Developmental toxicity of four model alkylating agents on japanese medaka fish (Oryzias latipes) embryos

Abstract As public and scientific interest increases in identifying in vitro alternatives to whole-animal testing, there is a need to characterize the responses of in vitro systems to defined chemical agents. In this paper we have utilized the medaka (Oryzias latipes) embryo culture system to examine the developmental toxicity of a series of chemically defined model alkylating agents. The medaka embryo culture system was used because it offers numerous advantages over other in vitro embryo systems. This study has examined the developmental toxicity of four model alkylating agents: methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS). These alkylating agents represent chemical models for compounds that are environmental and occupational pollutants. The embryotoxic parameters that were monitored include embryolethality and specific malformations. Medaka embryos in early organogenesis (stage 20) were exposed for 2 h to the test compound at concentrations ranging from 0.75 to 120 mM. The embryos were cultured at 20°C in Petri dishes and were assessed daily for viability and gross malformations until hatching. All agents tested produced concentration-dependent decreases in viability and increases in malformations. The developmental toxicity of the four alkylating agents examined is, in order of decreasing potency, MNU > ENU > MMS > EMS. A similar spectrum of malformations was produced following exposure to each alkylating agent; however, agents undergoing first-order nucleophilic substitution reactions were more potent developmental toxins than those agents which undergo second-order nucleophilic substitution reactions. Observed abnormalities included flexure, cardiac, optic, and cephalic malformations.