To further characterize the Drosophila-based prescreen to detect developmental toxicants, aminopterin (4-aminofolic acid [APN]; CAS No. 54-62-6), was studied. APN, a documented developmental toxicant and teratogen, was formerly used clinically as an antineoplastic agent. APN inhibits dihydrofolate reductase preventing the formation of folinic acid and stopping one carbon metabolism needed for the synthesis of DNA in rapidly dividing cells. Initially, 7 APN concentrations ranging from 4 - 80 ug/vial were evaluated. In a second experiment, 6 concentrations ranging from 0.01 - 2 ug/vial were utilized to determine if a threshold of developmental toxicity could be observed. Each experiment included a concurrent control. Drosophila were exposed throughout development (egg through third instar larva) in culture vials to medium containing APN. Each vial contained 1g of powdered medium and 5ml of distilled deionized water or a solution of test chemical in water. A mated, untreated, Oregon-R wild-type female (Mid-American Drosophila Stock Center, Bowling Green State University, Ohio) was added to each vial and allowed to oviposit for 20 hours, then removed. Emerging offspring were collected over 10 days, and examined microscopically (25x) for bent humeral bristles and wing blade notches; morphological defects shown to occur with an increased incidence in flies exposed to developmental toxicants. In each experiment, the incidence of the two defects at each concentration was compared to the controls using chi-square. The incidence of wing blade notches was significantly increased (all p< 0.001) at 2 ugg/vial, 17/138; 4 ug/vial, 20/170; 10 ug/vial, 25/96; 14.1 ug/vial, 9/81; 20 ugg/vial 13/82; 28.3 ug/vial 9/43; 40 ug/vial 18/61; and at 80 ug/vial, 10/25. One wing blade notch was observed among 414 control flies. Bristle defects were not increased by APN. Mortality of the offspring was increased and the first day of emergence was delayed at the two highest APN concentrations. The results with APN parallel the developmental toxicity reported in mammals and replicate and expand published Drosophila data. These findings provide additional support for increased utilization of this assay as a prescreen for the detection of developmental toxicants.
The Toxicologist. Society of Toxicology 39th Annual Meeting, March 19-23, 2000, Philadelphia, Pennsylvania