Cytogenetic effects of piperonyl butoxide and safrole in CHO-K1 cells

Recently, hepatocarcinogenicity in rats and mice was reported with regard to the methylenedioxyphenyl compound, piperonyl butoxide (PB), which is used as a synergist for pyrethrins and related insecticides. Induction of sister-chromatid exchanges (SCEs) and chromosomal aberrations (CAs) due to PB were investigated using CHO-K1 cells with or without rat liver S9 fraction (S9); at the same time, the effects of safrole (SF), a methylenedioxyphenyl compound and a weak hepatocarcinogen, were also examined. PB (0.25 and 0.3 mM) and SF (0.8 mM) caused a slight but significant increase in SCEs followed by a cell-cycle delay in the 3-h treatment without S9. In the presence of S9 (4.5%), the cytotoxicity of PB or SF was weakened greatly or slightly, the top dose capable of cell division was raised to 0.6 mM (2-fold) or 1 mM, respectively. PB with S9 induced SCE at doses of 0.4 and 0.5 mM, and caused endoreduplications (ERDs, 7%) at a dose of 0.6 mM, while SF caused a dose-related significant increase in SCE at all doses used (0.4–1 mM) with S9. Genotoxicity of the metabolites of PB or SF was cleared by changing the dose of S9 (1.5–9%) while holding the dose of each chemical constant. In the case of SF (0.6 mM), induction of SCE, ERD and cell-cycle delay intensified almost in a dose-effect relationship, and CAs and a high level of ERD (14%) were caused by a 9% dose of S9. The concentration of unchanged SF in the incubated medium was certainly in inverse proportion to the dose of S9. This strongly suggests that the metabolites of SF are genotoxic. In the case of PB (0.3 mM), no positive responses were produced in the cultures, even with a high level of S9, though the amount of unchanged PB left in the incubated medium was very slight. This indicates that the metabolites of PB may not be genotoxic. In conclusion, PB and SF are possible to somewhat induce SCE at high dose(s) in the absence of S9, and the genotoxic effects of SF are more intensified in the presence of S9 than in its absence, while PB is probably no genotoxic in the presence of sufficient metabolic activation.