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Comparative Study
. 2017 Sep 5:279:60-66.
doi: 10.1016/j.toxlet.2017.07.897. Epub 2017 Jul 29.

Genotoxicity induced by monomethylarsonous acid (MMA+3) in mouse thymic developing T cells

Huan Xu  1 , Sebastian Medina  1 , Fredine T Lauer  1 , Christelle Douillet  2 , Ke Jian Liu  1 , Miroslav Stýblo  2 , Scott W Burchiel  3
Affiliations
Comparative Study

Genotoxicity induced by monomethylarsonous acid (MMA+3) in mouse thymic developing T cells

Huan Xu et al. Toxicol Lett. .

Abstract

Drinking water exposure to arsenic is known to cause immunotoxicity. Our previous studies demonstrated that monomethylarsonous acid (MMA+3) was the major arsenical species presented in mouse thymus cells after a 30 d drinking water exposure to arsenite (As+3). MMA+3 was also showed to be ten times more toxic than As+3 on the suppression of IL-7/STAT5 signaling in the double negative (DN) thymic T cells. In order to examine the genotoxicity induced by low to moderate doses of MMA+3, isolated mouse thymus cells were treated with 5, 50 and 500nMMMA+3 for 18h in vitro. MMA+3 suppressed the proliferation of thymus cells in a dose dependent manner. MMA+3 at 5nM induced DNA damage in DN not double positive (DP) cells. Differential sensitivity to double strand breaks and reactive oxygen species generation was noticed between DN and DP cells at 50nM, but the effects were not seen at the high dose (500nM). A stronger apoptotic effect induced by MMA+3 was noticed in DN cells than DP cells at low doses (5 and 50nM), which was negated by the strong apoptosis induction at the high dose (500nM). Analysis of intracellular MMA+3 concentrations in DN and DP cells, revealed that more MMA+3 accumulated in the DN cells after the in vitro treatment. Collectively, these results suggested that MMA+3 could directly induce strong genotoxicity in the early developing T cells in the thymus. The DN cells were much more sensitive to MMA+3 induced genotoxicity and apoptosis than DP cells, probably due to the higher intracellular levels of MMA+3.

Keywords: Apoptosis; Differential sensitivity; Double negative T cells; Double positive T cells; Genotoxicity; Monomethylarsonous acid.

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Conflict of interest statement

Conflict of interest

None.

Figures

Fig. 1
Fig. 1
Proliferation of mouse primary thymus cells after exposure to 5, 50 and 500 nM MMA+3 for 18 h in vitro with/without IL-7.Isolated thymus cells were treated with MMA+3 in the medium with or without 50 ng/mL IL-7. The MTS assay used CellTiter 96® AQueous One Solution Reagent to measure the proliferation. Formazan production from the cell culture was analyzed from the reading of absorbance at 490 nm. *Significantly different compared to Cont (n = 3, p < 0.05). # Significantly different compared to the same doses in the medium with 50 ng/mL IL-7 (n = 3, p < 0.05). Results are Means ± SD.
Fig. 2
Fig. 2
DNA damage and double strand breaks in DN and DP cells after exposure to 5, 50 and 500 nM MMA+3 for 18 h. DN and DP cells were treated with MMA+3 and analyzed by Comet assay or γ-H2AX intracellular flow cytometry. A, DNA damage in DN and DP cells. B, relative γ-H2AX fluorescence normalized to control. *Significantly different compared to Cont or the low dose (5 nM) (n = 3, p < 0.05). # Significantly different compared to the DN cells at the same dose (n = 3, p < 0.05). Results are Means ± SD.
Fig. 3
Fig. 3
ROS production in DN and DP cells after exposure to 5, 50 and 500 nM MMA+3 for 18 h. Thymus cells were treated with MMA+3 and analyzed by CD4, CD8 cell surface markers and DHE staining. A, DN and DP cell gating and stained/unstained samples. B, relative DHE fluorescence normalized to control. *Significantly different compared to the low dose (5 nM) (n = 3, p < 0.05). # Significantly different compared to the DN cells at the same dose (n = 3, p < 0.05). Results are Means ± SD.
Fig. 4
Fig. 4
Apoptosis in DN and DP cells after exposure to 5, 50 and 500 nM MMA+3 for 18 h. Thymus cells were treated with MMA+3 and analyzed by CD4, CD8 cell surface markers and Annexin V/7-AAD staining. A, DN and DP cell gating, unstained, stained and positive controls.B,viable cells (Annexin V−, 7-AAD−). C, early apoptotic cells (Annexin V+, 7-AAD−). D, late apoptotic cells (Annexin V+, 7-AAD+). *Significantly different compared to Cont (n = 3, p < 0.05). # Significantly different compared to the DN cells at the same dose (n = 3, p < 0.05). Results are Means ± SD.
Fig. 5
Fig. 5
Intracellular MMA+3levels in DN and DP cells treated with MMA+3 in vitro for 18 h. Sorted DN and DP were treated with MMA+3 for 18 h. Intracellular MMA+3accumulation was measured by HG-CT-ICP-MS. ND, not detected. *Significantly different compared to Cont (n = 3, p < 0.05). # Significantly different compared to the DN cells at the same dose (n = 3, p < 0.05). Results are Means ± SD.
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