Skip to main content
SpringerLink
Log in

Use of scintillometric quantitation of unscheduled DNA synthesis in isolated rat hepatocytes for the screening of genotoxic agents

  • Published:
Cell Biology and Toxicology Aims and scope Submit manuscript

The induction of unscheduled DNA synthesis has been considered as a suitable endpoint for the screening of genotoxic agents. Experimentally, unscheduled DNA synthesis is most frequently measured by autoradiography. The purpose of this report was to examine the usefulness of the liquid scintillation counting technique in measuring unscheduled DNA synthesis response in isolated rat hepatocytes. The various liquid scintillation counting-based unscheduled DNA synthesis assay procedures were examined according to the following groupings: (1) procedures based on the acid precipitation of cellular macromolecules, (2) procedures based on isopycnic gradient centrifugation of solubilized cells, (3) procedures based on nuclei isolation in conjunction with other DNA purification methods, and (4) procedures based on the selective retention of hepatocellular DNA. Limited cases in which test chemicals gave positive unscheduled DNA synthesis response in liquid scintillation counting-based assays and negative unscheduled DNA synthesis response in autoradiography-based assays are presented. It is concluded that liquid scintillation counting-based unscheduled DNA synthesis assays represent an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

2-AAF:

2-acetylaminofluorene

2-AF:

2-aminofluorene

AFB1 :

aflatoxin B1

ARG:

autoradiography

DMN:

dimethylnitrosamine

LSC:

liquid scintillation counting

MMS:

methyl methanesulfonate

MNNG:

N-methyl-N′-nitro-N-nitrosoguanidine

4-NQO:

4-nitroquinoline-1-oxide

PCA:

perchloric acid

TCA:

trichloroacetic acid

UDS:

unscheduled DNA synthesis

References

  • AGRELO, C.E. and SEVEREN, B.J. (1981). A simplified method for measuring scheduled and unscheduled DNA synthesis in human fibroblasts. Toxicology 21:151–158.

    Google Scholar 

  • ALTHAUS, F. R., LAWRENCE, S. D., SATTLER, G. L. and PITOT, H. C. (1980). The effect of nicotinamide on unscheduled DNA synthesis in cultured hepatocytes. Biochem. Biophys. Res. Commun. 95:1063–1070.

    Google Scholar 

  • ALTHAUS, F.R., LAWRENCE, S.D., SATTLER, G.L., LONGFELLOW, D.G. and PITOT, H.C. (1982a). Chemical quantitation of unscheduled DNA synthesis in cultured hepatocytes as an assay for the rapid screening of potential chemical carcinogens. Cancer Res. 42:3010–3015.

    Google Scholar 

  • ALTHAUS, F.R., LAWRENCE, S.D., SATTLER, G.L. and PITOT, H.C. (1982b). DNA damage induced by the antihistaminic drug methapyrilene hydrochloride. Mutat. Res. 103:213–218.

    Google Scholar 

  • BIANCHI, V., ZANTEDESCHI, A., RONCHESE, F. and LEVIS, A.G. (1983). Induction of DNA repair synthesis by ultraviolet radiation and methylmethanesulfonate in cultured mouse lymphocytes. Cancer Lett. 18:21–27.

    Google Scholar 

  • BLOBEL, G. and POTTER, V.R. (1966). Nuclei from rat liver: Isolation method that combines purity with high yield. Science 154:1662–1665.

    Google Scholar 

  • BROUNS, R.E., POOT, M., De VRIND, R., HHOEK-KON, T.V. and HENDERSON, P.T. (1979). Measurement of DNA excision repair in suspensions of freshly isolated rat hepatocytes after exposure to some carcinogenic compounds: Its possible use in carcinogenicity screening. Mutat. Res. 64:425–432.

    Google Scholar 

  • BUDROE, J.D., SHADDOCK, J.G. and CASCIANO, D.A. (1984). A study of the potential genotoxicity of methapyrilene and related antihistamines using the hepatocyte/DNA repair assay. Mutat. Res. 135:131–137.

    Google Scholar 

  • CASCIANO, D.A., FARR, J.A., OLDHAM, J.W. and CAVE, M.D. (1978). 2-Acetylaminofluorene-induced unscheduled DNA synthesis in hepatocytes isolated from 3-methylcholanthrene treated rats. Cancer Lett. 5:173–178.

    Google Scholar 

  • CLARKSON, J.M. (1978). Enhancement of repair replication in mammalian cells by hydroxyurea. Mutat. Res. 52:273–284.

    Google Scholar 

  • EDENBERG, H.J. and HANAWALT, P.C. (1973). Time course of DNA repair replication in ultraviolet-irradiated HeLa cells. Biochim. Biophys. Acta 324:206–217.

    Google Scholar 

  • FREEMAN, S.E. and LARCOM, L.L. (1983). Inhibition of unscheduled DNA synthesis in human lymphocytes by chemical carcinogens. Chem.-Biol. Interactions 46:85–99.

    Google Scholar 

  • HOLME, J.A. and SODERLUND, E.J. (1984). Unscheduled DNA synthesis of rat hepatocytes in monolayer culture. Mutat. Res. 126:205–214.

    Google Scholar 

  • HSIA, M.T.S. (1986). Use of DNA damage and unscheduled DNA synthesis in isolated rat hepatocytes for screening potential chemical carcinogens. In: Safety Evaluation and Regulation of Chemicals, Vol. 3, pp. 106–107 (F. Homberger, ed.). S. Karger, Basel.

    Google Scholar 

  • HSIA, M.T.S. and KREAMER, B.L. (1979). Induction of unscheduled DNA synthesis in suspensions of rat hepatocytes by an environmental toxicant, 3,3′,4,4′-tetrachloroazoben-zene. Cancer Lett. 6:207–212.

    Google Scholar 

  • HSIA, M.T.S., KREAMER, B.L. and DOLARA, P. (1983). A rapid and simple method to quantitate chemically induced unscheduled DNA synthesis in freshly isolated rat hepatocytes facilitated by DNA retention on membrane filters. Mutat. Res. 122:177–185.

    Google Scholar 

  • HSIA, M.T.S., KREAMER, B.L. and DUDDY, S.K. (1985). Induction of DNA damage and repair synthesis in freshly isolated rat hepatocytes by the proallatocidin precocene II. Cancer Lett. 26:311–318.

    Google Scholar 

  • JOE, C.O., RANKIN, P.W. and BUSBEE, D.L. (1984). Human lymphocytes treated with r-7, t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene require low-density lipoproteins for DNA excision repair. Mutat. Res. 131:37–43.

    Google Scholar 

  • KANTER, P.M. and SCHWARTZ, H.S. (1979). A hydroxyapatite batch assay for quantitation of cellular DNA damage. Anal. Biochem. 97:77–84.

    Google Scholar 

  • KOHN, K.W., ERICKSON, L.C., EWIG, R.A.G. and ZWELLING, L.A. (1976). Fractionation of DNA from mammalian cells by alkaline elution. Biochemistry 15:4629–4637.

    Google Scholar 

  • KOHN, K.W., EWIG, R.A.G., ERICKSON, L.C. and ZWELLING, L.A. (1981). Measurement of strand breaks and cross-links by alkaline elution. In: DNA Repair: A Laboratory Manual of Research Procedures, Vol. 1, pp. 379–401 (ed. by E.C. Friedberg and P.C. Hanawalt). Dekker, New York.

    Google Scholar 

  • KREAMER, B.L., STAECKER, J.L., SAWADA, N., SATTLER, G.L., HSIA, M.T.S. and PITOT, H.C. (1986). Use of a low-speed iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro 22:201–211.

    Google Scholar 

  • LAISHES, B.A. and WILLIAMS, G.M. (1976). Conditions affecting primary cell cultures of functional adult rat hepatocytes II dexamethasone enhanced longevity and maintenance of morphology. In Vitro 12:821–832.

    Google Scholar 

  • LAKE, R.S., KROPKO, M.L., PEZZUTTI, M.R., SHOEMAKER, R.H. and IGEL, H.J. (1978). Chemical induction of unscheduled DNA synthesis in human skin epithelial cell cultures. Cancer Res. 38:2091–2098.

    Google Scholar 

  • LAKE, R.S., KROPKO, M.L., MC LACHLAN, S., PEZZUTTI, M., SHOEMAKER, R.H. and IGEL, H.J. (1980). Chemical carcinogen induction of DNA-repair synthesis inhuman peripheral blood monocytes. Mutat. Res. 74:357–377.

    Google Scholar 

  • LEE, I.P. and SUZUKI, K. (1979). Induction of unscheduled DNA synthesis in mouse germ cells following 1,2-dibromo-3-chloropropane (DBCP) exposure. Mutat. Res. 68:169–173.

    Google Scholar 

  • LIJINSKY, W., REUBER, M.D. and BLACKWELL, B.-N. (1980). Liver tumors induced in rats by oral administration of the antihistaminic methapyrilene hydrochloride. Science 209:817–819.

    Google Scholar 

  • LOHMAN, P.H.M., SLUYTER, M.L. and MATTHIJS, I.A.A. (1973). Repair replication in human cells studied by sodium iodide isopycnic centrifugation of DNA in a fixed-angle rotor. Anal. Biochem. 54:178–187.

    Google Scholar 

  • LOURY, D.J. and BYARD, J.L. (1983). Aroclor 1254 pretreatment enhances the DNA repair response to amino acid pyrolysate mutagens in primary cultures of rat hepatocytes. Cancer Lett. 20:283–290.

    Google Scholar 

  • LOWING, R.K., FRY, J.R., KING, L.J. and BRIDGE, J.W. (1979). The early effects of chemical carcinogens on adult rat hepatocytes in primary culture. II. Effects on unscheduled DNA synthesis, cell division and α-fetoprotein production. Chem.-Biol. Interactions 25:303–319.

    Google Scholar 

  • MARTELLI, A., CAJELLI, E. and GHIA, M. (1985). Scintillometric determination of unscheduled DNA synthesis in primary cultures of rat-liver cells: A hydroxyapatite batch assay. Mutat. Res. 143:257–261.

    Google Scholar 

  • MARTIN, C.N., MC DERMID, A.C. and GARNER, R.C. (1978). Testing of known carcinogens and noncarcinogens for their ability to induce unscheduled DNA synthesis in HeLa cells. Cancer Res. 38:2621–2627.

    Google Scholar 

  • MC CANN, J.E., CHOI, E., YAMASAKI, E. and AMES, B.N. (1975). Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc. Natl. Acad. Sci. USA 72:5135–5139.

    Google Scholar 

  • MC QUEEN, C.A. and WILLIAMS, G.M. (1981). Characterization of DNA repair elicited by carcinogens and drugs in the hepatocyte primary culture/DNA repair test. J. Toxicol. Environ. Health 8:463–477.

    Google Scholar 

  • MICHALOPOULOS, G., SATTLER, G.L., O'CONNOR, L. and PITOT, H.C. (1978). Unscheduled DNA synthesis induced by procarcinogens in suspensions and primary cultures of hepatocytes on collagen membranes. Cancer Res. 38:1866–1871.

    Google Scholar 

  • MITCHELL, A.D., CASCIANO, D.A., MELTZ, M.L., ROBINSON, D.E., SAN, R.H.C., WILLIAMS, G.M. and VON HALLE, E.S. (1983). Unscheduled DNA synthesis tests: A report of the U.S. Environmental Protection Agency Gene-Tox. Program. Mutat. Res. 123:363–410.

    Google Scholar 

  • MORLEY, C.G.D. and KINGDON, H.S. (1972). Use of 3H-thymidine for measurement of DNA synthesis in rat liver—A warning. Anal. Biochem. 45:298–305.

    Google Scholar 

  • National Toxicology Program (1983). Third Annual Report on Carcinogens: Summary. Public Health Service, U.S. Department of Health and Human Services, Research Triangle Park.

    Google Scholar 

  • OLDHAM, J.W., CASCIANO, D.A. and CAVE, M.D. (1980). Comparative induction of unscheduled DNA synthesis by physical and chemical agents in non-proliferating primary cultures of rat hepatocytes. Chem.-Biol. Interactions 29:303–314.

    Google Scholar 

  • OLSON, M.J., CASCIANO, D.A. and POUNDS, J.G. (1983). A method for rapid, sensitive quantitation of short-patch DNA repair in cultured rat hepatocytes. Mutat. Res. 119:381–386.

    Google Scholar 

  • PROBST, G.S. and NEAL, S.B. (1980). The induction of unscheduled DNA synthesis by antihistamines in primary hepatocyte cultures. Cancer Lett. 10:67–73.

    Google Scholar 

  • PROBST, G.S., HILL, L.E. and BEWSEY, B.J. (1980). Comparison of three in vitro assays for carcinogen-induced DNA damage. J. Toxicol. Environ. Health 6:333–349.

    Google Scholar 

  • PROBST, G.S., MC MAHON, R.E., HILL, R.E., THOMPSON, C.Z., EPP, J.K. and NEAL, S.B. (1981). Chemically induced unscheduled DNA synthesis in primary rat hepatocyte cultures: A comparison with bacterial mutagenicity using 218 compounds. Environ. Mutagenesis 3:11–32.

    Google Scholar 

  • RASMUSSEN, R.E. and PAINTER, R.B. (1964). Evidence for repair of ultraviolet damaged deoxyribonucleic acid in cultured mammalian cells. Nature 203:1360–1362.

    Google Scholar 

  • ROBISON, S.H., CANTONI, O., HECK, J.D. and COSTA, M. (1983). Soluble and insoluble nickel compounds induce DNA repair synthesis in cultured mammalian cells. Cancer Lett. 17:273–279.

    Google Scholar 

  • RUDDICK, J.A., WILLIAMS, D.T., HIERLIHY, L. and KHERA, K.S. (1976). [14C] Ethylenethiourea: Distribution, excretion, and metabolism in pregnant rats. Teratology 13:35–40.

    Google Scholar 

  • SHAW, J.L., BLANCO, J. and MUELLER, G.C. (1975). A simple procedure for isolation of DNA, RNA and protein fractions from cultured animal cells. Anal. Biochem. 65:125–131.

    Google Scholar 

  • SIRICA, A.E., HWANG, C.G., SATTLER, G.L. and PITOT, H.C. (1980). Use of primary cultures of adult rat hepatocytes on collagen gel-nylon mesh to evaluate carcinogen-induced unscheduled DNA synthesis. Cancer Res. 40:3259–3267.

    Google Scholar 

  • SNYDER, R.D. and REGAN, J.D. (1982). DNA repair in normal human and xeroderma pigmentosum group A fibroblasts following treatment with various methanesulfonates and the demonstration of a long-patch (U.V.-like) repair component. Carcinogenesis 3:7–14.

    Google Scholar 

  • SOTOMAYOR, R.E., CHAUHAN, P.S. and EHLING, U.H. (1982). Induction of unscheduled DNA synthesis in the germ cells of male mice after treatment with hydrazine or procarbazine. Toxicology 25:201–211.

    Google Scholar 

  • TONG, C., LASPIA, M.F., TELANG, S. and WILLIAMS, G.M. (1981). The use of adult rat liver cultures in the detection of the genotoxicity of various polycyclic aromatic hydrocarbons. Environ. Mutagenesis 3:477–487.

    Google Scholar 

  • TROSKO, J.E. and YAGER, J.D. (1974). A sensitive method to measure physical and chemical carcinogen-induced “unscheduled DNA synthesis” in rapidly dividing eukaryotic cells. Exptl. Cell Res. 88:47–55.

    Google Scholar 

  • TSUTSUI, T., IDE, K. and MAIZUMI, H. (1984). Induction of unscheduled DNA synthesis in cultured human oral keratinocytes by sodium fluoride. Mutat. Res. 140:43–48.

    Google Scholar 

  • WILLIAMS, G.M. (1977). Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures. Cancer Res. 37:1845–1851.

    Google Scholar 

  • WILLIAMS, G.M. (1978). Further improvements in the hepatocyte primary culture DNA repair test for carcinogens: Detection of carcinogenic biphenyl derivatives. Cancer Lett. 4:69–75.

    Google Scholar 

  • WILLIAMS, G.M., LASPIA, M.F. and DUNKEL, V.C. (1982). Reliability of the hepatocyte primary culture/DNA repair test in testing of coded carcinogens and noncarcinogens. Mutat. Res. 97:359–370.

    Google Scholar 

  • YAGER, J. D. and MILLER, J.A. (1978). DNA repair in primary cultures of rat hepatocytes. Cancer Res. 38:4385–4394.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Pesticide Toxicology Group Department of Entomology and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin

    M. T. Stephen Hsia

Authors
  1. M. T. Stephen Hsia
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stephen Hsia, M.T. Use of scintillometric quantitation of unscheduled DNA synthesis in isolated rat hepatocytes for the screening of genotoxic agents. Cell Biol Toxicol 3, 127–142 (1987). https://doi.org/10.1007/BF00058452

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00058452

Keywords

Search

Navigation