Characterization of a rat lymphocyte culture system for assessing sister chromatid exchange after in vivo exposure to genotoxic agents

Abstract

Lymphocyte culture systems have the major advantage of permitting the analysis of in vivo cytogenetic damage with minimal injury to the animal under study. This paper describes a rat lymphocyte culture system designed for the study of sister chromatid exchange (SCE) induced by in vivo exposure to genotoxic agents. A standard protocol was established in which 1 to 2 ml of blood are removed from rats by cardiac puncture, washed three times with phosphate-buffered saline (pH 7.4), and grown in RPMI 1640. 5-Bromodeoxyuridine (BrdU) (1.0 microM) is added after 24 hr, and cells are harvested after 56 hr of culture. Critical steps for successful blast transformation include washing the blood in buffered saline and the adding of 2.0 to 4.0 microgram phytohemagglutinin/ml to the culture medium. The use of low concentrations of BrdU (less than or equal to 5.0 microM) is recommended to maintain low baseline SCE levels and to avoid cytotoxicity. The mutagenic carcinogen, ethyl methanesulfonate (EMS), was used as a positive control agent and at a dose of 300 mg/kg caused a fourfold increase in SCE frequency. Twenty-eight days after EMS administration (30, 100, 300 mg/kg), lymphocytes from treated animals still displayed SCE levels at least 50% above baseline. This system provides a reliable means of investigating chemically induced SCE in the rat.