Combined effects of alpha particles and depleted uranium on Zebrafish (Danio rerio) embryos

Abstract

The combined effects of low-dose or high-dose alpha particles and depleted uranium (DU) in Zebrafish (Danio rerio) embryos were studied. Three schemes were examined-(i) [ILUL]: 0.44 mGy alpha-particle dose + 10 µg/l DU exposure, (ii) [IHUH]: 4.4 mGy alpha-particle dose + 100 µg/l DU exposure and (iii) [IHUL]: 4.4 mGy alpha-particle dose + 10 µg/l DU exposure-in which Zebrafish embryos were irradiated with alpha particles at 5 h post fertilization (hpf) and/or exposed to uranium at 5-6 hpf. The results were also compared with our previous work, which studied the effects of [ILUH]: 0.44 mGy alpha-particle dose + 100 µg/l DU exposure. When the Zebrafish embryos developed to 24 hpf, the apoptotic signals in the entire embryos, used as the biological endpoint for this study, were quantified. Our results showed that [ILUL] and [IHUL] led to antagonistic effects, whereas [IHUH] led to an additive effect. The effect found for the previously studied case of [ILUH] was difficult to define because it was synergistic with reference to the 100 µg/l DU exposure, but it was antagonistic with reference to the 0.44 mGy alpha-particle dose. All the findings regarding the four different schemes showed that the combined effects critically depended on the dose response to each individual stressor. We also qualitatively explained these findings in terms of promotion of early death of cells predisposed to spontaneous transformation by alpha particles, interacting with the delay in cell death resulting from various concentrations of DU exposure.

Fig. 1.

Experimental flow for treatment procedures of embryos. The experimental flow for the treatment procedures of embryos in the alpha-particle–irradiated and uranium-dosed group (I_XU_Y) for the three different experimental schemes (Conditions 1 to 3).

Fig. 2.

Normalized net mean numbers of apoptotic signals obtained for different groups of embryos. The normalized net mean numbers of apoptotic signals (N ± SE) obtained for embryos in the C, I_XU_Y, I_X and U_Y groups for the three to four replicates of the three experimental schemes (Condition 1 [I_LU_L], Condition 2 [I_HU_H] and Condition 3 [I_HU_L]). The results (for [I_LU_H]) from our previous study [34] were also included for comparison. *Cases with P ≤ 0.05 were considered statistically significant using Student's t-test by comparing with the corresponding C group of embryos under each experimental condition. ^aData extracted from Ref. [34].

Fig. 3.

Cell deaths induced by alpha particles and depleted uranium. Simplified schematic diagrams showing the patterns of cell deaths induced by different alpha-particle doses alone and by different DU concentrations exposure alone. Numbers in brackets represent the net number of apoptotic cells at 24 hpf (i.e. after subtracting the background number). Left columns: naturally aberrant cells; right columns: healthy cells if no external stressors; red color: cells affected by alpha particles; green color: cells affected by DU; open circles with dotted outlines: cells that undergo apoptosis earlier than 24 hpf; solid circles with colors other than white: cells that undergo apoptosis at 24 hpf; open circles with bold outlines: cells that undergo apoptosis after 24 hpf; white solid circles: normal healthy cells that were not affected by any stressors or those that could repair the damages successfully; pink solid circles: naturally aberrant cells that underwent apoptosis at 24 hpf. Representative images of stained embryos in C, I_L, I_H, U_L and U_H groups were also shown. Images of embryos were captured, using a fluorescent microscope with a magnification of ×40. Three images focusing on different sections of the embryos were captured and were then combined into one image.

Fig. 4.

Construction of the combined effects from the effects of single stressors. Simplified schematic diagrams showing the construction of combined effects from the effects of single stressors. Numbers in parentheses represent the net number of apoptotic cells at 24 hpf (i.e. after subtracting the background number). Left columns: naturally aberrant cells; right columns: healthy cells if no external stressors; red color: cells affected by alpha particles; green color: cells affected by DU; blue color: cells affected by both alpha particles and DU; open circles with dotted outlines: cells that undergo apoptosis earlier than 24 hpf; solid circles with colors other than white: cells that undergo apoptosis at 24 hpf; open circles with bold outlines: cells that undergo apoptosis after 24 hpf; white solid circles: normal healthy cells that were not affected by any stressors or those that could repair the damages successfully; pink solid circles: naturally aberrant cells that undergo apoptosis at 24 hpf.