on DNA methylationwas determined in 7- to 8-week-old female B6C3F1 mice that were notadministered MNU. The mice were divided into the following three treatmentgroups of seven animals each: (Group 1) 25 mmol/liter DCA, (Group 2) 25mmol/liter TCA, and (Group 3) control drinking water. The DCA and TCAsolutions were neutralized and administered in the drinking water until theanimals were euthanized after 11 days of exposure.

The mice were terminated by cervical dislocation and the liver was excised,weighed, and examined for tumors. A portion of the tumors was fixed in 10%phosphate-buffered formalin for histopathological analysis and the remainderrapidly frozen in liquid nitrogen and stored at -70°C. Pieces of liver after 11 daysand of noninvolved liver after 44 weeks of exposure to the chloroacetic acids werealso harvested and rapidly frozen in liquid nitrogen and stored at -70°C. Withrespect to tumors, we found at necropsy and harvested hepatocellular adenomasand carcinomas in TCA-treated mice, only hepatocellular adenomas in DCA-treated mice, and no tumors in the MNU-treated mice that did not receive eitherchloroacetic acid. Hepatocellular adenoma and carcinoma were distinguished inhematoxylin and eosin stained sections by previously published criteria (Ward etal, 1983; Anna et al. 1994; Pereira et al, 1997).

Isolation, purification and characterization of DNA. DNA was extractedfrom liver, noninvolved liver, and liver tumors using the TRIZOL Reagent'sMethod developed by Chomczynski (Chomczynski, 1993) and according to themanufacturer's specification (GIBCO BRL/Life Technologies, Inc., Gaithers-burg, MD). Approximately 100 mg of the tissue was homogenized in 1 ml ofTRIZOL Reagent. After incubation for 10 min at room temperature, 200 (xl ofchloroform was added to the homogenate. The mixture was then incubated atroom temperature for 5 min and centrifuged at 12,000g for 15 min at 4°C. Theupper aqueous phase was removed and 300 ml of 100% ethanol added to theinterphase and organic phase. The DNA was allowed to precipitate at 4°C for10 min and then pelleted by centrifugation at 2000# for 5 min at 4"C. Thepellet containing the genomic DNA was washed twice with 0.1 M sodiumcitrate in 10% ethanol for 30 min each and twice with 75% ethanol for 20 mineach, and then dissolved in 8 mM NaOH. The insoluble material was removedby centrifugation at 12,000g for 15 min at 4°C. The supernatant containing theDNA was transferred to a new tube and the pH adjusted to 7.4 with 0.1 MHepes. To remove residual RNA and proteins, the solubilized DNA wastreated with 100 /ig/ml RNase A (Sigma Chemical Co., St, Louis, MO) and200 jig/ml proteinase K (Sigma Chemical Co.) for 2 h at 37°C. Then 1 MNaOH was added and the DNA incubated at 50°C for 1 h. DNA purity wasassessed by using the ratio of A^A-^ (acceptable values higher than 1.9) andthe amount was determined at 260 nm.

Hydrolysis of DNA. The DNA was hydrolyzed according to the method ofWilson and Jones (1983). To the purified genomic DNA (20 to 50 jig), 100 fiJof 12 M (70%) perchloric acid (Aldrich Co., Milwaukee, WI) was addedfollowed by incubation al 100°C for 1 h. The time and temperature ofincubation were carefully controlled in order to avoid the possible deaminarionof 5MeC into thymine (Wilson and Jones, 1983; Valencia et al., 1985). Afterneutralization with 6 M KOH (230 fii to each 100 fji\ of perchloric acid), theprecipitate was removed by centrifugation (5 min at 400g). The supernatantwas filtered through a 0.2-/im pore size polypropylene syringe filter (4 mmdiameter, Whatman Inc., Clifton, NJ). The pH was adjusted to 4.5 by addinga few drops of 2 M HC1 and the sample stored at 4°C until analysis by HPLC.

Separation and quantitation of SNfeC and the four bases in DNA byHPLC. The hydrolysates of the DNA were analyzed by HPLC using aWaters HPLC system (Milford, MA) consisting of a Model 510 pump, a ModelU6K universal injector, a Model 481 Lambda Max UV/visible LC spectro-photometer, and a Model 730 Data Module. Separation was carried out with aWhatman PartiSphere C,8 column (250 mm X 4.6 mm i.d., 5 /xm particle)(Clifton, NJ). An isocratic mobile phase consisting of 100 mM ammoniumacetate (pH 4.25) containing 0.5% (v/v) acetonitrile was used at room tem-

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DNA HYPOMETHYLATION IN DCA/TCA TUMORS

DNA Methylation in Mouse Uver

141

DNA Methylation in Non-Involved Mouse Liver

A. 11 Days

FIG. 1. 5-Methylcytosine levels in DNA from liver of female B6C3F1 mice administered either DCA or TCA for 11 days (A) or 44 weeks (B). Results aremean ± SE of 7-10 animals/group. Statistical analysis was performed by one-way ANOVA followed by the Tukey test, with astensks indicating significantdifference from control liver (p value <0.05).

perature with a flow rate of 2 ml/rnin. The detection wavelength was 280 nmwith a sensitivity of 0.02 AUFS. The duration of the analysis was 15 min withthe column reequilibrated for at least 10 min between each run.

The five bases, cytosine, 5MeC, guanine, thymine, and adenine, in thehydrolyzed DNA were well separated by this HPLC procedure. The retentiontimes of cytosine, 5MeC, and guanine were 5.03, 6.49, and 7.40 min, respec-tively, with thymine and adenine eluted later. Peak identification was based onthe retention times as compared with standard solutions analyzed every 10runs. Complete baseline separation was obtained for all four bases and 5MeC.The absence of RNA in the purified DNA was confirmed by the absence ofuracil in the chromatograms. The percentage of 5MeC was calculated from thepeak areas according to the formula: % 5MeC = (5MeC/5MeC + cytosine) X100 (Loennechen et al, 1989).

Statistical evaluation. SigmaStat software, version 2.0 (Jandel Corp., SanRafael, CA), was used to perform the statistical analysis. The results wereanalyzed for statistical significance by one-way analysis of variance (ANOVA)followed by the Tukey test. A p value <0.05 was used to signify statisticalsignificance.

RESULTS

The percentage of cytosine presented as 5MeC in DNA wasdetermined in liver after 11 days or 44 weeks of exposure(Figs. 1A and IB). After 11 days of exposure to either DCA orTCA the level of 5MeC in DNA was decreased relative tountreated controls (Fig. 1A). However, after 44 weeks ofexposure to either chloroacetic acid, the level of 5MeC was notsignificantly different from control mice that received MNUbut did not receive either of the chloroacetic acids (Fig. IB).The level of 5MeC in the liver of control animals at the twotime points did not differ significantly, i.e., 3.70 ± 0.40 and3.28 ± 0.24 at 11 days and 44 weeks, respectively.

The level of 5MeC in DNA of hepatocellular adenomas fromanimals promoted by DCA was 2.36 ± 0.15, representing a36% decrease compared to noninvolved liver tissue from thesame animal (p value <0.05, Fig. 2). The level of 5MeC inadenomas from DCA-treated animals was similarly lower thanthe level in liver tissue from control animals administered only

MNU. We have previously demonstrated that termination ofexposure to DCA but not TCA resulted in a reduction in theyield of adenomas (Pereira and Phelps, 1996). When exposureto DCA was terminated 1 week prior to sacrifice, the level of5MeC in the adenomas returned to the more methylated state(3.56 ± 0.42) and was no longer different from noninvolvedliver from the same animal and liver from control animalsadministered only MNU (p value > 0.05, Fig. 2).

DNA Mettiylabon in DCA-Treated Mice

FIG. 2. 5-Methylcytosine levels in DNA from liveT of control mice andfrom noninvolved liver and adenomas of mice promoted with DCA for 44weeks. Control mice were administered MNU but not the chloroacetic acidsand were of the same age as the DCA-treated mice when euthanized. In theAdenoma-Recovery Group the exposure to DCA was terminated 1 week priorto euthanization. Results are mean ± SE for groups of 9-16 samples each. Theasterisk indicates significant difference from both the Non-Involved Liver andthe Adenoma-Recovery Groups (p values <0.05) by one-way ANOVA fol-lowed by die Tukey test. The Control, Noninvolved Liver, and Adenoma-Recovery Groups were not statistically different (p value >0.05).

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142 TAO ET AL.

DNA Methylatton in TCA-Treated Mice

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FIG. 3. 5-Methylcytosine levels in DNA from liver of control mice and fromnoninvolved liver, adenomas, and carcinomas of mice promoted with TCA for 44weeks. Control mice were administered MNU but not the chloroacetic acids andwere of the same age as the TCA-treated mice when euthanized. The Adenoma-Recovery and Carcinoma-Recovery Groups had the exposure to TCA terminated1 week prior to euthanization. Results are mean ± SE for groups of 8-16 sampleseach. The asterisk indicates significant difference from noninvolved liver (p value<0.05) by one-way ANOVA followed by the Tukey test The difference betweenthe TCA-Promoted Adenomas and die Adenoma-Recovery Groups and betweenthe TCA-Promoted Carcinomas and the Carcinoma-Recovery Groups were notstatistically significant (/> values >0.05).

The level of 5MeC in DNA from hepatocellular adenomasand hepatocellular carcinomas promoted by TCA were 2.19 ±0.20 and 1.77 ± 0.25, representing 40 and 51% decrease,respectively, relative to noninvolved tissue from the sameanimal (Fig. 3). These levels of 5MeC in adenomas and car-cinomas were also less than the level of 5MeC in liver fromcontrol animals administered only MNU. Termination of ex-posure to TCA 1 week prior to sacrifice did not change thelevel of 5MeC in either adenomas or carcinomas, remainingsignificantly lower than in noninvolved liver tissue. Althoughthe level of 5MeC in carcinomas was less than in adenomas, itwas not statistically significant. However, when the adenomasand carcinomas from animals that continued to receive TCAuntil euthanization were pooled with the corresponding tumorsfrom animals that ceased to receive TCA 1 week prior toeuthanization, the level of 5MeC in DNA from carcinomasbecame statistically less than from adenomas, i.e., 2.24 ± 0.15and 1.61 ± 0.18, respectively.

DISCUSSION

DCA and TCA are found in finished drinking water asby-products of chlorination (Uden and Miller, 1983; Krasner etal, 1989) and are also metabolites of the important industrialand commercial solvent trichloroethylene (Bruckner et al,1989; Dekant et al, 1984; IARC, 1979). The two chloroaceticacids have been shown to be carcinogenic in the liver of

B6C3F1 mice (Herren-Freund et al, 1987; Bull et al, 1990;Pereira, 1996) and to promote MNU-initiated liver tumors inthis strain of mice (Pereira and Phelps, 1996; Pereira et al,1997). Phenobarbital and choline-devoid, methionine-deficientdiet are tumor promoters in mouse liver that have been shownto decrease the level of 5MeC in hepatic DNA (Christman etal, 1993; Counts et al, 1996). We observed that both DCAand TCA decreased the level of 5MeC in hepatic DNA. How-ever, the level of 5MeC was significantly decreased after 11days but not after 44 weeks of exposure to either chloroaceticacid. Five days of exposure to either DCA or TCA has beenshown to enhance the level of cell proliferation in the liver offemale B6C3F1 mice, while longer exposures of 12 or moredays had no effect (Pereira, 1996). Therefore, DCA and TCAwould appear to induce DNA hypomethylation and to enhancecell proliferation only after exposures of short duration. En-hanced cell proliferation could result in a decrease in the levelof DNA methylation should it limit the time for the methyl-ation of daughter DNA between cycles of replication. Expo-sures between 11 days and 44 weeks need to be evaluated inorder to determine when the level of 5MeC in DNA returned tocontrol level and to evaluate the suggested association with cellproliferation. It is also possible that the reduced level of DNAmethylation after 11 days of exposure to the chloroacetic acidsresulted from hypomethylation of specific genes that are dif-ferent from those methylated at 44 weeks when DNA methyl-ation was no longer different from control liver.

Exposure to either DCA or TCA reduced the level of 5MeC inDNA of liver tumors relative to noninvolved and control liver. Wefurther observed for TCA-treated animals that the level of 5MeCappeared to decrease with the progression of adenomas to carci-nomas. In humans, the level of 5MeC in DNA also decreased withthe progression from benign to malignant neoplasm (Gama-Sosaet al, 1983). Hence, the carcinogenic and tumor promoting ac-tivity of DCA and TCA and the neoplastic progression of theresulting lesions would appear to be associated with a decrease inthe level of 5MeC in DNA.

While DCA and TCA have been demonstrated in B6C3F1mice to be carcinogenic and to promote the occurrence ofMNU-initiated liver tumors, the stability of the tumors uponcessation of exposure to them was different (Bull et al, 1990;Pereira and Phelps, 1996). Upon cessation of exposure, ade-nomas promoted by DCA regressed, while those promoted byTCA did not regress, but rather progressed to carcinomas(Pereira and Phelps, 1996). After cessation of exposure to DCAfor 1 week, the level of 5MeC in adenomas increased, beingsimilar to the level in noninvolved liver tissue. In contrast, aftercessation of exposure to TCA the level of 5MeC in adenomasand carcinomas did not change. It is possible that a longerrecovery period would result in an increased level of 5MeC intumors from TCA-treated animals. However, this is unlikelysince a much longer period of recovery of 21 weeks did notresult in the regression of tumors from TCA-treated animals(Pereira and Phelps, 1996). DNA hypomethylation in B6C3F1mouse liver induced by 4 weeks of exposure to phenobarbital

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DNA HYPOMETHYLATION IN DCA/TCA TUMORS 143

was reversed after 2 weeks of removal of phenobarbital, i.e.,the level of 5MeC was restored to that in control liver (Countset ai, 1996). Hence, DNA hypomethylation induced in mouseliver and tumors by some but not all tumor promoters wouldappear to be reversible. Furthermore, the restoration of DNAmethylation in adenomas upon removal of DCA but not TCAcorresponded to the regression of adenomas upon removal ofDCA, but not TCA (Bull et al, 1990; Pereira and Phelps,1996). This further supports the hypothesis that the pathogen-esis of the carcinogenic activity of DCA and TCA is different.

ACKNOWLEDGMENT

This work was supported in part by U.S. Environmental Protection AgencyGrant R 825384-01-0.

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