The occurrence of a large number of diverse arsenic species in the environment and in biological systems makes it important to compare their relative toxicity. The toxicity of arsenic species has been examined in vari...The occurrence of a large number of diverse arsenic species in the environment and in biological systems makes it important to compare their relative toxicity. The toxicity of arsenic species has been examined in various cell lines using different assays, making comparison difficult. We report real-time cell sensing of two human cell lines to examine the cytotoxicity of fourteen arsenic species: arsenite(As~Ⅲ), monomethylarsonous acid(MMA~Ⅲ) originating from the oxide and iodide forms, dimethylarsinous acid(DMA~Ⅲ), dimethylarsinic glutathione(DMAG~Ⅲ), phenylarsine oxide(PAO~Ⅲ), arsenate(AsV), monomethylarsonic acid(MMA~Ⅴ), dimethylarsinic acid(DMA~Ⅴ),monomethyltrithioarsonate(MMTTA~Ⅴ), dimethylmonothioarsinate(DMMTA~Ⅴ),dimethyldithioarsinate(DMDTA~Ⅴ), 3-nitro-4-hydroxyphenylarsonic acid(Roxarsone, Rox),and 4-aminobenzenearsenic acid(p-arsanilic acid, p-ASA). Cellular responses were measured in real time for 72 hr in human lung(A549) and bladder(T24) cells. IC50 values for the arsenicals were determined continuously over the exposure time, giving rise to IC50 histograms and unique cell response profiles. Arsenic accumulation and speciation were analyzed using inductively coupled plasma-mass spectrometry(ICP-MS). On the basis of the 24-hr IC50 values, the relative cytotoxicity of the tested arsenicals was in the following decreasing order: PAO~Ⅲ? MMA~Ⅲ≥ DMA~Ⅲ≥ DMAG~Ⅲ≈ DMMTA~Ⅴ≥ As~Ⅲ? MMTTA~Ⅴ〉 AsV〉 DMDTA~Ⅴ〉DMA~Ⅴ〉 MMA~Ⅴ≥ Rox ≥ p-ASA. Stepwise shapes of cell response profiles for DMA~Ⅲ, DMAG~Ⅲ,and DMMTA~Ⅴcoincided with the conversion of these arsenicals to the less toxic pentavalent DMA~Ⅴ. Dynamic monitoring of real-time cellular responses to fourteen arsenicals provided useful information for comparison of their relative cytotoxicity.展开更多
The IκB kinases IKKα and IKKβ and the IKK-related kinases TANK-binding kinase 1 (TBK1) and IKKε are the master regulators of the NF-κB signaling pathway. Although this pathway has been extensively studied in ma...The IκB kinases IKKα and IKKβ and the IKK-related kinases TANK-binding kinase 1 (TBK1) and IKKε are the master regulators of the NF-κB signaling pathway. Although this pathway has been extensively studied in mammals, less attention has been paid in crustaceans, which have significant economic value. Here, we report the cloning and functional studies of two IKK homologs, LvlKKβ and LvlKKε, from Pacific white shrimp, Litopenaeus vannamei. LvlKKβ and LvlKKα mRNAs are widely expressed in different tissues and are responsive to white spot syndrome virus (WSSV) infection. When overexpressed in Drosophila S2 cells, LvlKKβ but not LvlKKε activates the promoters of NF-κB pathway-controlled antimicrobial peptide genes (AMPs), such as the Penaeidins (PENs). In HEK 293T cells, both LvlKKβ and LvlKKε activate an NF-κB reporter. The silencing of LvlKKβ or LvlKKε using double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) decreases the expression of L. vannamei AMPs, including PENs, lysozyme and crustins. Intriguingly, LvlKKβ- or LvlKKε-silenced L. vannameiare resistant to WSSV infection. We hypothesized that successful infection with WSSV requires the activation of the IKK-NF-κB signaling pathway to modulate viral gene expression. We constructed luciferase reporters for 147 WSSV genes. By screening, we found that the WSSV051, WSSV059, WSSV069, WSSV083, WSSV090, WSSV107, WSSV244, WSSV303, WSSV371 and WSSV445 promoters can be activated by LvlKKβ or LvlKKε in Drosophila S2 cells. Taken together, our results reveal that LvlKKβ and LvlKKε may participate in the regulation of shrimp AMPs and that WSSV may subvert the L. vannamei IKK-NF-κB signaling pathway to facilitate viral gene expression.展开更多
基金supported by Alberta Health, Alberta Innovates, the Canada Research Chairs Programthe Canadian Institutes of Health Research (CIHR)the Natural Sciences and Engineering Research Council (NSERC) of Canada
文摘The occurrence of a large number of diverse arsenic species in the environment and in biological systems makes it important to compare their relative toxicity. The toxicity of arsenic species has been examined in various cell lines using different assays, making comparison difficult. We report real-time cell sensing of two human cell lines to examine the cytotoxicity of fourteen arsenic species: arsenite(As~Ⅲ), monomethylarsonous acid(MMA~Ⅲ) originating from the oxide and iodide forms, dimethylarsinous acid(DMA~Ⅲ), dimethylarsinic glutathione(DMAG~Ⅲ), phenylarsine oxide(PAO~Ⅲ), arsenate(AsV), monomethylarsonic acid(MMA~Ⅴ), dimethylarsinic acid(DMA~Ⅴ),monomethyltrithioarsonate(MMTTA~Ⅴ), dimethylmonothioarsinate(DMMTA~Ⅴ),dimethyldithioarsinate(DMDTA~Ⅴ), 3-nitro-4-hydroxyphenylarsonic acid(Roxarsone, Rox),and 4-aminobenzenearsenic acid(p-arsanilic acid, p-ASA). Cellular responses were measured in real time for 72 hr in human lung(A549) and bladder(T24) cells. IC50 values for the arsenicals were determined continuously over the exposure time, giving rise to IC50 histograms and unique cell response profiles. Arsenic accumulation and speciation were analyzed using inductively coupled plasma-mass spectrometry(ICP-MS). On the basis of the 24-hr IC50 values, the relative cytotoxicity of the tested arsenicals was in the following decreasing order: PAO~Ⅲ? MMA~Ⅲ≥ DMA~Ⅲ≥ DMAG~Ⅲ≈ DMMTA~Ⅴ≥ As~Ⅲ? MMTTA~Ⅴ〉 AsV〉 DMDTA~Ⅴ〉DMA~Ⅴ〉 MMA~Ⅴ≥ Rox ≥ p-ASA. Stepwise shapes of cell response profiles for DMA~Ⅲ, DMAG~Ⅲ,and DMMTA~Ⅴcoincided with the conversion of these arsenicals to the less toxic pentavalent DMA~Ⅴ. Dynamic monitoring of real-time cellular responses to fourteen arsenicals provided useful information for comparison of their relative cytotoxicity.
文摘The IκB kinases IKKα and IKKβ and the IKK-related kinases TANK-binding kinase 1 (TBK1) and IKKε are the master regulators of the NF-κB signaling pathway. Although this pathway has been extensively studied in mammals, less attention has been paid in crustaceans, which have significant economic value. Here, we report the cloning and functional studies of two IKK homologs, LvlKKβ and LvlKKε, from Pacific white shrimp, Litopenaeus vannamei. LvlKKβ and LvlKKα mRNAs are widely expressed in different tissues and are responsive to white spot syndrome virus (WSSV) infection. When overexpressed in Drosophila S2 cells, LvlKKβ but not LvlKKε activates the promoters of NF-κB pathway-controlled antimicrobial peptide genes (AMPs), such as the Penaeidins (PENs). In HEK 293T cells, both LvlKKβ and LvlKKε activate an NF-κB reporter. The silencing of LvlKKβ or LvlKKε using double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) decreases the expression of L. vannamei AMPs, including PENs, lysozyme and crustins. Intriguingly, LvlKKβ- or LvlKKε-silenced L. vannameiare resistant to WSSV infection. We hypothesized that successful infection with WSSV requires the activation of the IKK-NF-κB signaling pathway to modulate viral gene expression. We constructed luciferase reporters for 147 WSSV genes. By screening, we found that the WSSV051, WSSV059, WSSV069, WSSV083, WSSV090, WSSV107, WSSV244, WSSV303, WSSV371 and WSSV445 promoters can be activated by LvlKKβ or LvlKKε in Drosophila S2 cells. Taken together, our results reveal that LvlKKβ and LvlKKε may participate in the regulation of shrimp AMPs and that WSSV may subvert the L. vannamei IKK-NF-κB signaling pathway to facilitate viral gene expression.