Early induction of genetic instability and apoptosis by arsenic in cultured Chinese hamster cells

Abstract

In order to assess at what time from the beginning of exposure inorganic arsenic can give rise to genetic instability and trigger apoptosis, V79-C13 Chinese hamster cells were treated with 10 microM sodium arsenite for 24 h. Under these conditions, cell survival was >70% and cells showed neither an increase in chromosome aberration frequency nor a delay in cell cycle progression. Investigations, which were carried out every 6 h during the treatment, revealed an early appearance of genetically unstable cells, namely micronucleated, multinucleated and mononucleated 'giant' cells, as well as apoptotic cells. Indirect immunostaining using anti-beta-tubulin antibody showed severe alterations in spindle morphology after only 6 h treatment, when cells with small spindles whose poles were inside the metaphase plate appeared, and after 12 h treatment, when cells in which spindle assembly had completely failed were observed. These cells, unable to complete mitosis, underwent apoptosis. In fact, cells which turned out to be positive in the TdT-FragEL test had condensed chromatin arranged in metaphase-like plates; their maximum frequency was reached after 24 h treatment. A cytogenetic study was conducted at the end of the period of exposure to arsenic and after post-treatment incubation in fresh medium for up to 5 days. It showed that the percentage of cells with 21 chromosomes (modal number of the cell line) decreased, making way for aneuploid cells. Arsenic, therefore, induced early genetic instability or apoptosis in dividing cells. However, while apoptosis tended to cease when arsenic was removed from the culture medium, the acquired instability remained and propagated within the cell population.

Further evidence against a direct genotoxic mode of action for arsenic-induced cancer

“…The gold standard for assessing cytotoxicity should therefore be clonal survival. Most human cells have an ID 50 of 1-2 μM for a 24 h exposure to arsenite (Table 2) and Chinese hamster cells are about 8 to 10-fold more resistant (Table 1), consistent with other reports (Sciandrello, et al, 2002). Our data supports previous reports that the trivalent methylated metabolites are even more toxic than arsenite (Styblo et al 2000;Petrick et al, 2000).…”

Methylated trivalent arsenicals as candidate ultimate genotoxic forms of arsenic: Induction of chromosomal mutations but not gene mutations

“…This inference is supported by a previous study showing that DMA V induced aneuploidy in mouse bone marrow [Kashiwada et al, 1998] and disrupted spindles in V79 cells [Ochi et al, 1999;Ochi, 2000, Ochi, 2002. Our results identified DMA III and DMA V as the two metabolites of arsenic that are most likely responsible for the induction of aneuploidy by inorganic arsenic observed in other studies [Eastmond and Tucker, 1989;Vega et al, 1995;Ramirez et al, 1997;Sciandrello et al, 2002]. The mechanisms by which the dimethylated arsenicals may induce aneuploidy are under further investigation in our laboratory.…”

Relative sensitivity of fish and mammalian cells to sodium arsenate and arsenite as determined by alkaline single‐cell gel electrophoresis and cytokinesis‐block micronucleus assay

“…Micronuclei originate from chromosomal (or chromatid) fragments in addition to whole chromosomes containing abnormal centromeres that result in the failure of spindle fiber attachment. Given that arsenicals may interfere with microtubule formation [Sciandrello et al, 2002;Ramirez et al, 2003], it is likely that both the clastogenic and aneugenic potential of the arsenicals may have contributed to the observed MN frequencies in the two cell lines. In the absence of anti-kinetochore staining, however, it is difficult to establish the relative aneugenic potential of the arsenicals in these cell types.…”