Journal Article

Induction of <i>hprt</i> gene mutations in splenic T-lymphocytes from the rat exposed <i>in vivo</i> to DNA methylating agents is correlated with formation of <i>O</i><sup>6</sup>-methylguanine in bone marrow and not in the spleen

J.G. Jansen, A.J.L. de Groot, C.M.M. van Teijlingen, A.D. Tates, H. Vrieling and A.A. van Zeeland

in Carcinogenesis

Volume 17, issue 10, pages 2183-2191
Published in print October 1996 | ISSN: 0143-3334
Published online October 1996 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/17.10.2183
Induction of hprt gene mutations in splenic
                    T-lymphocytes from the rat exposed in vivo to DNA methylating
                    agents is correlated with formation of
                    O6-methylguanine in bone marrow and not in the
                    spleen

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The suitability of splenic T-lymphocytes as a substitute tissue for detection of genotoxic effects induced in vivo by chemical agents that are organ-specifically activated was tested in rats exposed to single doses at the potent lung-carcinogen 4-(methylnitrosamino)-1-(3-pyri-dyl)-1-butanone (NNK), acetoxymethylmethylnitrosamine (AMMN) or N-methyl-N-nitrosourea (MNU). NNK, AMMN and MNU methylate DNA most likely via the formation of a methanediazohydroxide ion that decomposes to a methyl diazoniumion. For all three agents, an increase in the levels of O6-methylguanine and 7-methylguanine in DNA of rat liver and lung was detected by reverse phase HPLC and electrochemical detection. Treatment with NNK did not result in the formation of O6-methylguanine and 7-methylguanine in DNA of bone marrow and spleen, corresponding with the absence of metabolic activation pathways for this compound in these tissues. For AMMN formation of both O6-methylguanine and 7-methylguanine was detectable in DNA of the spleen, whereas in DNA of bone marrow only very low frequencies of 7-methylguanine were found at a toxic dose. MNU induced O6-methylguanine and 7-methylguanine in both spleen and bone marrow. Using splenic T-lymphocytes from the rat no increase above control levels of the hprt mutant frequencies was found for NNK and AMMN for all exposure levels tested, 32 days after chemical exposure. For MNU a dose-dependent increase in hprt mutant frequency was found at exposure levels of 0.097 mmol/kg up to 0.582 mmol/kg. DNA sequence analysis was performed on PCR products of hprt cDNA from 39 MNU-induced 6-thioguanine-resistant T-lymphocyte clones. Single base pair substitutions were found in 25 of these mutants (64%), GC↑AT transitions being the predominant type of mutation (19 of 25; 76%). These mutations are probably caused by mispairing of O6methyl-guanine with thymine during DNA replication. The results indicate that formation of mutagenic lesions in the spleen is not correlated with an enhanced frequency of 6-thioguanine-resistant splenic T-lymphocyte clones from rats, 32 days after exposure in vivo to DNA damaging agents. This suggests that mutation-fixation in T-lymphocytes does not occur in the spleen but at other sites in the body such as bone marrow, after which these mutated cells migrate to the spleen.

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Subjects: Clinical Cytogenetics and Molecular Genetics

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