The 2-phenylbenzotriazole-type water pollutant PBTA-2 has cytochalasin B-mimetic activity

The 2-phenylbenzotriazole (PBTA)-type water pollutant, 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5- amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), has been recently identified in samples from the Nishitakase River in Kyoto, Japan, and shows potent mutagenic activities in Salmonella typhimurium in the presence of a microsomal metabolizing system (S9 mix). In the present study, we conducted the in vitro micronucleus (MN) test on PBTA-2 in the absence and presence of S9 mix in two Chinese hamster cell lines, CHL and V79-MZ. In the MN test, PBTA-2 was weakly positive in CHL cells and strongly positive in V79-MZ cells. Because the positive results were accompanied by a statistically significant increase in the number of polynuclear (PN) and/or mitotic (M) cells, we examined treated cells in metaphase to see if numerical chromosome aberrations were being induced. We found that PBTA-2 induces polyploidy in both CHL and V79-MZ cells. A detailed analysis of MN preparations showed that in CHL cells, PBTA-2 predominantly induces equal-sized binucleated cells. Rhodamine phalloidin staining revealed that PBTA-2 causes actin filament abnormalities in both cell lines similar to those caused by cytochalasin B. Cytochalasin B induced PN cells predominantly and dose dependently, and almost all the cells were equal-sized and binucleate. The results suggest that PBTA-2 has cytochalasin B-mimetic activity, although agents affecting actin filaments, such as cytochalasins, phallotoxins and chloropeptide, have been derived only from molds so far. This study also suggests that our MN test protocol may be used to identify chemicals that have cytochalasin B-mimetic activity as well as those that induce numerical aberrations.