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Induction of 6‐thioguanine‐resistant lymphocytes in fischer 344 rats following in vivo exposure to n‐ethyl‐n‐nitrosourea and cyclophosphamide
Author(s) -
Aidoo Anane,
LynCook Lascelles E.,
Mittelstaedt Roberta A.,
Heflich Robert H.,
Casciano Daniel A.,
Cole J.
Publication year - 1991
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.2850170302
Subject(s) - in vivo , microbiology and biotechnology , lymphocyte , mixed lymphocyte reaction , spleen , cyclophosphamide , biology , cell culture , nitrosourea , clastogen , andrology , chemistry , immunology , toxicity , genotoxicity , chemotherapy , immune system , medicine , t cell , genetics , organic chemistry
Abstract We have developed a limiting dilution clonal assay for determining the frequency of 6‐thioguanine‐resistant (TG r ) lymphocytes produced in rats by in vivo exposure to genotoxic agents. Spleen lymphocytes were isolated from female Fischer 344 rats and were cultured with 1 μg/ml of phytohemagglutinin (PHA) for 40 hr. Northern blot analysis revealed that this procedure resulted in increased hprt and β‐actin mRNA synthesis. Conditions for optimum cloning were established by culturing four PHA‐primed lymphocytes/well in 96‐well round‐bottom microtiter plates containing a medium supplemented with interleukin‐2. These cultures also contained autologous and/or TK6 feeder cells inactivated with different doses of irradiation. Lymphocyte cloning efficiencies (CEs) were highest in plates containing both irradiated TK6 cells (5 × 10 3 cells/well; 90 Gy) and irradiated autologous feeder cells (5 × 10 4 cells/well; 50 Gy). CE did not depend on the number of primed lymphocytes/well when four or fewer target cells/well were cloned. To measure the effects of chemical mutagens on the frequency of TG r lymphocytes, rats were given a single i.p. injection of 0–150 mg/kg of N‐ethyl‐N‐nitrosourea (ENU), a direct‐acting alkylating agent, or 0–50 mg/kg of cyclophosphamide (CP), an indirect acting alkylating agent. Lymphocytes were isolated, primed, and cloned at 4 weeks after CP treatment and at 1, 2, 4 and 6 weeks after ENU treatment. CE in these cultures ranged from 12% to 27%. Cultures were also established for measuring CE in the presence of 6‐thioguanine (TG) and these contained 5 × 10 3 irradiated TK6 cells and 5 × 10 4 primed rat lymphocytes/well. The frequency of TG r lymphocytes was calculated by correcting the CE in the presence of TG with the CE measured in its absence. ENU exposure produced a higher frequency of TG r lymphocytes than CP, but both chemicals produced a dose‐dependent increase in TG r cells. In addition, the frequency of ENU‐induced TG r lymphocytes increased with time after treatment. The TG r cells are presumed to be hprt mutants, but further analysis at the DNA level is required to establish this. The dose‐dependent responses obtained with both ENU and CP treatments suggest that rat lymphocytes are sensitive to direct‐ and indirect‐acting alkylating agents administered in vivo and that the rat lymphocyte assay is a useful complement to the in vivo/in vitro mouse assay for determining the mutagenicity of environmental toxicants.