Frameshift mutations induced by the acridine mustard ICR-191 in embryos and in the adult gill and hepatopancreas of rpsL transgenic zebrafish

To determine whether frameshift mutations can be detected in rpsL transgenic zebrafish (Brachydanio rerio), embryos, and adult fish were treated with 6-chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxyacridine (ICR-191). Embryos exposed to 0, 10, or 20 μM ICR-191 in a water bath for 18 h exhibited induced mutant frequencies (MFs) of 14 × 10−5, 16 × 10−5, and 25 × 10−5, respectively. Only embryos exposed to 20 μM ICR-191 showed a significant increase in MF. The mutational spectra differed between the control and ICR-191-treated groups and single G:C pair insertions, which are a marked characteristic of ICR-191 mutagenesis, were observed in both 10 and 20 μM-treated embryos. In adult fish treated with 1 μM ICR-191 in a water bath for 18 h, a significant increase in MFs was observed in both gill (12 × 10−5 and 44 × 10−5 in control and treated fish, respectively), and hepatopancreas (5 × 10−5 and 29 × 10−5, respectively) 2 weeks after exposure. Sequence analysis showed that 58% of mutations in gill and 94% of mutations in hepatopancreas were single G:C pair insertions, which is typical of mutations induced by ICR-191. Additionally, these mutations occurred predominantly at a single site (CC sequence at bps 140-141) in the rpsL gene. Three weeks after exposure, however, the increased MFs and prominent mutational spectra of ICR-treated fish were undetectable. These findings suggest that using our protocols the rpsL transgenic zebrafish mutation assay is more effective for adult fish than for embryos, but that frameshift mutations can be detected in both embryos and adults at appropriate sampling times after treatment with ICR-191.