Human embryonic cell growth assay for teratogens with or without metabolic activation system using microplate

In vitro microassay for the screening of teratogens was investigated on cancer chemotherapeutic agents sterigmatocystins and benzimidazoles using human embryonic palatal mesenchymal (HEPM) cells. Five thousand cells were inoculated into each well of 96-well microtiter plates, and cultivated for 24 hr, after which the media were changed with new ones that contained various amounts of chemicals; after cultivation for an additional 72 hr, the media were discarded, and cells attached to the tissue plate were fixed and stained with Giemsa's solution; the cell number then was counted by colony counter with three readings for each well. For the metabolic activation, the liver S9 obtained from rats pretreated with phenobarbital and 5,6-benzoflavone and cofactors (S9 mix) were added directly to the HEPM cell cultures along with chemicals. After 6 hr, the cultures were exchanged with a fresh medium and incubated for a further 72 hr. The final IC50 (the concentration that inhibits growth 50%) concentration-finding run had 7 to 11 concentration points (mean, three to four wells). Concentrations of the cancer chemotherapeutic agents that inhibited growth by 50% ranged from 0.001 to 10 micrograms/ml. Sterigmatocystins indicated strong inhibition; among three derivatives, O-acetyl sterigmatocystin was the most potent inhibitor. Benzimidazoles also exhibited an inhibitory action on HEPM cell growth; nitro and chloro groups at the 5 position in 2-(2-pyridyl)benzimidazole were found to be potent substituents. As for the activation of cyclophosphamide in the HEPM cell culture, IC50 was decreased to 1.0 ug/ml by the incubation with S9 mix for 6 hr under our experimental conditions, and sterigmatocystin was found to be activated by S9 mix.(ABSTRACT TRUNCATED AT 250 WORDS)