Quantification of Thioguanine-resistant Lymphocytes from Mice Irradiated in Vivo

Overview

Journal Environ Health Perspect

Date 1990 Aug 1

PMID 2272307

Authors

R Lorenz

T Gollner

K Hempel

Affiliations

Soon will be listed here.

Abstract

Adult mice were Co-60 gamma irradiated, and 7 months later splenocytes were isolated, cultured in microwells, and the frequency of hprt-deficient mutants was determined by measuring the cloning efficiency in media with 6-thioguanine. The mutant frequency at 2, 4, and 6 Gy was 1.6 x 10(-5), 4.4 x 10(-5), and 12.7 x 10(-5), respectively. The frequency of spontaneous mutants was 2.5 x 10(-6). The effect of metabolic cooperation on the cloning efficiency of thioguanine-resistant T-cells in selective medium was evaluated in co-cultures with wild-type T-cells. We found that the growth of hprt-deficient T-cells is supported in the presence of thioguanine-inactivated wild-type splenocytes up to a cell density of 5 x 10(5) cells per well. When cell density was higher, cell growth was inhibited. Possibilities and limitations of cloned lymphocytes for the analysis of somatic mutations that occur in vivo are discussed.

References

Voogd C, van der Stel J, Jacobs J . The mutagenic action of nitroimidazoles. I. Metronidazole, nimorazole, dimetridazole and ronidazole. Mutat Res. 1974; 26(6):483-90. DOI: 10.1016/s0027-5107(74)80049-7. View

Yotti L, Chang C, Trosko J . Elimination of metabolic cooperation in Chinese hamster cells by a tumor promoter. Science. 1979; 206(4422):1089-91. DOI: 10.1126/science.493994. View

Furth E, Thilly W, Penman B, Liber H, Rand W . Quantitative assay for mutation in diploid human lymphoblasts using microtiter plates. Anal Biochem. 1981; 110(1):1-8. DOI: 10.1016/0003-2697(81)90103-2. View

Jones I, Carrano A . A method to quantify spontaneous and in vivo induced thioguanine-resistant mouse lymphocytes. Mutat Res. 1985; 147(3):97-105. DOI: 10.1016/0165-1161(85)90022-6. View

Dempsey J, Morley A . Measurement of in vivo mutant frequency in lymphocytes in the mouse. Environ Mutagen. 1986; 8(3):385-91. DOI: 10.1002/em.2860080307. View

Hakoda M, Akiyama M, Kyoizumi S, Awa A, Yamakido M, Otake M . Increased somatic cell mutant frequency in atomic bomb survivors. Mutat Res. 1988; 201(1):39-48. DOI: 10.1016/0027-5107(88)90109-1. View

Jones I, Strout C, Crippen T . Factors that affect the frequency of thioguanine-resistant lymphocytes in mice following exposure to ethylnitrosourea. Environ Mutagen. 1987; 9(3):317-29. DOI: 10.1002/em.2860090311. View

Bols N, Mosser D, Boliska S . Transfer of purine metabolites between cells through the medium and via cell contacts in cocultures of HGPRT+ and HGPRT- cells. Exp Cell Res. 1987; 173(1):206-17. DOI: 10.1016/0014-4827(87)90346-6. View

Ammenheuser M, Ward Jr J, Whorton Jr E, Killian J, Legator M . Elevated frequencies of 6-thioguanine-resistant lymphocytes in multiple sclerosis patients treated with cyclophosphamide: a prospective study. Mutat Res. 1988; 204(3):509-20. DOI: 10.1016/0165-1218(88)90045-6. View

10.

Vrieling H, Niericker M, Simons J, van Zeeland A . Molecular analysis of mutations induced by N-ethyl-N-nitrosourea at the HPRT locus in mouse lymphoma cells. Mutat Res. 1988; 198(1):99-106. DOI: 10.1016/0027-5107(88)90045-0. View

11.

Jones I, Crippen T . Cloned mouse lymphocytes permit analysis of somatic mutations that occur in vivo. Somat Cell Mol Genet. 1987; 13(4):325-33. DOI: 10.1007/BF01534926. View

12.

Hooper M, Hardy K, Handyside A, Hunter S, Monk M . HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature. 1987; 326(6110):292-5. DOI: 10.1038/326292a0. View