Potential teratogenic effects of benomyl in rat embryos cultured in vitro.

A possible association between environmental exposure to benomyl and anophthalmia has been suggested. The aim of the present study was to investigate potential teratogenic effects of benomyl using the 9.5 day rat embryo culture method using rat and human serum. Explanted rat embryos were cultured in rat serum (n=121) or human serum (n=90) with differing concentrations of benomyl [170 nM to 13.6 microM], dissolved in ethanol (0.136%), at least five embryos per concentration being cultured. In addition, 18 embryos were cultured in both human and rat serum with the equivalent concentration of ethanol to act as a vehicle control. The cultured embryos were then measured and scored for growth and differentiation by two blinded observers. Embryotoxic effects were considered to be demonstrated by a decrease in parameters of growth such as crown rump length, yolk sac diameter and protein content, whereas embryopathic effects were considered to be those causing a decease in parameters of differentiation such as morphological score, somite number and optic development. Benomyl [> or =5 microM] produced a significant concentration dependent deterioration in morphological score, somite number and optic development. Gross toxic effects were noticed at concentrations of >12 microM in rat serum and >10microM in human serum as indicated by a significant effect on parameters measuring size (crown rump length; yolk sac diameter and protein content). This study provides evidence that benomyl is a potential developmental toxicant, affecting many parameters of differentiation, including optic development at levels below those that could be considered embryotoxic.

Gene transfer into intact fetal skeletal muscle grown in vitro.

The development of an organ culture system for growing prenatal intercostal muscle in vitro and its use to study gene function is described. Fetal skeletal muscle is relatively inaccessible during the key stages of its development, and this method enables DNA transfections and other manipulations to be carried out. The system allows cell proliferation and differentiation to continue and also maintains the morphology and fiber types of developing muscle. Gene transfer into cultured embryonic intercostal muscle was achieved by square-pulse electroporation of intact pieces of tissue. Expression of a marker gene (GFP) was found within 5 h and maintained for 2 days in muscle fibers and cells. The technique should enable the function of genes implicated in muscle development and disease to be studied at stages when access is difficult and in a controlled environment.