Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Mar;81(6):1692–1695. doi: 10.1073/pnas.81.6.1692

O4-ethyldeoxythymidine, but not O6-ethyldeoxyguanosine, accumulates in hepatocyte DNA of rats exposed continuously to diethylnitrosamine.

J A Swenberg, M C Dyroff, M A Bedell, J A Popp, N Huh, U Kirstein, M F Rajewsky
PMCID: PMC344984  PMID: 6584902

Abstract

In previous investigations into the mechanisms responsible for cell specificity in hepatocarcinogenesis, we have demonstrated that O6-methylguanine accumulates in the DNA of nonparenchymal cells (NPC) but is efficiently removed from hepatocellular DNA. O6-Alkylguanine may, therefore, be an important promutagenic lesion responsible for the induction of hepatic angiosarcomas after exposure to methylating agents, but other promutagenic DNA alkylation products--i.e., O4-alkylthymine--may be responsible for the initiation of hepatocellular carcinomas. F-344 male rats were provided drinking water containing diethylnitrosamine (DEN) at 40 ppm for 0, 2, 4, 8, 16, 28, 49, or 77 days, a regimen that selectively causes hepatocellular carcinomas. Hepatocytes and NPC were isolated by using low-speed differential centrifugation. DNA was purified by hydroxyapatite chromatography and hydrolyzed enzymatically, and O4-ethyldeoxythymidine (O4-EtdThd) and O6-ethyldeoxyguanosine (O6-EtdGuo) of hepatocyte and NPC DNA were quantitated by competitive radioimmunoassay using high-affinity monoclonal antibodies. O4-EtdThd accumulated in hepatocyte DNA during the first 28 days of DEN exposure, approximating a steady state at an O4-EtdThd-to-deoxythymidine molar ratio of approximately equal to 1 X 10(-5). This O4-EtdThd concentration was maintained from 28 to 77 days of DEN exposure. In contrast, O6-EtdGuo did not accumulate in hepatocyte DNA, its greatest concentration O6-EtdGuo-to-deoxyguanosine ratio (approximately equal to 3.7 X 10(-7) ) being detected after 2 days of exposure to DEN. O6-EtdGuo concentrations in hepatocyte DNA decreased with duration of exposure to DEN to an O6-EtdGuo-to-deoxyguanosine ratio of less than 2 X 10(-7) from 28 to 77 days. The data indicate that O4-EtdThd disappears from the DNA of hepatocytes less than 1/200th as fast as O6-EtdGuo. DNA from NPC contained approximately half as much O4-EtdThd as hepatocytes did, but greater than or equal to 2.5 times more O6-EtdGuo.

Full text

PDF
1692

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abbott P. J., Saffhill R. DNA-synthesis with methylated poly(dA-dT) templates: possible role of O4-methylthymine as a pro-mutagenic base. Nucleic Acids Res. 1977 Mar;4(3):761–769. doi: 10.1093/nar/4.3.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barbason H., Rassenfosse C., Betz E. H. Promotion mechanism of phenobarbital and partial hepatectomy in DENA hepatocarcinogenesis cell kinetics effect. Br J Cancer. 1983 Apr;47(4):517–525. doi: 10.1038/bjc.1983.82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bedell M. A., Lewis J. G., Billings K. C., Swenberg J. A. Cell specificity in hepatocarcinogenesis: preferential accumulation of O6-methylguanine in target cell DNA during continuous exposure to rats to 1,2-dimethylhydrazine. Cancer Res. 1982 Aug;42(8):3079–3083. [PubMed] [Google Scholar]
  4. Beland F. A., Dooley K. L., Casciano D. A. Rapid isolation of carcinogen-bound DNA and RNA by hydroxyapatite chromatography. J Chromatogr. 1979 Jun 1;174(1):177–186. doi: 10.1016/s0021-9673(00)87048-x. [DOI] [PubMed] [Google Scholar]
  5. Columbano A., Rajalakshmi S., Sarma D. S. Requirement of cell proliferation for the initiation of liver carcinogenesis as assayed by three different procedures. Cancer Res. 1981 Jun;41(6):2079–2083. [PubMed] [Google Scholar]
  6. Druckrey H., Preussmann R., Ivankovic S., Schmähl D. Organotrope carcinogene Wirkungen bei 65 verschiedenen N-Nitroso-Verbindungen an BD-Ratten. Z Krebsforsch. 1967;69(2):103–201. [PubMed] [Google Scholar]
  7. Farber E. Chemical carcinogenesis: a biologic perspective. Am J Pathol. 1982 Feb;106(2):271–296. [PMC free article] [PubMed] [Google Scholar]
  8. Goth R., Rajewsky M. F. Molecular and cellular mechanisms associated with pulse-carcinogenesis in the rat nerbous system by ethyinitrosourea: ethylation of nucleic acids and elimination rates of ethylated bases from the DNA of different tissues. Z Krebsforsch Klin Onkol Cancer Res Clin Oncol. 1974;82(1):37–64. doi: 10.1007/BF00304382. [DOI] [PubMed] [Google Scholar]
  9. Goth R., Rajewsky M. F. Persistence of O6-ethylguanine in rat-brain DNA: correlation with nervous system-specific carcinogenesis by ethylnitrosourea. Proc Natl Acad Sci U S A. 1974 Mar;71(3):639–643. doi: 10.1073/pnas.71.3.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kleihues P., Doerjer G., Swenberg J. A., Hauenstein E., Bücheler J., Cooper H. K. DNA repair as regulatory factor in the organotropy of alkylating carcinogens. Arch Toxicol Suppl. 1979;(2):253–261. doi: 10.1007/978-3-642-67265-1_21. [DOI] [PubMed] [Google Scholar]
  11. Kleihues P., Margison G. P. Exhaustion and recovery of repair excision of O6-methylguanine from rat liver DNA. Nature. 1976 Jan 15;259(5539):153–155. doi: 10.1038/259153a0. [DOI] [PubMed] [Google Scholar]
  12. Leonard T. B., Dent J. G., Graichen M. E., Lyght O., Popp J. A. Comparison of hepatic carcinogen initiation-promotion systems. Carcinogenesis. 1982;3(8):851–856. doi: 10.1093/carcin/3.8.851. [DOI] [PubMed] [Google Scholar]
  13. Lewis J. G., Swenberg J. A. Differential repair of O(6)-methylguanine in DNA of rat hepatocytes and nonparenchymal cells. Nature. 1980 Nov 13;288(5787):185–141. doi: 10.1038/288185a0. [DOI] [PubMed] [Google Scholar]
  14. Lewis J. G., Swenberg J. A. Effect of 1,2-dimethylhydrazine and diethylnitrosamine on cell replication and unscheduled DNA synthesis in target and nontarget cell populations in rat liver following chronic administration. Cancer Res. 1982 Jan;42(1):89–92. [PubMed] [Google Scholar]
  15. Lewis J. G., Swenberg J. A. The kinetics of DNA alkylation, repair and replication in hepatocytes, Kupffer cells, and sinusoidal endothelial cells in rat liver during continuous exposure to 1,2-dimethylhydrazine. Carcinogenesis. 1983;4(5):529–536. doi: 10.1093/carcin/4.5.529. [DOI] [PubMed] [Google Scholar]
  16. Lindamood C., 3rd, Bedell M. A., Billings K. C., Swenberg J. A. Alkylation and de novo synthesis of liver cell DNA from C3H mice during continuous dimethylnitrosamine exposure. Cancer Res. 1982 Oct;42(10):4153–4157. [PubMed] [Google Scholar]
  17. Margison G. P., Curtin N. J., Snell K., Craig A. W. Effect of chronic N,N-diethylnitrosamine on the excision of O6-ethylguanine from rat liver DNA. Br J Cancer. 1979 Nov;40(5):809–813. doi: 10.1038/bjc.1979.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Margison G. P., Kleihues P. Chemical carcinogenesis in the nervous system. Preferential accumulation of O6-methylguanine in rat brain deoxyribonucleic acid during repetitive administration of N-methyl-N-nitrosourea. Biochem J. 1975 Jun;148(3):521–525. doi: 10.1042/bj1480521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Margison G. P., Margison J. M., Montesano R. Accumulation of O6-methylguanine in non-target-tissue deoxyribonucleic acid during chronic administration of dimethylnitrosamine. Biochem J. 1977 Sep 1;165(3):463–468. doi: 10.1042/bj1650463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Montesano R. Alkylation of DNA and tissue specificity in nitrosamine carcinogenesis. J Supramol Struct Cell Biochem. 1981;17(3):259–273. doi: 10.1002/jsscb.380170307. [DOI] [PubMed] [Google Scholar]
  21. Montesano R., Brésil H., Planche-Martel G., Margison G. P., Pegg A. E. Effect of chronic treatment of rats with dimethylnitrosamine on the removal of O6-methylguanine from DNA. Cancer Res. 1980 Feb;40(2):452–458. [PubMed] [Google Scholar]
  22. Müller R., Rajewsky M. F. Enzymatic removal of O6-ethylguanine versus stability of O4-ethylthymine in the DNA of rat tissues exposed to the carcinogen ethylnitrosourea: possible interference of guanine-O6 alkylation with 5-cytosine methylation in the DNA of replicating target cells. Z Naturforsch C. 1983 Nov-Dec;38(11-12):1023–1029. doi: 10.1515/znc-1983-11-1224. [DOI] [PubMed] [Google Scholar]
  23. Müller R., Rajewsky M. F. Immunological quantification by high-affinity antibodies of O6-ethyldeoxyguanosine in DNA exposed to N-ethyl-N-nitrosourea. Cancer Res. 1980 Mar;40(3):887–896. [PubMed] [Google Scholar]
  24. Pegg A. E., Balog B. Formation and subsequent excision of O6-ethylguanine from DNA of rat liver following administration of diethylnitrosamine. Cancer Res. 1979 Dec;39(12):5003–5009. [PubMed] [Google Scholar]
  25. Pegg A. E. Formation and metabolism of alkylated nucleosides: possible role in carcinogenesis by nitroso compounds and alkylating agents. Adv Cancer Res. 1977;25:195–269. doi: 10.1016/s0065-230x(08)60635-1. [DOI] [PubMed] [Google Scholar]
  26. Pegg A. E., Perry W., Bennett R. A. Effect of partial hepatectomy on removal of O6-methylguanine from alkylated DNA by rat liver extracts. Biochem J. 1981 Jul 1;197(1):195–201. doi: 10.1042/bj1970195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pegg A. E., Wiest L., Foote R. S., Mitra S., Perry W. Purification and properties of O6-methylguanine-DNA transmethylase from rat liver. J Biol Chem. 1983 Feb 25;258(4):2327–2333. [PubMed] [Google Scholar]
  28. Pitot H. C., Sirica A. E. The stages of initiation and promotion in hepatocarcinogenesis. Biochim Biophys Acta. 1980 May 6;605(2):191–215. doi: 10.1016/0304-419x(80)90004-9. [DOI] [PubMed] [Google Scholar]
  29. Rabes H. M., Kerler R., Wilhelm R., Rode G., Riess H. Alkylation of DNA and RNA by [14C]dimethylnitrosamine in hydroxyurea-synchronized regenerating rat liver. Cancer Res. 1979 Oct;39(10):4228–4236. [PubMed] [Google Scholar]
  30. Rajewsky M. F. Changes in DNA synthesis and cell proliferation during hepatocarcinogenesis by diethylnitrosamine. Eur J Cancer. 1967 Nov;3(4):335–342. doi: 10.1016/0014-2964(67)90016-3. [DOI] [PubMed] [Google Scholar]
  31. Rajewsky M. F., Dauber W., Frankenberg H. Liver carcinogenesis by diethylnitrosamine in the rat. Science. 1966 Apr 1;152(3718):83–85. doi: 10.1126/science.152.3718.83. [DOI] [PubMed] [Google Scholar]
  32. Rajewsky M. F. Possible determinants for the differential susceptibility of mammalian cells and tissues to chemical carcinogens. Arch Toxicol Suppl. 1980;3:229–236. doi: 10.1007/978-3-642-67389-4_17. [DOI] [PubMed] [Google Scholar]
  33. Rajewsky M. F. Proliferative parameters of mammalian cell systms and their rôle in tumor growth and carcinogenesis. Z Krebsforsch Klin Onkol Cancer Res Clin Oncol. 1972;78(1):12–30. doi: 10.1007/BF00284309. [DOI] [PubMed] [Google Scholar]
  34. Scherer E., Timmer A. P., Emmelot P. Formation by diethylnitrosamine and persistence of O4-ethylthymidine in rat liver DNA in vivo. Cancer Lett. 1980 Jul;10(1):1–6. doi: 10.1016/0304-3835(80)90058-0. [DOI] [PubMed] [Google Scholar]
  35. Singer B., Bodell W. J., Cleaver J. E., Thomas G. H., Rajewsky M. F., Thon W. Oxygens in DNA are main targets for ethylnitrosourea in normal and xeroderma pigmentosum fibroblasts and fetal rat brain cells. Nature. 1978 Nov 2;276(5683):85–88. doi: 10.1038/276085a0. [DOI] [PubMed] [Google Scholar]
  36. Singer B., Fraenkel-Conrat H., Kuśmierek J. T. Preparation and template activities of polynucleotides containing O2- and O4-alkyluridine. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1722–1726. doi: 10.1073/pnas.75.4.1722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Singer B., Spengler S., Bodell W. J. Tissue-dependent enzyme-mediated repair or removal of O-ethyl pyrimidines and ethyl purines in carcinogen-treated rats. Carcinogenesis. 1981;2(10):1069–1073. doi: 10.1093/carcin/2.10.1069. [DOI] [PubMed] [Google Scholar]
  38. Singer B., Sági J., Kuśmierek J. T. Escherichia coli polymerase I can use O2-methyldeoxythymidine or O4-methyldeoxythymidine in place of deoxythymidine in primed poly(dA-dT).poly(dA-dT) synthesis. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4884–4888. doi: 10.1073/pnas.80.16.4884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Swann P. F., Mace R. Changes in O6-methylguanine disappearance from rat liver DNA during chronic dimethylnitrosamine administration. A possible similarity between the system removing O6-methylguanine from DNA in rat liver and in Escherichia coli adapted to N-methyl-N'-nitro-N-nitrosoguanidine. Chem Biol Interact. 1980 Aug;31(2):239–245. doi: 10.1016/0009-2797(80)90012-5. [DOI] [PubMed] [Google Scholar]
  40. Swenberg J. A., Bedell M. A., Billings K. C., Umbenhauer D. R., Pegg A. E. Cell-specific differences in O6-alkylguanine DNA repair activity during continuous exposure to carcinogen. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5499–5502. doi: 10.1073/pnas.79.18.5499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tomatis L., Cefis F. The effects of multiple and single administration of dimethylnitrosamine to hamsters. Tumori. 1967 Sep-Oct;53(5):447–451. doi: 10.1177/030089166705300502. [DOI] [PubMed] [Google Scholar]
  42. Weisburger J. H., Madison R. M., Ward J. M., Viguera C., Weisburger E. K. Modification of diethylnitrosamine liver carcinogenesis with phenobarbital but not with immunosuppression. J Natl Cancer Inst. 1975 May;54(5):1185–1188. doi: 10.1093/jnci/54.5.1185. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES