Cytotoxicity of environmentally relevant concentrations of aluminum in murine thymocytes and lymphocytes

Abstract

The effects of low concentrations of aluminum chloride on thymocytes and lymphocytes acutely dissociated from young mice were studied using flow cytometry with a DNA-binding dye. We demonstrate a rapid and dose-dependent injury in murine thymocytes and lymphocytes resulting from exposure to aluminum, as indicated by an increase in the entry into the cell of the DNA-binding dye, propidium iodine. A 60-minute exposure to 10 μM AlCl(3) caused damage of about 5% of thymocytes, while 50% were injured after 10 minutes at 20 μM. Nearly all thymocytes showed evidence of damage at 30 μM AlCl(3) after only 5 minutes of incubation. In lymphocytes, injury was observed at 15 μM AlCl(3) and less than 50% of cells were injured after a 60-minute exposure to 20 μM. Injury only rarely proceeded to rapid cell death and was associated with cell swelling. These results suggest that aluminum has cytotoxic effects on cells of the immune system.

Figure 1

Histograms showing the effects of aluminum exposure to thymocytes. PI fluorescence intensity (x-axis) is plotted against cell count (y-axis). Histogram (a) shows untreated thymocytes where most of the cells have low PI intensity, which is characteristic of healthy cells whose membranes exclude PI. In (b) thymocytes were exposed to 2% ethanol, and a large number of cells show very high PI intensity. These are dead cells, whose plasma membrane has lost integrity. (c) and (d) show the gradual increase in PI intensity in thymocytes exposed to 20 μM of AlCl₃ at five- and 25-minute exposure. The number of dead cells did not increase with AlCl₃ exposure. There is, however, a shift in the distribution of healthy cells to the right, indicating an increased uptake of PI, reflecting cell damage. For purposes of quantitation, all cells falling under the bar labeled R1 are considered injured.

Figure 2

Dose and time dependence of AlCl₃-induced injury in thymocytes. The cells were treated with a range of concentrations of AlCl₃ (0–40 μM) at various time points. Thymocytes were considered to be damaged when the level of fluorescence intensity of PI in the cells was higher than the level in untreated cells. Values are mean ± SD obtained from six independent measurements (based on Student's paired t-test). The concentration curves are all statistically significantly different at the P < .05 level by ANOVA analysis. There are no significant changes with time between 25, 40, and 60 minutes, nor between 10 and 15 minutes, but all other time differences are significant.

Figure 3

Dose and time dependence of lymphocyte injury with exposure to various concentrations of AlCl₃ (0–40 μM) at various time points. Other conditions were as described in the legend to Figure 2. All concentration curves are significantly different from each other at the P < .05 level by ANOVA with the exception of 0 and 10 minutes. There were no significant changes with time between 25, 40, and 60 minutes, nor between 10 and 15 minutes, but all other time periods were significantly different at the P < .05 level by ANOVA.

Figure 4

Thymocytes were exposed to 0 μM (a, c) and 20 μM (b, d) AlCl₃ at 0 and 20 minutes. Various staining patterns signify different cell populations. Region Q3 includes live cells (PI-negative and Annexin V-negative), whereas region Q4 contains apoptotic cells (PI-negative and Annexin V-positive). Dead cells are represented in Q2 region (both PI- and Annexin V-positive), while quadrant Q1 shows damaged cells (PI-positive and Annexin V-negative). Upon a 20-minute exposure to 20 μM AlCl₃ the cell population from the Q3 region moved to Q1 region, without a clear shift to the Q4 area first. This fact indicates that thymocytes are not undergoing the apoptotic process. Contour plots (e, f) show fluorescence intensity with regard to forward scatter and side scatter in the control (e) and in the presence of 20 μM AlCl₃ for 20 minutes (f). The increase in forward scatter on exposure to AlCl₃ is indicative of an increase in size (i.e., swelling).