Induction of centrosome injury, multipolar spindles and multipolar division in cultured V79 cells exposed to dimethylarsinic acid: role for microtubules in centrosome dynamics

Role for microtubules in the induction of multiple microtubule organizing centers (MTOCs) and multipolar spindles by dimethylarsinic acid (DMAA), a methylated derivative of inorganic arsenics, was investigated with respect to the effects of microtubule disruption and reorganization. DMAA induced multiple signals of gamma-tubulin, a well-characterized component of MTOCs in the centrosome, in a manner specific to mitotic cells. The multiple signals of gamma-tubulin were co-localized with multipolar spindles caused by DMAA. Disruption of microtubules by nocodazole (NOZ) suppressed the appearance of centrosome injury caused by DMAA while disorganization of actin microfilaments by cytochalasin D did not. Post-treatment incubation of cells in which multiple signals of gamma-tubulin caused by DMAA had been coalesced to one or two dots by NOZ caused the reappearance of mitotic cells with multiple signals of gamma-tubulin, in conjunction with reorganization of the microtubules. These results suggest a role for microtubules in the dynamic behavior of the mitotic centrosome. DMAA induced aberrant cytokinesis, such as tripolar and quadripolar division, in a concentration-dependent manner. These results, together with the findings of earlier studies, suggest that the centrosome is the primary target for the induction of multipolar spindles by DMAA and the resultant induction of multinucleation and multipolar division.     

Induction of multiple microtubule-organizing centers, multipolar spindles and multipolar division in cultured V79 cells exposed to diethylstilbestrol, estradiol-17beta and bisphenol A

Inducibility of multipolar spindles and multipolar division by diethylstilbestrol (DES) and estradiol-17beta (E2) was investigated in terms of the mechanism of induction of aneuploidy by the estrogens. DES, E2 and bisphenol A (Bp-A), a structural analogue of DES, caused mitotic arrest and aberrant spindles, such as tripolar and multipolar spindles, in a concentration-dependent manner. Gamma-tubulin, a well-characterized component of microtubule-organizing centers (MTOCs), was co-localized with the aberrant spindles induced by estrogens and Bp-A. The number of gamma-tubulin signals in the mitotic cells coincided with that of the aberrant spindles and rose with an increasing concentration of the chemicals. The incidence and location of gamma-tubulin in interphase cells were not influenced by the chemicals. These results suggest that multiple MT nucleating sites were induced by the estrogens and Bp-A during the transition from interphase to the mitotic phase. DES, E2 and Bp-A induced multipolar division in a concentration-dependent process associated with the induction of aneuploidy.     

Role of mitotic motors, dynein and kinesin, in the induction of abnormal centrosome integrity and multipolar spindles in cultured V79 cells exposed to dimethylarsinic acid.

The role of microtubule-based motors in the induction of abnormal centrosome integrity by dimethylarsinic acid (DMAA) was investigated with the use of monastrol, a specific inhibitor of mitotic kinesin, and vanadate, an inhibitor of dynein ATPase. Cytoplasmic dynein co-localized with multiple foci of gamma-tubulin in mitotic cells arrested by DMAA. Disruption of microtubules caused dispersion of dynein while multiple foci of gamma-tubulin were coalesced to a single dot. Vanadate also caused dispersion of dynein, which had been co-localized with multiple foci of gamma-tubulin by DMAA, without affecting spindle organization. However, the dispersion of dynein did not prohibit the induction of abnormal centrosome integrity by DMAA. Inhibition of mitotic kinesin by monastrol resulted in monoastral cells with non-migrated centrosomes in the cell center. Monastrol, when applied to mitotic cells with abnormal centrosome integrity, rapidly reduced the incidence of cells with the centrosome abnormality. Moreover, monastrol completely inhibited reorganization of abnormal centrosomes that had been coalesced to a single dot by microtubule disruption. These results suggest that abnormal centrosome integrity caused by DMAA is not simply due to dispersion of fragments of microtubule-organizing centers, but is dependent on the action of kinesin. In addition, the results suggest that kinesin plays a role not only in the induction of mitotic centrosome abnormality, but also in maintenance.     

Comparison of HepG2 feeder cells generated by exposure to gamma-rays, X-rays, UV-C light or mitomycin C for ability to activate 7,12-dimethylbenz[a]anthracene in a cell-mediated Chinese hamster V79/HGPRT mutation assay

The cell-mediated Chinese hamster V79/HGPRT mutagenicity assay is an established in vitro testing method. Although gamma-irradiated human HepG2 hepatoma cells have been used recently for chemical activation, an alternative is now needed due to scheduled retirement of the available gamma-source. X-irradiation, 254 nm UV-C light and mitomycin C were examined as possible HepG2 mitotic inhibitors, and treated cells compared for activation of 7, 12-dimethylbenz[a]anthracene (DMBA). In colony-forming assays, V79 and HepG2 cells differed in sensitivity to DMBA, with V79 survival declining sharply between 1-2.5 microM (LD50=1.75 microM) while HepG2 survival decreased gradually, beginning at 0.01 microM DMBA (LD50=0.045 microM). When HepG2 feeder cells generated by each method were included in V79/HGPRT mutation assays, activation of 1 microM DMBA was found to vary according to the mitotic inhibitor used, with mutation frequencies decreasing in the order 4000 rads gamma-rays>25 microg/ml mitomycin C>4000 rads X-rays>25 J/m2 UV-C light. Only assays containing gamma-irradiated HepG2 cells generated an increase (2-3-fold) in mutation frequency when DMBA exposure was extended from 24 to 48 h. The effect of HepG2 preincubation with either Aroclor 1254 or DMBA on feeder cell activation of DMBA was also assessed using concentrations of Aroclor 1254 (10 microg/ml) or DMBA (1.0 microM) which were found to produce optimum induction of ethoxyresorufin-O-deethylase (EROD) activity (3.1-fold and 2-fold increases, respectively). Compared to results obtained with uninduced HepG2 cells, assays incorporating HepG2 cells activated by either Aroclor 1254 or DMBA produced slightly increased V79/HGPRT mutation frequencies after 24 h of exposure to mutagen; however, a 48 h incubation with mutagen in the presence of HepG2 preincubated with either Aroclor 1254 or DMBA resulted in higher mutation frequencies regardless of the mitotic inhibitor treatment. EROD activity was also induced 1.4-fold following exposure of HepG2 cells to mitomycin C alone. Although gamma-irradiation remains the treatment of choice for producing metabolically active HepG2 feeder cells, comparison of the alternatives tested suggests that mitomycin C would be a convenient and suitable replacement.     

Spindle disturbances in mammalian cells. I. changes in the quantity of free sulfhydryl groups in relation to survival and c-mitosis in V79 chinese hamster cells after treatment with colcemid,diamide, carbaryl and methyl mercury

Asynchronously growing V79 Chinese hamster cells were treated with colcemid, diamide, carbaryl and methyl mercury, which are all known to be spindle disturbing agents. For each compound the dose response for c-mitosis, survival and level of free sulfhydryl groups was investigated under comparable conditions. Diamide, carbaryl and methyl were all found to give a significant increase of c-mitosis at a dose giving a decrease of non-protein sulfhydryl groups (NPSH, mainly glutathione) of 30–40% suggesting that a decrease of this magnitude may have a predictive value for spindle disturbances. Despite this similarity at concentrations close to the respective thresholds it was found that the c-mitotic activity at higher concentrations was not a simple function of average NPSH decrease. Diamide, which rapidly oxidizes glutathione to glutathione disulfide, was a less efficient c-mitotic agent than carbaryl and methyl mercury in relation to average NPSH decrease at higher concentrations. Protein bound sulfhydryl groups (PSH) were not significantly affected with diamide and carbaryl at their lowest c-mitotic concentrations while methyl mercury caused a significant decrease already at concentrations below the lowest c-mitotic concentration. With colcemid a significant decrease of average NPSH (14%) and PSH (12%) was observed only with concentrations giving close to 100% c-mitotic cells. Concentrations giving more than 20% c-mitosis gave a pronounced decrease of survival with carbaryl, diamide and methyl mercury while no toxic effects were obtained with colcemid, not even with concentrations giving close to 100% c-mitosis. Carbaryl, diamide and methyl mercury caused increased glutathione peroxidase activity indicating that these compounds cause increased lipid peroxidation. The possible connection between peroxidative damage of membranes and c-mitosis is discussed.