Dead or dying: the importance of time in cytotoxicity assays using arsenite as an example

Komissarova, Elena V.; Saha, Sam K.; Rossman, Toby G.

doi:10.1016/j.taap.2004.06.010

Cited by 52 publications

(41 citation statements)

References 48 publications

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“…Death was also associated with translocation of AIF, and suppression of AIF translocation by inhibition of p38 and CaMK-II, and by CsA and NAC, suggest this is a downstream event in Cd 2þ -induced death. The MTT assay was reported to show a delayed response in toxicity compared to a clonal survival assay after exposure to arsenite (Komissarova et al, 2005), indicating that it may be less sensitive for acute toxicity. It is surprising, therefore, that the MTT assay indicates lower viability of cells in serum-replete media than does flow cytometry analysis (e.g., Fig.…”

Section: Discussionmentioning

confidence: 99%

Initiation of caspase‐independent death in mouse mesangial cells by Cd²⁺: Involvement of p38 kinase and CaMK‐II

Liu

Templeton

2008

Journal Cellular Physiology

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Cadmium (Cd) is a toxic metal with multiple effects on cell signaling and cell death. We studied the effects of Cd(2+) on quiescent mouse mesangial cells in serum-free conditions. Cadmium induces cell death over 6 h through annexin V+ states without or with causing uptake of propidium iodide, termed apoptotic and apoptosis-like death, respectively. Little or no necrosis is observed, and cell death is caspase-independent and associated with nuclear translocation of the apoptosis-inducing factor, AIF. We previously showed that Cd(2+) increased phosphorylation of Erk and CaMK-II, and CaMK-II activation increased cell death in an Erk-independent manner. Here we demonstrate that Cd(2+) increases Jnk and p38 kinase phosphorylation, and inhibition of p38-but not of Jnk-increases cell viability by suppressing apoptosis in preference to apoptosis-like death. Neither p38 kinase nor CaMK-II inhibition protects against a decrease in mitochondrial membrane potential, psi, indicating that kinase-mediated death is either independent of, or involves events downstream of a mitochondrial pathway. However, both the antioxidant N-acetyl cysteine (NAC) and the mitochondrial membrane-stabilizing agent cyclosporine A (CsA) partially preserve psi, suppress activation of p38 kinase, and partially protect the cells from Cd(2+)-induced death. Whereas the effect of CsA is on apoptosis, NAC acts on apoptosis-like death. Inhibition of glutathione synthesis exacerbates a Cd(2+)-dependent increase in cellular peroxides and favors apoptosis-like death over apoptosis. The caspase-independence of these modes of cell death is not due to an absence of this machinery in the mesangial cells: when they are exposed to Cd(2+) for longer periods in the presence of serum, procaspase-3 and PARP are cleaved and caspase inhibition is protective. We conclude that Cd(2+) can kill mesangial cells by multiple pathways, including caspase-dependent and -independent apoptotic and apoptosis-like death. Necrosis is not prominent. Activation of p38 kinase and of CaMK-II by Cd(2+) are associated with caspase-independent apoptosis that is not dependent on mitochondrial destabilization.

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Section: Discussionmentioning

confidence: 99%

Initiation of caspase‐independent death in mouse mesangial cells by Cd²⁺: Involvement of p38 kinase and CaMK‐II

Liu

Templeton

2008

Journal Cellular Physiology

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“…In vitro Recovery (hrs) *While trypan blue exclusion assay is widely used and often recommended for viability checks before the Comet assay, it is a less rigorous estimation of cell survival than the clonal survival assay [Komissarova et al, 2005]. Therefore it is possible that the treatment [CP, H 2 O 2 , formaldehyde, or DMA(V)] could have caused a lower viability when measured in more sensitive assays, such as the clonal survival assay, than in the trypan blue exclusion assay.…”

Section: In Vitro Dosingmentioning

confidence: 99%

Arsenate and dimethylarsinic acid in drinking water did not affect DNA damage repair in urinary bladder transitional cells or micronuclei in bone marrow

Wang

Kligerman

Holladay

et al. 2009

Environ and Mol Mutagen

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Arsenic is a human skin, lung, and urinary bladder carcinogen, and may act as a cocarcinogen in the skin and urinary bladder. Possible modes of action of arsenic carcinogenesis/cocarcinogenesis include oxidative stress induction and inhibition of DNA damage repair. We investigated the effects of arsenic in drinking water on DNA damage repair in urinary bladder transitional cells and on micronucleus formation in bone marrow. F344 rats were given 100 ppm arsenate [As(V)] or dimethylarsinic acid [DMA(V)] in drinking water for 1 week. The in vivo repair of cyclophosphamide (CP)-induced DNA damage resulting from a single oral gavage of CP, and the in vitro repair of hydrogen peroxide (H(2)O(2))- or formaldehyde-induced DNA damage, resulting from adding H(2)O(2) or formaldehyde into cell medium, were measured by the Comet assay. DMA(V) effects were not observed on either CP-induced DNA damage induction or on DNA repair. Neither DMA(V) nor As(V) increased the H(2)O(2)- or formaldehyde-induced DNA damage, and neither inhibited the repair of H(2)O(2)-induced DNA damage. Neither DMA(V) nor As(V) increased the micronucleus frequency, nor did they elevate micronucleus frequency resulting from CP treatment above the level observed by the treatment with CP alone. These results suggest that arsenic carcinogenesis/cocarcinogenesis in the urinary bladder may not be via DNA damage repair inhibition. To our knowledge this is the first report of arsenic effects on DNA damage repair in the urinary bladder.

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“…We have previously shown that, because of delayed apoptosis, clonal survival assays are the most accurate method of assessing cytotoxicity induced by arsenite, followed by MTT or neutral red assays performed 72 hours after exposure (Komissarova et al, 2005). Because lymphoblasts grow in suspension, cytotoxicity cannot easily be measured by these methods.…”

Section: Short-term Cytotoxicity Measurementsmentioning

confidence: 99%

“…Because lymphoblasts grow in suspension, cytotoxicity cannot easily be measured by these methods. For convenience, and because we are interested in comparative rather than actual cytotoxicity, we have chosen to assess cytotoxicity here by counting cells unstained with trypan blue after a 24-h exposure to arsenite, knowing that this is a measure of necrosis and does not detect cells undergoing apoptosis (Komissarova et al, 2005).…”

Section: Short-term Cytotoxicity Measurementsmentioning

confidence: 99%

Gene expression levels in normal human lymphoblasts with variable sensitivities to arsenite: Identification of GGT1 and NFKBIE expression levels as possible biomarkers of susceptibility

Komissarova

Uddin

et al. 2008

Toxicology and Applied Pharmacology

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Drinking arsenic-contaminated water is associated with increased risk of neoplasias of the skin, lung, bladder and possibly other sites, as well as other diseases. Earlier, we showed that human lymphoblast lines from different normal unexposed donors showed variable sensitivities to the toxic effects of arsenite. In the present study, we used microarray analysis to compare the basal gene expression profiles between two arsenite-resistant (GM02707, GM00893) and two arsenite-sensitive lymphoblast lines (GM00546, GMO00607). A number of genes were differentially expressed in arsenite-sensitive and arsenite-resistant cells. Among these, γ-glutamyltranspeptidase 1 (GGT1) and NFB inhibitor-epsilon (NFKBIE) showed higher expression levels in arsenite-resistant cells. RT-PCR analysis with gene-specific primers confirmed these results. Reduction of GGT1 expression level in arsenite resistant lymphoblasts with GGT1-specific siRNA resulted in increased cell sensitivity to arsenite. In conclusion, we have demonstrated for the first time that expression levels of GGT1 and possibly NFKBIE might be useful as biomarkers of genetic susceptibility to arsenite. Expression microarrays can thus be exploited for identifying additional biomarkers of susceptibility to arsenite and to other toxicants.

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Dead or dying: the importance of time in cytotoxicity assays using arsenite as an example

Cited by 52 publications

References 48 publications

Initiation of caspase‐independent death in mouse mesangial cells by Cd²⁺: Involvement of p38 kinase and CaMK‐II

Initiation of caspase‐independent death in mouse mesangial cells by Cd²⁺: Involvement of p38 kinase and CaMK‐II

Arsenate and dimethylarsinic acid in drinking water did not affect DNA damage repair in urinary bladder transitional cells or micronuclei in bone marrow

Gene expression levels in normal human lymphoblasts with variable sensitivities to arsenite: Identification of GGT1 and NFKBIE expression levels as possible biomarkers of susceptibility

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Dead or dying: the importance of time in cytotoxicity assays using arsenite as an example

Cited by 52 publications

References 48 publications

Initiation of caspase‐independent death in mouse mesangial cells by Cd2+: Involvement of p38 kinase and CaMK‐II

Initiation of caspase‐independent death in mouse mesangial cells by Cd2+: Involvement of p38 kinase and CaMK‐II

Arsenate and dimethylarsinic acid in drinking water did not affect DNA damage repair in urinary bladder transitional cells or micronuclei in bone marrow

Gene expression levels in normal human lymphoblasts with variable sensitivities to arsenite: Identification of GGT1 and NFKBIE expression levels as possible biomarkers of susceptibility

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Initiation of caspase‐independent death in mouse mesangial cells by Cd²⁺: Involvement of p38 kinase and CaMK‐II

Initiation of caspase‐independent death in mouse mesangial cells by Cd²⁺: Involvement of p38 kinase and CaMK‐II