Toxicity of Four Alkylating Agents on in Vitro Rat Embryo Differentiation and Development

The relative developmental toxicity of four direct acting, alkylatingagents was determined in primary cultures of differentiatingrat embryo midbrain (CNS) and limb bud (LB) cells and comparedwith that observed in the rat whole embryo postimplantationculture system. The alkylating agents tested include methylnitrosourea(MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS),and ethyl methanesulfonate (EMS). These alkylating agents havebeen shown to produce developmental toxicity following eitherin vitro or in vivo exposure. Viability for both CNS and LBwas assessed by a neutral red dye assay. Differentiation ofCNS cells was assessed by hematoxylin staining of neurons; differentiationof LB cells was assessed by Alcian blue staining of extracellularproteoglycans. Relative potencies of these compounds in thecell culture system were not the same as those observed in theembryo culture system. Whereas rank order of potency in thecell culture system, for viability and differentiation, wasMMS > MNU > ENU > EMS, rank order in the embryo culturesystem, for embryo lethality and malformations, was MNU >ENU > MMS > EMS. Effective concentrations for cell cultureviability and differentiation by MNU and ENU in cell culturewere about three to nine times higher than comparable valuespreviously reported for embryos, while effective concentrationsfor MMS and EMS were two to seven times lower than those observedin the embryos. Differences in potency between the two culturesystems may be related to differences in formation and repairof DNA adducts, as well as differences in culture conditions.