TABLE 8.
In vitro genotoxicity tests with arsenic species.
| Reference | Test system | Cells | Concentration/treatment time | Results | Additional information |
|---|---|---|---|---|---|
| Ding et al. (2009) |
8‐oxodG measurement by HPLC‐ED and in situ immune fluorescence; PARP‐1 activity by in situ immune fluorescence |
Human keratinocyte cell line (HaCaT) |
Sodium arsenite 2 μM (24 h) |
Enhancement of UVR‐induced 8‐oxodG and suppression of UVR‐induced PARP‐1 activation (lowest dose tested 0.2 μM) |
No data on cytotoxicity. Exposure to 1–3 μM arsenite: no increase of 8‐oxodG Inhibition of PARP‐1 activity significantly increases UVR‐induced 8‐OHdG formation Mass spectrometry analysis reveals that arsenite can occupy the first zinc finger of PARP‐1 (PARPzf) |
| Nollen et al. (2009) | Western blotting and gene expression analysis of cell extracts | Tert‐immortalised human diploid skin fibroblasts (VH10hTert) |
Sodium arsenite 1, 5, 10 μM (24 h) MMA(III) 0.1, 1, 1.3 μM (24 h) |
Inhibition of XPC expression and protein levels for both arsenicals. Diminished association of XPC to sites of local UVC damage Viability > 80% |
Cell viability: cell number and colony forming ability Inhibition of XPE expression |
| Ying et al. (2009) |
γ‐H2AX foci Pulse field gel electrophoresis (PFGE) |
Chinese hamster cells (AA8) | Sodium arsenite from 5 to 50 μM (24 h) |
Positive: concentrations as low as 5 μM could induce gH2AX foci (IC50 = 18.6 μM) |
Cell viability: clonogenic assay 50 μM arsenite produces a similar amount of DSBs as a 6 Gy dose of ionising radiation as estimated by PFGE. The occurrence of homologous recombination (HR) confirmed by Rad 51 foci and cell recombination assay HR defective cells are hypersensitive to arsenite Arsenite inhibits SSB repair |
| Catanzaro et al. (2010) | Alkaline comet assay | Primary cultures of rat astrocytes |
Sodium arsenite 2.5, 5, 10, 30 μM (24 h) |
Positive: significant increase of DNA breaks (tail length) at 10 μM for 24 h (> 70% viability) |
Cell viability: trypan blue exclusion test Cells treated with 30 μM for 24 h were all ghost (small heads and large tails), likely apoptotic cells Absence of ROS production by CM‐H2DCFDA |
| Dopp et al. (2010) | Alkaline comet assay | Non‐methylating cells: UROtsa transformed human urothelial cells; methylating cells: primary human hepatocytes | As(III), MMA(III), iAs(V), MMA(V) for 1 h; range of doses tested not reported |
Positive: significant induction of DNA breaks (tail moment): primary human hepatocytes at 5 μM MMA(III) (LC50 = 20μM) and 50 μM iAs(III) (not cytotoxic up to 5 mM); UROtsa cells at 5 μM MMA(III) (LC50 = 83μM) and 5 μM iAs(III) (LC50 = 5000 μM) for 1 h Negative: in human hepatocytes iAs(V) and MMA(V) did not induce DNA breaks at doses up to 500 μM; they were not tested in urothelial cells |
Cell viability: trypan blue exclusion test Induction of free radicals, measured by the thiobarbituric acid test, showed cell‐type specific differences |
| Kligerman et al. (2010) | Chromosome aberration assay | Freshly prepared splenic lymphocyte cultures | MMA(III), 0.3 and 0.5 μM (12–17 h) | Positive: significant increase of CA at both concentrations. MMA(III) (0.5 μM) positive when cells were treated in late G1‐ or S‐phase; negative when treatment was confined to the G0‐ or G1‐phase of the cell cycle |
Replication index indicates that both concentrations are cytotoxic DNA lesions produced by MMA(III)I that can lead to cytogenetic damage are short‐lived |
| Bartel et al. (2011) |
MN assay Alkaline unwinding |
Human A549 lung cells |
Arsenite, MMA(III), DMA(III), MMA(V), DMA(V), thio‐DMA(V) (1, 24 h) |
Positive: significant increase of MN frequency at non‐ cytotoxic/slightly cytotoxic concentrations for 24 h (< IC70): MMA(III) (0.5, 1 μM), thio‐DMA(V) (5 μM), MMA(V) (250 μM) and DMA(V) (250 μM) At cytotoxic concentrations significant increases also for arsenite (≥ 50 μM for 24 h) (≥ IC70) Negative: thio‐DMA(V) did not induce DNA strand breaks up to high cytotoxic doses (100 μM for 1 h, 75 μM for 24 h) |
Cell viability: cell number and colony forming ability. Based on the effective cellular arsenic concentrations, the cytotoxic order was: thio‐DMA(V) ∼ arsenite ∼ MMA(III) > DMA(III) MMA(V) ∼ DMA(V) Thio‐DMA(V) and especially DMA(III) increased the formation of multinucleated cells (significant effects at cytotoxic concentrations, 15 μM, IC70 = 7.2 μM) |
| Ebert et al. (2011) | DNA repair enzymes cleavage assay, gene expression and western blotting of cell extracts | A549 human epithelial lung adenocarcinoma cells |
Arsenite: up to 100 μM DMA(III): up to 7.5 μM MMA(III): up to 7.5 μM MMA(V): up to 500 μM DMA(V): up to 500 μM 24 h exposure followed by preparation of cell extracts |
Specific inhibitory effects on OGG1 activity by DMA(V), on LigIII by arsenite, on XRCC1 by MMA(V) (starting at ≥ 3.2 μM cellular arsenic; cytotoxicity < 30%); the trivalent methylated metabolites effective only at cytotoxic concentrations |
Cytotoxicity, cell number and colony forming ability Cytotoxic effects correlate with cellular uptake in the decreasing order DMA(III), MMA(III), arsenite, MMA(V), DMA(V) |
| Lai et al. (2011) |
Alkaline comet assay Micronucleus assay |
Mouse embryonic fibroblasts (MEF) Pol β+/+ and Pol β −/− | Sodium arsenite: 2.5, 5, 10, μM (24 h) |
Positive: accumulation of DNA breaks (comet rate%) at 2.5, 5 and 10 μM in both cell lines, more pronounced in Pol β −/−cells Positive: significantly increased number of micronuclei at 2,5, 5 and 10 μM in both cell lines, more pronounced in Pol β−/− cells Viability > 80% Pol β +/+IC50 = 58.3 μM Pol β−/‐IC50 = 46.6 μM |
Cell viability: MTT assay Comet rate (%) = (total number of cells with tails/total number of counted cells) x 100. Slower DNA repair kinetics in Pol β−/− cells |
| Naranmandura, Xu, et al. (2011) | Intracellular ROS generation measured by oxidation‐sensitive fluorescent probe (DCFH‐DA) | At liver RLC‐16 cells | MMA(III) (IC50 = 1 μM), DMA(III) (IC50 = 2 μM), iAsIII (IC50 = 18 μM) (24 h) | MMA(III), ROS generated primarily in mitochondria; DMA(III), ROS generation in other organelles; iAS(III), no ROS generation |
Cytotoxicity: MMA(III) (IC50 = 1μM) > DMA(III) (IC50 = 2μM) > iAs(III) (IC50 = 18 μM) Mitochondria are the primary target for MMA(III)‐induced cytotoxicity |
| Naranmandura, Carew, et al. (2011) | Alkaline comet assay | Human bladder cancer cells (EJ‐1) | iAsIII (75 μM), DMA(III) (12 μM), Thio‐DMA(V) (here named DMMTA(V)) (17 μM) (3–6‐24 h). All tested at IC50 | Positive: iAs(III), significant increase of DNA breaks (tail length) after 6 and 24 h exposure; DMA(III) and thio‐DMA(V) after 3,6 and 24 h exposure; all tested at IC50 (MTS assay) |
Concomitant measurement of ROS levels: iAs(III), increase only after 24 h exposure; DMA(III) and thio‐DMA(V) after 3, 6, 24 h exposure iAs(III), increased GSH levels; DMA(III) and thio‐DMA(V), reduction of GSH(5μM for 24 h). iAs(III) increased expression of p21 and p53; DMA(III) and thio‐DMA(V) decreased expression of p21 and p53 (dose dependent) |
| Singh et al. (2011) |
Alkaline comet assay Mitochondrial DNA mutation assay by multiplex PCR |
Immortalised normal prostate epithelial cells (RWPE‐1) | Sodium arsenite: 100 pg/mL (90 days) |
Positive: increased DNA breaks (tail length) as compared to control cells (no tails) No major deletions or insertions in the sequence of mitochondrial DNA; insertion mutation (a ‘G’) in the mitochondrial ATPase gene |
Cell viability: cell count Chronic exposure to 100 pg/mL: Increase in cell proliferation (cells become resistant to As) |
| Jiang et al. (2013) |
Alkaline comet assay Micronucleus assay |
Human lung adenocarcinoma cells (A549) | Sodium arsenite: 5, 10, 15, 20, 25 μM (24 h) |
Positive: significant increase of DNA breaks (tail moment) in the full concentration range. (cell viability > 70% up to 15μM) Positive: significant (p < 0.05) increase of MN frequency from 10 μM (cell viability > 70%) |
Cell viability measured by the MTT assay and Trypan blue exclusion. Induction of cell cycle arrest at G2/M phase and apoptosis. |
| Leffers et al. (2013) | Micronucleus assay | Immortalised human urothelial cells (UROtsa) |
Sodium arsenite: 0.1, 0.5, 1, 2, 3.5 μM (48 h) DMA(V): 50, 100, 170, 200 μM (48 h) Thio‐DMA(V): 0.1, 0.5, 1, 2 μM (48 h) |
Positive: Arsenite: significant increase (p < 0.001) of induced MN from 0.5 to 3.5 μM (including a subtoxic concentration range) DMA(V) and thio‐DMA(V): significant increase (p < 0.001) of bi‐ and multi‐nucleated cells from 100 to 200 μM (DMA(V)) and at 2 μM (thio‐DMA(V)), at incipient cytotoxic concentrations. 30% reduction of CFA: Arsenite 2 μM; DMA(V) 150 μM; thio‐DMA(V) 1.5 μM |
Cell viability: cell number and colony forming ability. Arsenite: No increase in the number of bi‐ or multinucleated cells. |
| Tokar et al. (2013) |
Cell transformation (agar assay and invasion assay) ROS generation by the immuno‐spin trapping analysis of protein radicals (immunochemical quantification) |
Arsenic methylation‐proficient TRL1215 liver cell line and methylation‐deficient RWPE1 prostate cell line | MMA(III): 0.25–1.0 μM (20 weeks or more) | Positive: increased matrix metal‐ loproteinase secretion, colony formation and invasion at 18–22 weeks in both cell lines |
Similar alterations in arsenic and oxidative stress adaptation factors in both cell lines MMA(III) and iAs cause an acquired malignant phenotype in methylation‐deficient cells, yet iAs does not induce oxidative DNA damage |
| Bach et al. (2014) | FPG‐modified comet assay | Mouse embryo fibroblasts (MEF) Ogg1 +/+ and Ogg1 −/− |
Sodium arsenite: Short‐term studies: up to 20 μM for 24 or 48 h; Long‐term studies: 0.5, 1 and 2 μM for up to 17 weeks |
Positive: significant levels of oxidative DNA lesions (%DNA in tail) in Ogg1 −/− cells at subtoxic concentrations (> 80% survival) in both short‐term (5 μM for 1.5, 3, 24 h) and long‐term (0.5 μM for 4, 10, 17 weeks) studies; in Ogg1 wt at 20 μM for 24 h and 1 and 2 μM for 17 weeks |
Cell viability: Beckman counter method (up to 80 μM for 24 or 48 h) Analysis of arsenic metabolism in the cells; similar in MEF wt and Ogg1 −/− |
| Ebert et al. (2014) | Alkaline unwinding with FPG | Immortalised human urothelial cells (UROtsa) | Sodium arsenite and thio‐DMA(V): up to 5 μM (1, 24 and 48 h) |
Negative: arsenite and thio‐DMA(V) significant increase of DNA breaks only at cytotoxic concentration (5 μM) Arsenite and thio‐DMA(V): CFA, IC70 = 5.2 μM |
Cell viability: cell number and colony forming ability Inhibition of damage‐induced poly (ADP)‐ribosylation: arsenite at high concentration (3.5 μM), thio‐DMA(V): at 35,000‐fold lower concentration Thio‐DMAV or arsenite induced significant S phase and G2/M phase arrest; higher induction of apoptosis by thio‐DMA(V) |
| Li et al. (2014) | Alkaline comet assay | Rat pheochromocytoma cell line (PC12) | Sodium arsenite: 5, 20 μM for 24 h | Positive: increase of DNA breaks (%tail DNA) at both doses, dose‐related (cell viability 5 μM, 97%; 20 μM, 81%) |
Cell viability: MTT assay Increased ROS levels |
| Rehman et al. (2014) | Intracellular ROS generation measured by oxidation‐sensitive fluorescent probe (DCFH‐DA) | Human myeloid leukaemia HL‐60 cells |
MMA(III), DMA(III) and iAs(III): (1 μM for 12 h) MMA(III): (IC50 3 μM) DMA(III): (IC50 2 μM) iAs(III): (IC50 10 μM) |
MMA(III) and DMA(III) but not iAs(III) increased oxidative stress, loss of mitochondrial membrane potential and apoptosis | |
| Unterberg et al. (2014) | Micronucleus assay | Non‐tumorigenic urothelial cell line (UROtsa) |
Arsenite and thio‐DMA(V): 0.005, 0.01, 0.1, 1, 10, 100, 250, 500 nM (7, 14 and 21 days) |
Positive: Arsenite, 100 nM, 14 days and 250 and 500 nM all exposure times (100 nM subtoxic dose, > 80% CFA). Thio‐DMA(V): negative in the full concentration range. No cytotoxicity up to 500 μM |
Cell viability: cell number and colony forming ability. Induction of global DNA methylation |
| Xie et al. (2014) |
Chromosomal aberration assay Neutral comet assay and gH2AX foci forming assay |
Human primary bronchial fibroblast (NHBF) and epithelial (NHBE) cells. |
Sodium arsenite: 0.5, 1, 5, 10 μM (24 and 120 h) |
Positive: increase in chromosome damage in fibroblasts but not in epithelial cells, significant at 5, 10 μM with both exposure times High cytotoxicity at both concentrations (≤ 50% CFA) Induction of aneuploidy and mitotic abnormalities in fibroblasts only. Positive: induction of DNA double strand breaks in both fibroblast and epithelial cells Range 1–10 μM, 24 h, 120 h) including sub‐toxic doses in the case of epithelial cells |
Cell viability: colony forming ability |
| Meyer et al. (2015) | Micronucleus assay | Human liver HepG2 cells | DMA(V): 0, 1, 10, 100, 500 μM (48 h) | Positive: significant increase of MN at 100 and 500 μM, IC70 155 μM | |
| Benhusein et al. (2016) | Alkaline comet assay | HepG2 Liver Cells | Arsenate, sodium arsenite and DMA(V): 10 μM for 24 h |
Positive: increase of DNA breaks (tail moment) with arsenate and arsenite Negative: with DMA(V) Cell viability: 85%–90% |
Cell viability: trypan blue exclusion test Increase of cellular levels of glutathione with arsenate and arsenite |
| Okamura and Nohara et al. (2016) | γ‐H2AX by western blotting | Mouse B lymphoma cells (A20) | Sodium arsenite: 10 μM (up to 14 days) | Positive: γ‐H2AX was increased following exposure for 8 and 14 days | Activation of the p53‐p21 pathway. Decreased expression of DNA repair genes. Increased expression of cytidine deaminases. Increased Bcl6 mutations (point mutations, deletions and insertions). Induction of senescence |
| Xu et al. (2016) | Alkaline and hOGG1‐modified (FLARE) comet assay | Isolated mouse bone marrow, spleen, thymus cells | Sodium arsenite or MMA(III): 5, 50 and 500 nM (4 h) | Positive: Arsenite: bone marrow cells significant increase of DNA breaks (% DNA in tail) from 5 to 500 nM, spleen and thymus cells only at 500 nM. MMA(III): cells from all the three organs showed significant increase of DNA breaks starting at 5 μM |
No data on cytotoxicity MMA(III) is more genotoxic than iAs(III) in vitro |
| Holcomb et al. (2017) | NER capacity by slot‐blot assay | Human lung fibroblasts (IMR‐90 cells) and primary mouse keratinocytes | Sodium arsenite: 10–20‐40 μM (24 h) |
Concentration‐dependent inhibition of the removal of 6–4 PPs and CPDs in both cell types Significant reduction of XPC protein levels. Significant effects at 20 and 40 μM Viability: 10 μM ~ 80%; 20 μM ~ 70%; 40 μM ~ 50% (from the graph) |
Cell viability: trypan blue exclusion test |
| Jiang et al. (2017) | In vitro binding assay, streptavidin agarose affinity assay, western blotting, fluorescence microscopy, mass spectrometry, clonogenic survival | HEK293T human embryonic kidney epithelial cells and HeLa cell | Sodium arsenite: 5 μM (cell exposure for 24 h) | Arsenite binding to FANCL in vitro and in cells. Reduction of FANCD2 recruitment to chromatin and DNA damage sites in cells upon ICL induction. Increased cell sensitivity toward ICL‐inducing agents | No data on arsenite cytotoxicity are presented |
| Xu et al. (2017) |
Alkaline comet assay and γ‐H2AX foci forming assay ROS detection by flow cytometry |
Isolated mouse thymus cells. DN, double negative, do not express CD4 or CD8; DP, double positive, express both cell markers (obtained by cell sorting) | MMA(III): 5, 50 and 500 nM (18 h) | Positive: MMA(III) significant increase of DNA breaks (%DNA in tail) in DN cells in the whole concentration range; DP cells at 50 and 500 nM. Significant increase of gH2AX fluorescence and reactive oxygen species level (DN cells, 50 and 500 nM; DP cells, 500 nM) | Cell viability: MTS assay. No quantitative data provided. Low (5 nM, ~ 80% survival) to moderate (500 nM ~ 50% srv)) cytotoxicity (from the graph) |
| Kopp et al. (2018) | γ‐H2AX by western blotting |
Human hepatoblastoma cells (HepG2) Human epithelial colorectal adenocarcinoma cells (LS‐174T) |
Sodium arsenite: 1, 10, 25, 50, 75, 100 μM (24 h) Arsenic pentoxide: 10, 50, 100, 250, 500 μM (24 h) |
Positive: significant increase (p < 0.01) of gH2AX levels at subtoxic doses for both arsenicals Arsenite: in HepG2 cells, LOAEC 25 μM, %RCC = 92; LS‐174T, LOAEC 10 μM, %RCC = 100. Arsenic pentoxide: in HepG2 cells, LOAEC 250 μM, %RCC = 88; LS‐174T: LOAEC 100 uM, %RCC = 96 |
Cell viability: comparison of DNA content (related to the number of cells) in treated versus control cells and expressed as relative cell count, %RCC |
| Ganapathy et al. (2019) | Aneuploidy test | Human lung epithelial BEAS‐2B cells and keratinocytes | Sodium arsenite: 0.5 μM (2 months) | Positive: significant increase of cells with aneuploidy (~ 2% in untreated cells versus ~ 14% in arsenite‐treated cells) |
No data on cytotoxicity are presented. 100 cells scored for each treatment, 5 experiments per point Perturbation of mitosis via activation of Akt and upregulation of Plk1. Suppression of Akt or Plk1 inhibited aneuploidy |
Abbreviations: 8‐OHdG, 8‐hydroxy‐2′‐deoxyguanosine; 8‐oxodG, 8‐hydroxydeoxyguanosine; ADP, adenosine diphosphate; Akt, protein kinase B or PKB; As(III), arsenite; BEAS‐2B, bronchial epithelial airway sensitive‐2B (normal human bronchial epithelial cell line); CA, chromosomal aberration; CD4 / CD8, cluster of differentiation 4 and 8; CFA, colony‐forming ability; CM‐H2DCFDA, chloromethyl derivative of H2DCFDA; CPD, cyclobutane pyrimidine dimer; DCFH‐DA, 2′,7′ dichloro‐dihydrofluorescein diacetate; DMA(III), dimethylarsinous acid; DMA(V), dimethylarsinic acid; DN, double negative; DNA, deoxyribonucleic acid; DP, double positive; DSB, double‐strand break; FANCD2, fanconi anaemia complementation group D2; FANCL, fanconi anaemia complementation group L; FLARE, fragment length analysis using repair enzymes; FPG, formamidopyrimidine DNA glycosylase; gH2AX, serine139‐phosphorylated histone H2AX; GSH, glutathione; Gy, Grey; HaCaT, human epidermal keratinocyte line; HeLa cells, human cell line named after Henrietta Lacks; HEK293T, human embryonic kidney epithelial cells; HepG2, human hepatoblastoma cells; hOGG1, human 8‐oxoguanine DNA glycosylase 1; HPLC‐ED, high‐performance liquid chromatography with electrochemical detection; HR, homologous recombination; iAs, inorganic arsenic; iAs(V), arsenate; IC50, inhibitory concentration 50%; ICL, inter‐strand DNA cross‐link; IMR‐90 cells, human lung fibroblasts; LC50, lethal dose killing 50% of the animals; LigIII, DNA ligase III; LOAEC, lowest‐observed‐adverse‐effect‐concentration; LS‐174T, human epithelial colorectal adenocarcinoma cell; MEF, mouse embryo fibroblast; MMA(III), monomethylarsonous acid; MMA(V), monomethylarsonic acid; MN, micronucleus; MTS, 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide; NER, nucleotide excision repair; NHBE, normal human bronchial epithelial cells; NHBF, normal human bronchial fibroblast cells; OGG1, 8‐oxoguanine DNA glycosylase 1; PARPzf, the first zinc finger of PARP‐1; PARP‐1, poly (adenosine diphosphate‐ribose) polymerase 1; PCR, polymerase chain reaction; PFGE, pulse field gel electrophoresis; pg, picogram(s); PLK1, polo‐like kinase 1; Pol β, DNA polymerase beta; PP, photoproduct; RCC, reactive cell count; ROS, reactive oxygen species; RWPE‐1, immortalised normal prostate epithelial cells; SSB, single‐strand break; UROtsa, immortalised human urothelial cells; VH10hTert, tert‐immortalised human diploid skin fibroblasts; thio‐DMA(V), thio‐dimethylarsinic acid; u‐tiAs, total urinary inorganic arsenic (sum of inorganic arsenic and its methylated metabolites MMA and DMA); UVC, ultraviolet C light; UVR, ultraviolet radiation; wt, wild‐type; XPC, Xeroderma pigmentosum complementation group C; XPE, Xeroderma pigmentosum complementation group E; XRCC1, X‐ray repair cross‐complementing protein 1.