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Mouse Peritoneal Lymphocytes, a New Target for Analyzing Induction of Sister Chromatid Exchanges on in Vivo Exposure to a Genotoxic Agent

Section of Cell Biology and Cytogenetics, Biological Research Center, Japan Tobacco, Inc., 23 Nakogi, Hatano, Kanagawa 257, Japan

The availability of use of mouse peritoneal lymphocytes as target cells for analyzing sister chromatid exchanges (SCE) upon exposure to a genotoxic drug, cyclophosphamide, was investigated using female ICR mice.

Use of these cells overcame the difficulty in use of mouse lymphocyte cultures, recovering sufficient metaphase cells. The greatest advantage of use of peritoneal lymphocytes was that about 1–2 x 10⁶ lymphocytes/mouse could easily be recovered from the peritoneal cavity in high purity. Their mitogenic responses were good when Escherichia coli lipopolysac-charide, in combination with 2-mercaptoethanol, was used as mitogens, but they were less when purified phytohemagglutinin was used. In the presence of lipopolysaccharide (60 µg/ml) and 2-mercaptoethanol (22–88 µM), the maximum incidence of second division metaphases (>50%) and the highest mitotic index (>4%) were observed 36–40 h after stimulation. Under these conditions, the base-line SCE showed the constant level. The range of intrastrain variations in the base-line SCE was 0.24–0.36/chromosome. The distribution histograms of SCE/chromosome did not fit a single Poisson model, suggesting that these cells are heterogeneous with respect to the base-line SCE.

Single s.c. injections 1 h before harvest of doses of 0.75–3.0 mg of cyclophosphamide per kg evoked positive responses, and injections of over 0.375 mg/kg had linear dose-dependent effects. On harvest of cells for up to 192 h after the injection, the maximal induction of SCE attained 1 h after exposure was found to return time dependently to the control level at 192 h. After the initial rapid reduction in the cell number, cellular recovery, measured as the mitotic index and the number of peritoneal exudate cells recovered, returned to the control level within 48 h, without a significant increase thereafter. After maintaining cells under the liquidholding experiment for various times in vitro following a single exposure to cyclophosphamide for 1 h in vivo, the reduction of their SCE and recovery of their mitotic index were more rapid than those of cells in the time-course experiment. These findings suggest that the association of the recruitment of less- and/or nondamaged cells from their precursors with reduction of the SCE is slight. Repair(s) and, to a lesser extent, selective loss of more damaged cells may be the main factors contributing to the early reduction response of the SCE frequency. The relations of these factors are discussed.

¹ To whom requests for reprints should be addressed.

Received 12/13/85. Revised 3/ 5/86. Accepted 3/17/86.