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Loss of heterozygosity on chromosome 6 in dichloroacetic acid and trichloroacetic acid-induced liver tumors in female B6C3F1 mice
ELSEVIER
CANCER LETTERS
Cancer Letters 108 (1996) 257-261
Loss of heterozygosity on chromosome 6 in dichloroacetic acid and trichloroacetic acid-induced liver tumors in female B6C3Fl mice Lianhui Tao*, Kewa Li, Paula M. Kramer, Michael A. Pereira Medical
College
of Ohio, Center for Environmental
Medicine,
Department
of Pathology,
3000 Arlington
Ave., Toledo,
OH 43614,
USA
Received 31 July 1996; revision received 31 July 1996; accepted 16 August 1996
Abstract Dichloroacetic acid (DCA) and trichloroacetic oethylene (TCE) and are found in chlorinated
acid (TCA) are major metabolites of tetrachloroethylene (PCE) and trichlordrinking water. All four chlorinated compounds are liver carcinogens in
B6C3Fl mice. It haspreviouslybeenreportedthat approximately20% of hepatictumorsinducedby PCE exhibitedloss of heterozygosity(LOH) on chromosome 6, suggesting the presenceof a tumor suppressor gene.In the current investigation, we determinedwhetherTCA or DCA alsoinducedLOH on chromosome 6. Liver tumorswereinitiatedin 15dayold female B6C3Fl mice with N-methyl-N-nitrosourea (MNU) and promoted with 20 mmol/l DCA or TCA in their drinking water. Twenty-four and thirty-seven liver tumors promoted by DCA and TCA, respectively, were examined for LOH using 4 polymorphic loci on chromosome 6. Ten of 37 (27%) tumors (7 of 27 carcinomas and 3 of 10 adenomas) promoted by
TCA exhibitedLOH at leastfor two loci on chromosome 6. All 10tumorsthat exhibitedLOH, lost the C57BL/6Jalleleat both the D6mit9 andD6mit323loci, while two alsolostat leastoneof the C3H/HeJalleles.No LOH on chromosome 6 was observed in the 24 liver tumorspromotedby DCA. The LOH on chromosome 6 in TCA but not in DCA-promotedtumors supports it as an active metabolite of PCE and demonstrates different pathogenesis at least for some of the DCA and TCA-
promotedliver cancer. Keywords:
Lossof heterozygosity;Alleles; Trichloroaceticacid; Dichloroaceticacid; Mouseliver tumors
1. Introduction Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are major metabolites of the important industrial and commercial solvents, trichloroethylene (TCE) and tetrachloroethylene (PCE) [1,2]. The two chloroacetic acids are also two of the most common organic contaminants found in chlorinated drinking *Corresponding author. Tel.: +I 419 381 3877; fax: +l 419 3813089. 0304-3835/96/$12.00 PZZ SO304-3835(96)04451-5
water [3,4]. They are formed as reaction byproducts during chlorine disinfection of water containing humic acid and other natural organic compounds [4-71. All four chlorinated compounds(DCA, TCA, PCE, and TCE) have been shown to be peroxisome proliferators [ 1,8,9] and to induce and/or promote hepatocellular adenomasand carcinomas in B6C3Fl mice [lo-141. The mechanismand pathogenesisof the carcinogenic activity of DCA and TCA in mouseliver appearsto be different [ 13,141.For example, in female B6C3Fl mice, liver tumors induced by
0 1996 Elsevier Science Ireland Ltd. All rights reserved
DCA were predominantly eosinophilic and contained glutathione S-transferase-(~GST-a), while those induced by TCA were basophilic and lacked GST-n. Oncogene activation and loss of tumor suppressor genesor inactivation are consideredto represent key events in the pathogenesis of liver tumorx. Livet tumors induced by DCA, TCA, PCE, and TCE have beenshownnot to contain a unique mutation spectrum in the H-r-asand K-r-usoncogenes,but rather mutation in the 61st codon of the H-l-as oncogenecommon to spontaneousliver tumors in B6C?FI mice [ 15,161. It has previously been reported that 20% of hepatic tumors (4 of 20) induced by PCE exhibited loss of heterozygosity (LOH) on chromosome6, suggesting the presenceof a tumor suppressorgene [ 171.In the present study, we examined chromosome6 for lossof heterozygosity in liver tumors from 36C3Fl mice that were initiated with N-methyl-iV-nitrosourea (MNU). and promoted by DCA or TCA. Our goal was to determine whether liver tumors promoted by DCA or TCA had a similar LOH on chromosome6 asthose induced by PCE and to further determine molecular differences in the pathogenesisof hepatocarcinogenesis by the two chloroacetic acids.
2. Materials
and methods
The sourceof the tumors usedin this study hasbeen published [14]. Briefly, female B6C3F1 mice at 15 days of age were administered by intraperitoneal injection either 25 mgikg N-methyl-N-nitrosourea (MNU; Sigma, St. Louis, MO). The pups started to receive in their drinking water 20.0 mmol/l of either DCA (Eastman Kodak, Rochester. NY) or TC’A (Fisher Scientific, Pittsburgh. PA) neutralized with sodium hydroxide to pH 6.5-7.5. The chloroacetic acids were administered to the mice continuously until sacrificed by carbon dioxide asphyxiation after 52 weeks of exposure. At necropsy. the liver was excised. weighed and examined for tumors larger than IO mm in diameter. A portion of the tumors were fixed in 10% phosphate-buffered formalin for histopathological analysis and the rest rapidly frozen in liquid nitrogen and stored at -7WC for LOH am lysis. The formalin fixed tumor specimenswere pro. cessed,embeddedin paraffin, sectionedat 5 pm and stained with hematoxylin and eosin. The histopathological diagnosisof hepatocellular adenomaand car-
cinema was determined using previously published criteria [ lS.lS]. High-molecular-weight DNA was extracted from frozen tumors and normal liver using TRIAZOL Reagent’& Method developed by Chormczyrnskc [ 191. according to the manufacturer’s specitication (GIBCO BRL/Life Technologies. Gaithersburg. MD). Buffer preparation and DNA extraction were performed in a pre-PCR area of the laboratory, ph) xically separatedfrom the post-PCR area. Pipette tip5 usedfor the preparation of buffer. solutionsand DNA extraction all contained fitters to prevent cross-cot:tamination between samples.Four polymorphic locii on chromosome6 (D6mit1, D6mit9. D6mit204 and D6mit.W) were selected basedon previously published information [ 17.201 and obtained fron\ Research Genetics, Inc. (Huntsville. Al..) ‘Ttio (ii the loci were from the proximal end. D&lit{ and D6mitL04, and Iwo t’rom the middle. D6mit9 and D6mit32.3. of chromosome6 (Fig. 1 I. The polymorphic loci were amplified by PCR in 511 ~1 volumes of a mixture containing 1 x PCK buffer ( 10 mM Tris-HCI, pH X.3, 50 mM KCl, I.5 mM MgCl1.O. 1 mg/ml gelatin). 200 r/lM of each deoxynu-
) D6mit 1, D6mit 204
D6mit 9, D6mit 323
Fig. I. Diagam ot chromosome 6. The chromosome number I> shown in the circle representing the centromere. The name and position of the polymorphic loci are shown to the right of the chromosome.
L. Tuo et al. I Cancer
cleotide triphosphate, 0.2 FM primer, 0.5 pg of genomic DNA as the template and 1.25 unit AmpliTaq DNA polymerase. After denaturation at 94°C for 120 s, the reactions were cycled 45 times through 60 s of 94”C, 60 s of 60°C and 120 s of 72°C. Following the final cycle, additional 10 min extension was performed to complete polymerization of all strands (GeneAmp PCR System 9600, Perkin Elmer Corporation, Norwalk, CT). The PCR products were then stored at 4°C until analyzed. PCR products were analyzed on 2.5% agarose gel (ultra pure agarose, GIBCO BRLfLife Technologies, Inc., Gaithersburg, MD) containing ethidium bromide in 0.5 :K TBE and visualized with UV light. These conditions were employed as the alleles differed by 10 base pairs or more. The DNA ladder was 4x 174 RF DNA/HaeIII fragments ranging from 72 to 1353 bp (GIBCO BRL/Life Technologies, Gaithersburg, MD, USA). To evaluate the allelic dosage, the signal intensities of amplified polymorphic alleles were quantified by Foto/Eclipse Image System with AppCollege (version 4.0) Analytic Software (Fotodyne Inc., New Berlin, WI). The percent reduction of an allele was calculated as 100 minus the percentage of the ratio of intensity of the abnormal allele to that of the normal allele in tumor DNA divided by the ratio of the two alleles in normal liver DNA. LOH was demonstrated by a greater than 50% reduction in the intensity of one of the parental bands or of both bands. 3. Results Ten hepatocellular adenomas and 27 hepatocellular carcinomas promoted by TCA, and 23 hepatocellular adenomas and 1 hepatocellular carcinoma promoted by DCA, were examined for LOH using the 4 polymorphic loci on chromosome 6. Typical results from 2.5% agarose gel electrophoresis that depict LOH, are displayed in Fig. 2. Lane 2 contains the PCR product for the D6mit9 locus of DNA from normal liver. Lanes 3.. 5,6,7,9, 10 and 11 contain the PCR product for this locus from tumors promoted by TCA and demonstrate the loss of the C57BL/6J allele. Lanes 4 and 8 are from TCA-promoted tumors that do not demonstrate loss at this locus. Table 1 summarizes the allele loss on chromosome 6. Ten of 37 (27%) liver tumors (3 of 10 hepatocellular adenomas and 7 of 27 hepatocellular carcino-
Letters
108 (1996)
257-261
259
mas) promoted by TCA exhibited LOH for at least the two loci in the middle of chromosome 6 (D6mit9 and D6mit323). All 10 of these tumors lost the C57BL/6J allele at both the D6mit9 and the D6mit323 loci, while two tumors also lost at least one of the C3H/HeJ alleles at these loci. Seven of Table
1
Summary tumors Tumor
of allele loss on chromosome
no.
Proximal D6mit
Adenomas 196 Tl” 196 T2 218 Tl 263 Tl 263 T2 264 T2 264 T3 264 T4 270 Tl 371 Tl Carcinomas 13 T1 13 T2 47 Tl 47 T2 48 Tl 18 Tl 109 Tl I10 Tl 141 TI 142 Tl 142 T2 170 T2 198 Tl 241 Tl 241 T2 243 Tl 264 Tl 268 Tl 268 T3 269 Tl 294 Tl 312 TI 328 Tl 371 Tl 372 Tl 382 Tl 394 TI
H
H+B
sites 1
D6mit
H
Middle
sites
D6mit
9
D6mit
H+B
B
B
B
B
H+B
B
B
B
B
B
B
B
B
B
B
B
B H+B B
B H+B B
H
H+B
204
6 in TCA-promoted
B
liver
323
“Tl, T2, T3 and T4 are different tumors from the same mouse. B indicates loss of C57BL/6J allele; H, loss of C3H/HeJ.
Fig. 7. .4nalyus of TCA-promoted liver tumors Ior lob> of heteroLygosity of the D6mit9 locus on chromosome 6. The PCK products were electrophoresed on 2.5% agarose gel. The tirht lane from the left is the DNA ladder #IX 173 RF DNA/HaeIIi fragment< 172-1353 bp), and lane 2 is DNA from controt liver. The upper ( 141 bp) and lower (119 bpl bands arc C57BLi6J and C3H/HeJ allele\. rc\peclively. Lanes 3. 5. h. 7, 9. 10 and 1 I arc example\ of tumors that exhibited LOH of the C57BL/6J allele and lanes 4 and X are exanlpies of tumor\ that did not exhibit LOH ai either all&
these IO tumors that lost the two loci in the middle of chromosome 6. also lost at least one allele it one of the two loci at the proximal end of thih chromosome (D6mit I and D6mit 204). No LOH on chromosome 4 was observed in the 24 tumors (23 hepatocellular adenomas and 1 hepatocellular carcinoma) promoted by DCA. The incidence of LOH in the tumors promoted by TCA (10 of 37) was significantly greater (Pvalue ‘I 0.01) than in those promoted by DCA (0 of 24). In adenomas, LOH was observed only in those promoted by TCA (3 of 10) and not by DCA (0 of 23 ). 4. Discussion
Dichloroacetic acid and trichloroacetic acid are common organic contaminants found in chlorinated drinking water in concentration ranging from 34 to 160 pg/l and are metabolites of the important industrial and commercial solvents, TCE and PCE [i-3]. Although the two chloroacetic acidshave beenshown to be carcinogenic in the liver of B6C3Fl mice 1IO--14], differences in the hematoxylin and eosin tincture and GST-(content of altered hepatocyte foci and tumors induced by them, have suggesteddifferent mechanismsand pathogenesisfor their hepatticarci-
nogenic activity [ 13,141. Our observation that only liver tumors promoted by TCA and not by DCA. h;id a lossof heterozygosity of a portion of chromosome6 further supportsdifferent pathogenesisfor the hepatocarcinogenicactivity of thesetwo very similar chloroacetic acids The fact that only TCA and not DCA-promoted tumors demonstratedLOH, alsoindicatesthat the loss&dependent upon the TCA and not the result of MNU-initiation. The LOH in 3 of IO hepatoceilular adenomaspromoted by TCA. would indicate that the loss of this portion of chromosome 6 occurs early in the hepatocarcinogenesisby TCA. Loss of heterozygosity in chromoscime6 suggests the presenceof an as yet, unidentified tumor suppressor gene.Human chromosome3~25-26 hasanalogy to mousechromosome6 { 171.A possiblecandidate for this tumor suppressorgene is the von Hippel-Lindau !VHL) diseasetumor suppressorgene found in this portion of the humanchromosome. It haspreviously been reported that 20% of hepatic tumors (4 of 20) induced by PCE exhibited LOH on chromosome6 [ 171.The similar percentage of LOH on chromosome 6 in TCA-promoted tumors (27%). supportsTCA as anactive metabolite of PCE. Since TCA is also a metabolites of TCE, it would be of interest to determine whether tumors induced by TCE also possessthis LOH on chromosome6. supporting TCA as an active metabolite. However, the majority of tumors in DCA. TCA and PCE treated mice do not exhibit LOH on chromosome6. so that other molecular activity is involved in their hepato<.arcinogenesis. References j I 1 Bruckner. J.V.. Udvis. B.D. and Blancato. J.N. (19X9) Metabolism. toxicity, and carcinogefiicity of trichloroethylenc CRC Crit. Rev. Toxicol.,. 20. 31-50. 171 Ware, G.W.( 1988) Tetrachloroethylene. Kev. Environ. Con tam. Toxicot. 106. 175-188. i.;J Elcombe, C.R.. Rose, M.S. and PraLt. 1.S. 11985) Biochemr..cai. histological, and nltrastructtiral changqs in rat and mouse liver following the administration of trichloroethylene: possible relevance to species differences in hepatocarcinogenicity. Toxicol. Appl. Pharmacol.. 79. 365.376. 141 Uden. P.C. an& Miller. J.W. 11983) Chlorinated acids and chloral in drinking water. J. Am. Water Works Assoc.. 75. 524 -527. 15 / Krasner. S.W.. McGuire. M.J.. Jacangelo. J.G.. Patania. N.L.. Reagen, K:M. and Ajeta. E.M. (1989) The occurrence ot’
L. Tao et al. i Cancer
[6]
[7]
[8]
[9]
[lo]
[I 11
[12]
[ 131
disinfection by-products in U.S. drinking water, J. Am. Water Works Assoc., 81, 41-53. Miller, J.W. and Uden, P.C. (1983) Characterization of nonvolatile aqueous chlorination products of humic substances, Environ. Sci. Technol., 17, 150-157. Coleman, E.W., Munch, J.W., Kaylor, W.H., Streicher, R.P., Ringhand, H.P. and Meier, J.R. (1984) Gas chromatography/ mass spectroscopy analysis of mutagenic extracts of aqueous chlorinated humic acid. A comparison of the byproducts to drinking water contaminants, Environ. Sci. Technol., 18, 674-678. Goldsworthy, T.L. and Popp, J.A. (1987) Chlorinated hydrocarbon-induced peroxisomal enzyme activity in relation to species and organ carcinogenicity, Toxicol. Appl. Pharmacol., 88, 225-233. Odum, J., Green, T., Foster, J.R. and Hext, P.M. (1988) The role of trichloroacetic acid and peroxisome proliferation in the differences in carcinogenicity of perchloroethylene in the mouse and rat, Toxicol. Appl. Pharmacol., 92, 103-l 12. Herren-Freund, S.L., Pereira, M.A., Khoury, M.D. and Olson, G. (1987) The carcinogenicity of trichloroethylene and its metabolites, tricbloroacetic acid and dichloroacetic acid, in mouse liver, Toxicol. Appl. Pharmacol., 90, 183-189. Bull, R.J., Sanchez, I.M., Nelson, M.A., Larson, J.L. and Lansing, A.J. (1990) Liver tumor induction in B6C3Fl mace by dichloroacetate and trichloroacetate, Toxicology. 63.341-359. DcAngelo, A.B., Daniel, F.B., Stober, J.A. and Olson, G.R. (1991) The carcinogenicity of dichloroacetic acid in the male BhC3Fl mouse, Fundam. Appl. Toxicol., 16, 337-347. Pereira, M.A. (1996) Carcinogenic activity of dichloroacetic acsd and trichloroacetic acid in the liver of female B6C3Fl mice, Fundam. Appl. Toxicol., 31, 192-199.
Letters
108 (1996)
257-261
261
[14] Pereira, M.A. and Phelps, J.B. (1996) Promotion by dichloroacetic acid and trichloroacetic acid of N-methyl-iV-nitrosourea-initiated cancer in the liver of female B6C3Fl mice, Cancer Lett., 102, 133-141. [I51 Anna, C.H., Maronpot, R.R., Pereira, M.A., Foley, J.F.. Malarkey, D.E. and Anderson, M.W. (1994) Ras proto-oncogene activation in dichloroacetic acid-, trichloroethyleneand tetrachloroethylene-induced liver tumors in B6C3Fl mice, Carcinogenesis, 1.5, 2255-2261. [16] Ferreira-Gonzalez, A., DeAngelo, A.B., Nasim, S. and Garrett, CT. (1995) Ras oncogene activation during hepatocarcinogenesis in B6C3Fl male mice by dichloroacetic acid and trichloroacetic acid, Carcinogenesis, 16, 495-500. [17] Davis, L.M., Caspary, W.J., Shakallah, S.A., Maronpot, R., Wiseman, R., Barrett, J.C., Elliott, R. and Hozier, J.C. (1994) Loss of heterozygosity in spontaneous and chemically induced tumors of B6C3Fl mouse, Carcinogenesis, 1.5, 1637-1645. [18] Ward, J.M., Rice, J.M., Creasia, D., Lynch, P. and Riggs, C. (1983) Dissimilar patterns of promotion by diQ-ethylhexyl)phthalate and phenobarbital of hepatocellular neoplasia initiated by diethylnitrosamine in B6C3Fl mice, Carcinogenesis, 4, 1021-1029. [ 191 Chomczynski, P. (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples, Biotechniques, 15, 532-535. [20] Copeland, N.G., Jenkins, N.A. and Gilberg, D.J. (1993) A genetic linkage map of the mouse: current applications and future prospects, Science, 262, 57-66. [21] Marsman, D.S. and Popp, J.A. (1994) Biological potential of basophilic hepatocellular foci and hepatic adenoma induced by the peroxisome proliferator, Wy-14,643, Carcinogenesis, 15,111-117.
CANCER LETTERS
Cancer Letters 108 (1996) 257-261
Loss of heterozygosity on chromosome 6 in dichloroacetic acid and trichloroacetic acid-induced liver tumors in female B6C3Fl mice Lianhui Tao*, Kewa Li, Paula M. Kramer, Michael A. Pereira Medical
College
of Ohio, Center for Environmental
Medicine,
Department
of Pathology,
3000 Arlington
Ave., Toledo,
OH 43614,
USA
Received 31 July 1996; revision received 31 July 1996; accepted 16 August 1996
Abstract Dichloroacetic acid (DCA) and trichloroacetic oethylene (TCE) and are found in chlorinated
acid (TCA) are major metabolites of tetrachloroethylene (PCE) and trichlordrinking water. All four chlorinated compounds are liver carcinogens in
B6C3Fl mice. It haspreviouslybeenreportedthat approximately20% of hepatictumorsinducedby PCE exhibitedloss of heterozygosity(LOH) on chromosome 6, suggesting the presenceof a tumor suppressor gene.In the current investigation, we determinedwhetherTCA or DCA alsoinducedLOH on chromosome 6. Liver tumorswereinitiatedin 15dayold female B6C3Fl mice with N-methyl-N-nitrosourea (MNU) and promoted with 20 mmol/l DCA or TCA in their drinking water. Twenty-four and thirty-seven liver tumors promoted by DCA and TCA, respectively, were examined for LOH using 4 polymorphic loci on chromosome 6. Ten of 37 (27%) tumors (7 of 27 carcinomas and 3 of 10 adenomas) promoted by
TCA exhibitedLOH at leastfor two loci on chromosome 6. All 10tumorsthat exhibitedLOH, lost the C57BL/6Jalleleat both the D6mit9 andD6mit323loci, while two alsolostat leastoneof the C3H/HeJalleles.No LOH on chromosome 6 was observed in the 24 liver tumorspromotedby DCA. The LOH on chromosome 6 in TCA but not in DCA-promotedtumors supports it as an active metabolite of PCE and demonstrates different pathogenesis at least for some of the DCA and TCA-
promotedliver cancer. Keywords:
Lossof heterozygosity;Alleles; Trichloroaceticacid; Dichloroaceticacid; Mouseliver tumors
1. Introduction Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are major metabolites of the important industrial and commercial solvents, trichloroethylene (TCE) and tetrachloroethylene (PCE) [1,2]. The two chloroacetic acids are also two of the most common organic contaminants found in chlorinated drinking *Corresponding author. Tel.: +I 419 381 3877; fax: +l 419 3813089. 0304-3835/96/$12.00 PZZ SO304-3835(96)04451-5
water [3,4]. They are formed as reaction byproducts during chlorine disinfection of water containing humic acid and other natural organic compounds [4-71. All four chlorinated compounds(DCA, TCA, PCE, and TCE) have been shown to be peroxisome proliferators [ 1,8,9] and to induce and/or promote hepatocellular adenomasand carcinomas in B6C3Fl mice [lo-141. The mechanismand pathogenesisof the carcinogenic activity of DCA and TCA in mouseliver appearsto be different [ 13,141.For example, in female B6C3Fl mice, liver tumors induced by
0 1996 Elsevier Science Ireland Ltd. All rights reserved
DCA were predominantly eosinophilic and contained glutathione S-transferase-(~GST-a), while those induced by TCA were basophilic and lacked GST-n. Oncogene activation and loss of tumor suppressor genesor inactivation are consideredto represent key events in the pathogenesis of liver tumorx. Livet tumors induced by DCA, TCA, PCE, and TCE have beenshownnot to contain a unique mutation spectrum in the H-r-asand K-r-usoncogenes,but rather mutation in the 61st codon of the H-l-as oncogenecommon to spontaneousliver tumors in B6C?FI mice [ 15,161. It has previously been reported that 20% of hepatic tumors (4 of 20) induced by PCE exhibited loss of heterozygosity (LOH) on chromosome6, suggesting the presenceof a tumor suppressorgene [ 171.In the present study, we examined chromosome6 for lossof heterozygosity in liver tumors from 36C3Fl mice that were initiated with N-methyl-iV-nitrosourea (MNU). and promoted by DCA or TCA. Our goal was to determine whether liver tumors promoted by DCA or TCA had a similar LOH on chromosome6 asthose induced by PCE and to further determine molecular differences in the pathogenesisof hepatocarcinogenesis by the two chloroacetic acids.
2. Materials
and methods
The sourceof the tumors usedin this study hasbeen published [14]. Briefly, female B6C3F1 mice at 15 days of age were administered by intraperitoneal injection either 25 mgikg N-methyl-N-nitrosourea (MNU; Sigma, St. Louis, MO). The pups started to receive in their drinking water 20.0 mmol/l of either DCA (Eastman Kodak, Rochester. NY) or TC’A (Fisher Scientific, Pittsburgh. PA) neutralized with sodium hydroxide to pH 6.5-7.5. The chloroacetic acids were administered to the mice continuously until sacrificed by carbon dioxide asphyxiation after 52 weeks of exposure. At necropsy. the liver was excised. weighed and examined for tumors larger than IO mm in diameter. A portion of the tumors were fixed in 10% phosphate-buffered formalin for histopathological analysis and the rest rapidly frozen in liquid nitrogen and stored at -7WC for LOH am lysis. The formalin fixed tumor specimenswere pro. cessed,embeddedin paraffin, sectionedat 5 pm and stained with hematoxylin and eosin. The histopathological diagnosisof hepatocellular adenomaand car-
cinema was determined using previously published criteria [ lS.lS]. High-molecular-weight DNA was extracted from frozen tumors and normal liver using TRIAZOL Reagent’& Method developed by Chormczyrnskc [ 191. according to the manufacturer’s specitication (GIBCO BRL/Life Technologies. Gaithersburg. MD). Buffer preparation and DNA extraction were performed in a pre-PCR area of the laboratory, ph) xically separatedfrom the post-PCR area. Pipette tip5 usedfor the preparation of buffer. solutionsand DNA extraction all contained fitters to prevent cross-cot:tamination between samples.Four polymorphic locii on chromosome6 (D6mit1, D6mit9. D6mit204 and D6mit.W) were selected basedon previously published information [ 17.201 and obtained fron\ Research Genetics, Inc. (Huntsville. Al..) ‘Ttio (ii the loci were from the proximal end. D&lit{ and D6mitL04, and Iwo t’rom the middle. D6mit9 and D6mit32.3. of chromosome6 (Fig. 1 I. The polymorphic loci were amplified by PCR in 511 ~1 volumes of a mixture containing 1 x PCK buffer ( 10 mM Tris-HCI, pH X.3, 50 mM KCl, I.5 mM MgCl1.O. 1 mg/ml gelatin). 200 r/lM of each deoxynu-
) D6mit 1, D6mit 204
D6mit 9, D6mit 323
Fig. I. Diagam ot chromosome 6. The chromosome number I> shown in the circle representing the centromere. The name and position of the polymorphic loci are shown to the right of the chromosome.
L. Tuo et al. I Cancer
cleotide triphosphate, 0.2 FM primer, 0.5 pg of genomic DNA as the template and 1.25 unit AmpliTaq DNA polymerase. After denaturation at 94°C for 120 s, the reactions were cycled 45 times through 60 s of 94”C, 60 s of 60°C and 120 s of 72°C. Following the final cycle, additional 10 min extension was performed to complete polymerization of all strands (GeneAmp PCR System 9600, Perkin Elmer Corporation, Norwalk, CT). The PCR products were then stored at 4°C until analyzed. PCR products were analyzed on 2.5% agarose gel (ultra pure agarose, GIBCO BRLfLife Technologies, Inc., Gaithersburg, MD) containing ethidium bromide in 0.5 :K TBE and visualized with UV light. These conditions were employed as the alleles differed by 10 base pairs or more. The DNA ladder was 4x 174 RF DNA/HaeIII fragments ranging from 72 to 1353 bp (GIBCO BRL/Life Technologies, Gaithersburg, MD, USA). To evaluate the allelic dosage, the signal intensities of amplified polymorphic alleles were quantified by Foto/Eclipse Image System with AppCollege (version 4.0) Analytic Software (Fotodyne Inc., New Berlin, WI). The percent reduction of an allele was calculated as 100 minus the percentage of the ratio of intensity of the abnormal allele to that of the normal allele in tumor DNA divided by the ratio of the two alleles in normal liver DNA. LOH was demonstrated by a greater than 50% reduction in the intensity of one of the parental bands or of both bands. 3. Results Ten hepatocellular adenomas and 27 hepatocellular carcinomas promoted by TCA, and 23 hepatocellular adenomas and 1 hepatocellular carcinoma promoted by DCA, were examined for LOH using the 4 polymorphic loci on chromosome 6. Typical results from 2.5% agarose gel electrophoresis that depict LOH, are displayed in Fig. 2. Lane 2 contains the PCR product for the D6mit9 locus of DNA from normal liver. Lanes 3.. 5,6,7,9, 10 and 11 contain the PCR product for this locus from tumors promoted by TCA and demonstrate the loss of the C57BL/6J allele. Lanes 4 and 8 are from TCA-promoted tumors that do not demonstrate loss at this locus. Table 1 summarizes the allele loss on chromosome 6. Ten of 37 (27%) liver tumors (3 of 10 hepatocellular adenomas and 7 of 27 hepatocellular carcino-
Letters
108 (1996)
257-261
259
mas) promoted by TCA exhibited LOH for at least the two loci in the middle of chromosome 6 (D6mit9 and D6mit323). All 10 of these tumors lost the C57BL/6J allele at both the D6mit9 and the D6mit323 loci, while two tumors also lost at least one of the C3H/HeJ alleles at these loci. Seven of Table
1
Summary tumors Tumor
of allele loss on chromosome
no.
Proximal D6mit
Adenomas 196 Tl” 196 T2 218 Tl 263 Tl 263 T2 264 T2 264 T3 264 T4 270 Tl 371 Tl Carcinomas 13 T1 13 T2 47 Tl 47 T2 48 Tl 18 Tl 109 Tl I10 Tl 141 TI 142 Tl 142 T2 170 T2 198 Tl 241 Tl 241 T2 243 Tl 264 Tl 268 Tl 268 T3 269 Tl 294 Tl 312 TI 328 Tl 371 Tl 372 Tl 382 Tl 394 TI
H
H+B
sites 1
D6mit
H
Middle
sites
D6mit
9
D6mit
H+B
B
B
B
B
H+B
B
B
B
B
B
B
B
B
B
B
B
B H+B B
B H+B B
H
H+B
204
6 in TCA-promoted
B
liver
323
“Tl, T2, T3 and T4 are different tumors from the same mouse. B indicates loss of C57BL/6J allele; H, loss of C3H/HeJ.
Fig. 7. .4nalyus of TCA-promoted liver tumors Ior lob> of heteroLygosity of the D6mit9 locus on chromosome 6. The PCK products were electrophoresed on 2.5% agarose gel. The tirht lane from the left is the DNA ladder #IX 173 RF DNA/HaeIIi fragment< 172-1353 bp), and lane 2 is DNA from controt liver. The upper ( 141 bp) and lower (119 bpl bands arc C57BLi6J and C3H/HeJ allele\. rc\peclively. Lanes 3. 5. h. 7, 9. 10 and 1 I arc example\ of tumors that exhibited LOH of the C57BL/6J allele and lanes 4 and X are exanlpies of tumor\ that did not exhibit LOH ai either all&
these IO tumors that lost the two loci in the middle of chromosome 6. also lost at least one allele it one of the two loci at the proximal end of thih chromosome (D6mit I and D6mit 204). No LOH on chromosome 4 was observed in the 24 tumors (23 hepatocellular adenomas and 1 hepatocellular carcinoma) promoted by DCA. The incidence of LOH in the tumors promoted by TCA (10 of 37) was significantly greater (Pvalue ‘I 0.01) than in those promoted by DCA (0 of 24). In adenomas, LOH was observed only in those promoted by TCA (3 of 10) and not by DCA (0 of 23 ). 4. Discussion
Dichloroacetic acid and trichloroacetic acid are common organic contaminants found in chlorinated drinking water in concentration ranging from 34 to 160 pg/l and are metabolites of the important industrial and commercial solvents, TCE and PCE [i-3]. Although the two chloroacetic acidshave beenshown to be carcinogenic in the liver of B6C3Fl mice 1IO--14], differences in the hematoxylin and eosin tincture and GST-(content of altered hepatocyte foci and tumors induced by them, have suggesteddifferent mechanismsand pathogenesisfor their hepatticarci-
nogenic activity [ 13,141. Our observation that only liver tumors promoted by TCA and not by DCA. h;id a lossof heterozygosity of a portion of chromosome6 further supportsdifferent pathogenesisfor the hepatocarcinogenicactivity of thesetwo very similar chloroacetic acids The fact that only TCA and not DCA-promoted tumors demonstratedLOH, alsoindicatesthat the loss&dependent upon the TCA and not the result of MNU-initiation. The LOH in 3 of IO hepatoceilular adenomaspromoted by TCA. would indicate that the loss of this portion of chromosome 6 occurs early in the hepatocarcinogenesisby TCA. Loss of heterozygosity in chromoscime6 suggests the presenceof an as yet, unidentified tumor suppressor gene.Human chromosome3~25-26 hasanalogy to mousechromosome6 { 171.A possiblecandidate for this tumor suppressorgene is the von Hippel-Lindau !VHL) diseasetumor suppressorgene found in this portion of the humanchromosome. It haspreviously been reported that 20% of hepatic tumors (4 of 20) induced by PCE exhibited LOH on chromosome6 [ 171.The similar percentage of LOH on chromosome 6 in TCA-promoted tumors (27%). supportsTCA as anactive metabolite of PCE. Since TCA is also a metabolites of TCE, it would be of interest to determine whether tumors induced by TCE also possessthis LOH on chromosome6. supporting TCA as an active metabolite. However, the majority of tumors in DCA. TCA and PCE treated mice do not exhibit LOH on chromosome6. so that other molecular activity is involved in their hepato<.arcinogenesis. References j I 1 Bruckner. J.V.. Udvis. B.D. and Blancato. J.N. (19X9) Metabolism. toxicity, and carcinogefiicity of trichloroethylenc CRC Crit. Rev. Toxicol.,. 20. 31-50. 171 Ware, G.W.( 1988) Tetrachloroethylene. Kev. Environ. Con tam. Toxicot. 106. 175-188. i.;J Elcombe, C.R.. Rose, M.S. and PraLt. 1.S. 11985) Biochemr..cai. histological, and nltrastructtiral changqs in rat and mouse liver following the administration of trichloroethylene: possible relevance to species differences in hepatocarcinogenicity. Toxicol. Appl. Pharmacol.. 79. 365.376. 141 Uden. P.C. an& Miller. J.W. 11983) Chlorinated acids and chloral in drinking water. J. Am. Water Works Assoc.. 75. 524 -527. 15 / Krasner. S.W.. McGuire. M.J.. Jacangelo. J.G.. Patania. N.L.. Reagen, K:M. and Ajeta. E.M. (1989) The occurrence ot’
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