Differential response of human T-lymphocytes to arsenic and uranium

Abstract

Elevated levels of arsenic and uranium have been detected in water sources near abandoned uranium mines in the Southwest. Evidence suggests uranium exposure increases the likelihood of immune dysfunction and this study investigates the impact of arsenic and uranium on human immune cell lines. Concentration-dependent cytotoxicity occurred following exposure to arsenite, whereas cells remained viable after 48 -h treatment with up to 100 μM uranyl acetate despite uptake of uranium into cells. Arsenite stimulated an oxidative stress response as detected by Nrf-2 nuclear accumulation and induction of HMOX-1 and NQO1, which was not detected with up to 30 μM uranyl acetate. Cellular oxidative stress can promote DNA damage and arsenite, but not uranium, stimulated DNA damage as measured by pH2AX. Arsenic enhanced the cytotoxic response to etoposide suggesting an inhibition of DNA repair, unlike uranium. Similarly, uranium did not inhibit PARP-1 activity. Because uranium reportedly stimulates oxidative stress, DNA damage and cytotoxicity in adherent epithelial cells, the current study suggests distinct cell type differences in response to uranium that may relate to generation of oxidative stress and associated downstream consequences. Delineating the actions of uranium across different cell targets will be important for understanding the potential health effects of uranium exposures.

Keywords: Arsenic; DNA damage; Immunotoxicity; Oxidative stress; T-lymphocyte; Uranium.

Figure 1.. Viability is reduced by AS but not UA despite accumulation of UA in Jurkat cells.

Jurkat cells were exposed to AS or UA for 24 and 48 hr and viability was measured as described in Materials and Methods. (A) A dose dependent decrease in viability was observed with arsenic exposure at 24 hr (solid line) or 48h (dashed lines). (B) No decrease in viability was observed in cells exposed to UA up to 100 μM for 24 or 48 hr. Jurkat cells were exposed to AS (1 μM or 10 μM) or UA (3 μM or 30 μM) for 24 hr and intra-cellular accumulation of arsenic (C) or uranium (D) was measured by ICP-MS. NT=no treatment control. Graphs represent mean ± SEM of at least 3 independent experiments. *p≤0.05, ***p≤0.001

Figure 2.. AS, but not UA, stimulated Nrf-2 nuclear localization.

Nuclear translocation of Nrf-2 was measured by western blot analysis. Results are expressed as a ratio of nuclear Nrf-2 to total protein and values shown are normalized to the untreated control (NT). The graph represents mean ± SEM of 3 independent experiments. **p≤0.01

Figure 3.. AS, but not UA, exposure increases HMOX1 and NQO1 expression and subsequent HO-1 protein production.

Jurkat cells were treated with the indicated concentrations of AS or UA for 6 hr and RNA was harvested for qRT-qPCR. (A) HMOX1 expression increased significantly upon exposure to 10 μM AS, but not UA. (B) NQO1 expression increased significantly in cells exposed to 10 μM AS, but not UA. (C) HO-1 protein production increased significantly after 6 hours in response to 10 μM AS. HO-1 levels were normalized to β-tubulin and reported values are relative to the untreated control (NT). No response was observed with UA. (D) Representative western blot of HO-1 protein. Graphs represent mean ± SEM of at least 3 independent experiments. *p≤0.05, **p≤0.01, ***p≤0.001

Figure 3.. AS, but not UA, exposure increases HMOX1 and NQO1 expression and subsequent HO-1 protein production.

Jurkat cells were treated with the indicated concentrations of AS or UA for 6 hr and RNA was harvested for qRT-qPCR. (A) HMOX1 expression increased significantly upon exposure to 10 μM AS, but not UA. (B) NQO1 expression increased significantly in cells exposed to 10 μM AS, but not UA. (C) HO-1 protein production increased significantly after 6 hours in response to 10 μM AS. HO-1 levels were normalized to β-tubulin and reported values are relative to the untreated control (NT). No response was observed with UA. (D) Representative western blot of HO-1 protein. Graphs represent mean ± SEM of at least 3 independent experiments. *p≤0.05, **p≤0.01, ***p≤0.001

Figure 4.. DNA damage in response to metal treatment.

Jurkat cells were treated with metals at the indicated times and concentrations (μM). (A) Representative western blot showing pH2AX in Jurkat cells in response to metal treatment. (B) Bar graph depicting quantification of western blots by densitometry. pH2AX levels were normalized to β-tubulin and reported values are relative to the untreated control (NT). Graph represents mean ± SEM of 3 independent experiments. (C) Catalase mitigates Arsenic induced DNA damage. Jurkat cells were pretreated with catalase (Cat) for 30 min when indicated and treated with metal (10μM AS, 100μM UA) for 6h. Cells were fixed post-metal exposure and stained with pH2AX. Representative images for each treatment group are shown. Scale bar is 25 μm. (D) Bar graph depicting quantification of pH2AX intensity. Bars represent mean ± SEM of the intensity per nuclei of at least 3 images from each treatment group from 3 independent experiments, normalized to TBHP control. **p≤0.01.

Figure 4.. DNA damage in response to metal treatment.

Jurkat cells were treated with metals at the indicated times and concentrations (μM). (A) Representative western blot showing pH2AX in Jurkat cells in response to metal treatment. (B) Bar graph depicting quantification of western blots by densitometry. pH2AX levels were normalized to β-tubulin and reported values are relative to the untreated control (NT). Graph represents mean ± SEM of 3 independent experiments. (C) Catalase mitigates Arsenic induced DNA damage. Jurkat cells were pretreated with catalase (Cat) for 30 min when indicated and treated with metal (10μM AS, 100μM UA) for 6h. Cells were fixed post-metal exposure and stained with pH2AX. Representative images for each treatment group are shown. Scale bar is 25 μm. (D) Bar graph depicting quantification of pH2AX intensity. Bars represent mean ± SEM of the intensity per nuclei of at least 3 images from each treatment group from 3 independent experiments, normalized to TBHP control. **p≤0.01.

Figure 4.. DNA damage in response to metal treatment.

Jurkat cells were treated with metals at the indicated times and concentrations (μM). (A) Representative western blot showing pH2AX in Jurkat cells in response to metal treatment. (B) Bar graph depicting quantification of western blots by densitometry. pH2AX levels were normalized to β-tubulin and reported values are relative to the untreated control (NT). Graph represents mean ± SEM of 3 independent experiments. (C) Catalase mitigates Arsenic induced DNA damage. Jurkat cells were pretreated with catalase (Cat) for 30 min when indicated and treated with metal (10μM AS, 100μM UA) for 6h. Cells were fixed post-metal exposure and stained with pH2AX. Representative images for each treatment group are shown. Scale bar is 25 μm. (D) Bar graph depicting quantification of pH2AX intensity. Bars represent mean ± SEM of the intensity per nuclei of at least 3 images from each treatment group from 3 independent experiments, normalized to TBHP control. **p≤0.01.

Figure 5.. AS, but not UA, sensitizes and reduces PARP-1 activity in Jurkat cells when combined with the DNA damaging agent etoposide.

Jurkat cells were treated for 2 hr with 5 μM ETOP and then exposed to 10 μM AS or 30 μM UA for 24 and 48 hr and viability measured as described in Materials and Methods. (A) AS but not UA treatment sensitized Jurkat cells to DNA damage induced by ETOP. (B) Jurkat cells were treated with AS (1 or 10 μM), or UA (3 or 10 μM) for 24 hr. One well of each concentration was then treated with 25 μM ETOP for 4 hr before protein was collected and samples were analyzed for PAR using the PARP ELISA kit according to the manufacturer’s instructions. AS, but not UA treatment decreased the activity of PARP-1 in response to ETOP induced DNA damage. Graphs represent mean ± SEM of at least 3 independent experiments. *p≤0.05, ** p≤0.01, ***p≤0.001