Developmental toxicity in zebrafish (Danio rerio) exposed to uranium: A comparison with lead, cadmium, and iron

Abstract

Populations of plants and animals, including humans, living in close proximity to abandoned uranium mine sites are vulnerable to uranium exposure through drainage into nearby waterways, soil accumulation, and blowing dust from surface soils. Little is known about how the environmental impact of uranium exposure alters the health of human populations in proximity to mine sites, so we used developmental zebrafish (Danio rerio) to investigate uranium toxicity. Fish are a sensitive target for modeling uranium toxicity, and previous studies report altered reproductive capacity, enhanced DNA damage, and gene expression changes in fish exposed to uranium. In our study, dechorionated zebrafish embryos were exposed to a concentration range of uranyl acetate (UA) from 0 to 3000 μg/L for body burden measurements and developmental toxicity assessments. Uranium was taken up in a concentration-dependent manner by 48 and 120 h post fertilization (hpf)-zebrafish without evidence of bioaccumulation. Exposure to UA was not associated with teratogenic outcomes or 24 hpf behavioral effects, but larvae at 120 hpf exhibited a significant hypoactive photomotor response associated with exposure to 3 μg/L UA which suggested potential neurotoxicity. To our knowledge, this is the first time that uranium has been associated with behavioral effects in an aquatic organism. These results were compared to potential metal co-contaminants using the same exposure paradigm. Similar to uranium exposure, lead, cadmium, and iron significantly altered neurobehavioral outcomes in 120-hpf zebrafish without inducing significant teratogenicity. Our study informs concerns about the potential impacts of developmental exposure to uranium on childhood neurobehavioral outcomes. This work also sets the stage for future, environmentally relevant metal mixture studies. Summary Uranium exposure to developing zebrafish causes hypoactive larval swimming behavior similar to the effect of other commonly occurring metals in uranium mine sites. This is the first time that uranium exposure has been associated with altered neurobehavioral effects in any aquatic organism.

Keywords: Development; Metals; Neurobehavior; Uranium; Zebrafish.

Fig. 1.. Uranium uptake by developing zebrafish.

Concentrations of uranium detected by ICP-MS analyses in 48 hpf and 120 hpf zebrafish exposed from 6 hpf to 30, 300, or 3,000 μg/L UA. As exposure concentration increased, the concentration detected in zebrafish also increased. The UA did not bioaccumulate between 48 hpf and 120 hpf. A two-way ANOVA followed by a Sidak’s post-hoc multiple comparison test was used to compare samples. Graph represents mean ± SEM of at least 3 independent replicates (ns = not significant, **p ≤ 0.01).

Fig. 2.. UA exposure did not affect embryonic photomotor response (EPR).

Dechorionated zebrafish embryos (n=32-68) were exposed to 0, 0.1, 3, 10, 30, 100, 300, 1,000, and 3,000 μg/L UA from 6 hpf, and the EPR assay was conducted at approximately 24 hpf. Normal zebrafish embryos exhibit baseline activity in IR light prior to the first light flash (Background), an immediate burst of tail bending in response to the first light flash (Excitation) that rapidly decays to immobility, and little or no movement in response to the second light flash (Refractory). The K-S test (p < 0.05, delta > 50%) was used to determine that there were no alterations from normal behavior before and after the two light pulses in UA-exposed zebrafish. Any dead or malformed animals were excluded from the analysis.

Fig. 3.. UA exposure altered larval photomotor response (LPR).

Developmental exposure of zebrafish (n=32-68) to UA was associated with hypoactivity in the light (quiescent) and dark (active) periods. (A) LPR assay consisted of three 3-minute periods of alternating light and dark periods. Dechorionated zebrafish embryos exposed to 10, 30, 100, 300, and 3,000 μg/L UA had a significant decrease (p < 0.05, Relative Ratio ≤ −10%) in photo-motor activity in the light phase, and embryos exposed to 3, 30, and 100 μg/L UA had a significant decrease in the dark phase (p < 0.05, Relative Ratio ≥ 10%). (B) Table shows area under the curve (AUC), AUC Relative Ratio (%), p value (calculated by the K-S test), and the detected significant effect, if any, calculated during the third light-dark cycle of the assay.

Fig. 4.. Comparison of developmental toxicity of UA with lead, cadmium, and iron.

(A) Developmental exposure of zebrafish to lead and iron was associated with hypoactivity in the light (quiescent) and dark (active) phases, and exposure to cadmium resulted in hypoactivity in the light phase. The lowest effect levels (LEL) in the dark and light phases, and the lowest sample sizes of all concentrations for each chemical are indicated in the table. (B) Developmental exposures to uranyl acetate, lead, cadmium, and iron did not cause teratogenic effects till 120 hpf. “Any effect” includes all 18 developmental endpoints described in the Teratogenicity Assessments section of the Methods (Abbreviations: MO24 = mortality at 24 hpf, MORT = mortality at 120 hpf).