In vitro assays of in vivo exposure to cyclophosphamide: induction of sister-chromatid exchanges in peripheral lymphocytes, bone-marrow cells and in cultured cells exposed to plasma.

The in vivo genotoxic potential of cyclophosphamide (CY) was assessed by sister-chromatid exchange (SCE) induction after removal and in vitro culture of circulating peripheral lymphocytes and bone marrow from CY exposed male, Fischer 344 rats. Plasma was simultaneously obtained and assessed for genotoxic activity by incubation with 4 cultured mammalian cell lines: HepG2, H4-II-E (H4), V79 and IMR-90. These 4 cell types were used to help discriminate the role of metabolism in generating SCE-inducing factors in plasma. HepG2 and H4 have been shown to metabolize certain xenobiotics while V79 and IMR-90 do not. An in vivo dose response to CY at doses of 0, 5, 10, 20, 30 and 50 mg CY/kg assayed 1 h post-i.p. injection was performed. Phytohemagglutinin (PHA)-stimulated lymphocytes showed a dose-related increase in SCE up to 66.7 SCE/cell at 20 mg/kg (30 mg/kg was cytotoxic). Bone marrow also showed an SCE increase to 34.8 SCE/cell at 10 mg/kg (higher doses were cytotoxic). Plasma induced a dose-dependent SCE increase in the 4 cultured cell lines at all tested doses indicating the presence of direct-acting SCE-inducing metabolites of CY. A time course study using 20 mg CY/kg indicated peak plasma levels of CY genotoxic activity at approximately 0.5-1 h post-injection. By 3 h, the level of genotoxic activity in plasma was considerably reduced. Lymphocytes, however, showed a cumulative increase in SCE to 74.7 SCE/cell after 3 h of exposure. These in vivo exposure--in vitro assay techniques may be useful and facile systems with which to develop an integrative testing system for assessing the in vivo genotoxicity of a chemical.