Cancer Letters

Cancer Letters

Volume 158, Issue 2, 1 October 2000, Pages 185-193
Cancer Letters

Hypomethylation and overexpression of c-jun and c-myc protooncogenes and increased DNA methyltransferase activity in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors

https://doi.org/10.1016/S0304-3835(00)00518-8Get rights and content

Abstract

Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are mouse liver carcinogens. Methylation of the c-jun and c-myc genes, expression of both genes and DNA methyltransferase (DNA MTase) activity were determined in liver tumors initiated by N-methyl-N-nitrosourea and promoted by DCA and TCA in female B6C3F1 mice. Hypomethylated and over-expression of c-jun and c-myc genes were found in DCA- and TCA-promoted liver tumors. DNA MTase activity was increased in tumors while decreased in non-involved liver. Thus, DCA- and TCA-promoted carcinogenesis appears to include decreased methylation and increased expression of c-jun and c-myc genes in the presence of increased DNA MTase activity.

Introduction

Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are common organic contaminants of drinking water formed as by-products during chlorine disinfection [1], [2]. They are also metabolites of trichloroethylene, a common industrial and commercial solvent and an environmental contaminant found in ground water and at hazardous waste sites [3]. TCA is also a major metabolite of tetrachloroethylene [4]. Hence, there exists human exposure to DCA and TCA either from chlorinated drinking water or from metabolism of trichloroethylene and tetrachloroethylene.

DCA and TCA in B6C3F1 mice have been shown to induce hepatocellular adenomas and carcinomas [3], [5], [6], [9], [10] and to promote N-methyl-N-nitrosourea (MNU)-initiated foci of altered hepatocytes and liver tumors [7], [8]. However, the mechanism for their carcinogenicity in mouse liver is unclear. Mouse liver tumors induced by DCA and TCA do not contain a unique mutation spectrum in the ras oncogene relative to spontaneous tumors [11], [12], [13], [14], [15]. Due to their very weak genotoxicity [16], [17] and their ability to promote liver tumors in mice [7], [8], they likely induce liver cancer by a non-genotoxic mechanism. DCA and TCA have been shown to increase cell proliferation in mouse liver [5], [6] that is a proposed mechanism for non-genotoxic carcinogens [18], [19], [20]. Increased cell proliferation in the liver is associated with increased expression of the immediate-early protooncogenes, c-fos, c-jun and c-myc [21], [22]. It has been reported that c-jun and c-myc proteins are increased in DCA and TCA-induced foci of altered hepatocytes and tumors in female mice [9], [23]. Furthermore, in mouse liver 5 days of exposure to DCA and TCA also increased the level of the mRNA and protein of both protooncogenes [24].

Methylation of DNA as 5-methylcytosine (5-MeC) in the promoter regions of genes including c-jun and c-myc can regulate their expression [25], [26], [27], [28], [29]. DNA is methylated by DNA methyltransferase (DNA MTase) with S-adenosyl methionine (SAM) as the methyl donor [27], [28], [29], [30], [31]. Hypomethylation of genes including c-jun and c-myc is a frequent early event of carcinogenesis in both humans and rodents [25], [26], [27], [28], [29], [31], [32], [33], [34]. Hypomethylation has been reported to decrease further with the progression from benign to metastatic neoplasm [31], [32], [33], [34]. Interestingly, the activity of DNA MTase has been reported to be increase in tumors of humans and laboratory animals even though the DNA is hypomethylated [27], [28], [29], [30], [31].

In mouse liver, subchronic exposure to non-genotoxic carcinogens including phenobarbital, choline–methionine deficient diet, DCA, TCA and trichloroethylene have been reported to decrease the methylation DNA [24], [32], [33], [34], [35], [36], [37], [38], [39]. Liver tumors induced by a choline–methionine deficient diet in both mice and rats exhibited hypomethylation and overexpression of the H-ras, c-myc and c-fos genes [25], [36], [37], [38], [39]. We have previously demonstrated that mouse liver tumors initiated by MNU and promoted by either DCA or TCA contained hypomethylated DNA [34]. The study reported here demonstrates that liver tumors from DCA and TCA-treated mice have decreased methylation in the promoter regions of the c-jun and c-myc protooncogenes and increased expression of their mRNA and proteins. We also reported that DNA MTase activity is increased in the liver tumors of mice initiated with MNU and promoted with DCA and TCA.

Section snippets

Chemicals

DCA and TCA were obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI). Ribonuclease A type III-A and proteinase K were from Sigma Chemical Co., Inc. (St. Louis, MO). TRIzol Reagent was purchased from Gibco BRL/Life Technologies, Inc. (Gaithersburg, MD). Oligonucleotide probes for c-jun and c-myc were obtained from Oncogene Research Products (Cambridge, MA). Restriction endonucleases, HpaII, XbaI and EcoO109I were from New England BioLabs (Beverly, MA). Hybond-N+ nylon membranes [α-32P]dCTP

Methylation status in the promoter region of the c-jun and c-myc protooncogenes

The methylation status in the promoter regions of the c-jun and c-myc genes in liver tumors and non-involved liver from MNU-initiated and DCA- and TCA-promoted mice was evaluated by HpaII restriction enzyme digestion followed by Southern blot analysis (Fig. 1A,B). HpaII does not cut CCGG sites when the internal cytosine is methylated. Therefore, the ability to cut DNA indicates that the internal cytosine is not methylated while the inability of HpaII to cut DNA indicates that the cytosine is

Discussion

DCA and TCA are important environmental contaminants and metabolites of trichloroethylene and tetrachloroethylene [1], [2], [3], [4] that in B6C3F1mice are carcinogenic in the liver [5], [6], [7], [8], [9], [10] and promote MNU-initiated liver tumors [7], [8]. It is likely that their carcinogenic mechanism is non-genotoxic and involves enhancement of cell proliferation and/or prevention of apoptosis [3], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. During cell proliferation

Acknowledgements

This research was supported in part by U.S. Environmental Protection Agency Grant No. R 825384-01-0.

References (47)

  • L. Tao et al.

    Effect of dichloroacetic acid and trichloroacetic acid on DNA methylation in liver and tumors of female B6C3F1 mice

    Toxicol. Sci.

    (1998)
  • R.L.P. Adams et al.

    Microassay for DNA methyltransferase

    J. Biochem. Biophys. Methods

    (1991)
  • P.K. Vogt et al.

    Jun: oncogene and transcription factor

    Adv. Cancer Res.

    (1990)
  • P.A. Jones et al.

    The role of DNA methylation in cancer

    Adv. Cancer Res.

    (1990)
  • W.J. Chen et al.

    Concentration changes of halogenated disinfection by-products in a drinking water distribution system

    J. Am. Water Works Assoc.

    (1998)
  • P.C. Uden et al.

    Chlorinated acids and chloral in drinking water

    J. Am. Water Works Assoc.

    (1983)
  • Trichloroethylene

  • G.W. Ware

    Tetrachloroethylene

    Rev. Environ. Contam. Toxicol.

    (1988)
  • J.R. Latendresse et al.

    Dissimilar characteristics of N-methyl-N-nitrosourea-initiated foci and tumors promoted by dichloroacetic acid or trichloroacetic acid in the liver of female B6C3F1 mice

    Toxicol. Pathol.

    (1997)
  • C.H. Anna et al.

    Ras protooncogene activation in dichloroacetic acid-, trichloroethylene- and tetrachloroethylene-induced liver tumors in B6C3F1 mice

    Carcinogenesis

    (1994)
  • A. Ferreira-Gonzalez et al.

    Ras oncogene activation during hepatocarcinogenesis in B6C3F1 male mice by dichloroacetic and trichloroacetic acids

    Carcinogenesis

    (1995)
  • M. Schroeder et al.

    Dichloroacetic acid reduces Ha-ras codon 61 mutations in liver tumors from female B6C3F1 mice

    Carcinogenesis

    (1997)
  • S.A. Leavitt et al.

    Assessment of the mutagenicity of dichloroacetic acid in lacI transgenic B6C3F1 mouse liver

    Carcinogenesis

    (1997)
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