A carbamate herbicide causes microtubule and microfilament disruption and nuclear fragmentation in fibroblasts

Abstract

Isopropyl( N-(3-chlorophenyl)carbamate (CIPC) is a widely used herbicide thought to inhibit mitosis by selectively perturbing either the microtubules or microtubule organizing centers of susceptible plant species. Its effects on microtubule and microfilament organization in mouse 3T3 fibroblasts are analyzed here by immunofluorescence techniques using anti-tubulin and anti-actin antibodies. We report that relatively high levels of CIPC (but not higher than the usual levels employed in plant studies) inhibit fibroblast growth, cause the disappearance of cytoplasmic microtubules and also lead to disorientation of microfilament bundles. These responses develop within 24 h of exposure of 3T3 cells to CIPC and persist as long as the herbicide is present in the culture medium. More importantly, cells returned to drug-free medium following 5–7 days exposure to CIPC resume rapid division but show several remarkable morphological responses. In particular they develop multipolar spindles during mitosis and show an unequal partitioning of nuclei between daughter cells. Consequently a high proportion of interphase cells contains multiple and multilobed nuclei. Such fibroblast cultures exposed to CIPC and then returned to drug-free medium may provide chromosome-deficient cells of value for genetic mapping and recombinant studies. On the other hand if similar responses to CIPC exposure and withdrawal occur in vivo then the herbicide may pose a genetic risk to man that is not revealed by usual testing procedures. The mechanism(s) by which CIPC produces these responses remains unclear. Fibroblasts cultured in the continued presence of the known microtubule-specific drugs colchicine and nocodazole (10 −6 M) show microtubule disassembly within 30 min followed by cell rounding and detachment. By contrast CIPC-treated cells lose microtubule integrity more slowly and remain highly elongated and firmly glass adherent even with prolonged treatment. These dissimilar morphological responses, as well as the failure of CIPC to affect microtubule assembly in vitro, indicate that tubulin is not the exclusive target of CIPC in animal cells. The distribution of assembled microtubules into the arms of multipolar mitotic spindles is clearly observed following withdrawal of CIPC. The abnormality could reflect an irreversible effect of CIPC on microtubule organizing centers. However, the redistribution of cytoplasmic microfilaments into dense, discontinuous submembranous bundles in the presence of CIPC and the failure of microfilaments to resume an entirely normal distribution following herbicide withdrawal suggests that microfilaments are additional targets of herbicide action.