Cadmium kills hair cells in zebrafish larvae in a dose-dependent manner. (A) Treating 5-days post-fertilization (dpf) zebrafish with cadmium chloride doses ranging from 0 to 120 μM for 3 h showed an increasing amount of hair cell death with increasing cadmium chloride dose. Data are displayed as mean ± standard deviation. ****p < 0.0001 as compared to 0 μM cadmium chloride control by ANOVA and Dunnett’s post hoc test. Fish were fixed and stained with an anti-parvalbumin antibody and hair cells from six neuromasts from each fish were counted (OP1, M2, IO4, O2, MI2, and MI1) and averaged. n = 10 fish for the 0, 15, 60 and 120 μM cadmium chloride groups and n = 7 for the 30 μM cadmium chloride group. (B) Representative images of the MI1 neuromast following treatment with either 0 (left) or 60 (right) μM cadmium chloride.

Cadmium toxicity is dependent on functional mechanotransduction activity. (A)cdh23^tj264 mutants, which lack functional mechanotransduction activity, are resistant to cadmium-induced hair cell death. (B) Representative images of the O2 Neuromast in wild-type siblings (top) and cdh23 mutants (bottom) fish following treatment with either 0 (left) or 60 (right) μM cadmium chloride. (C) Treatment with 200 μM Benzamil, a mechanotransduction channel blocker, significantly blocks cadmium-induced hair cell death as compared to fish treated with the same amount of DMSO alone (a 1:200 dilution). (D) Representative images of the O2 Neuromast in DMSO-treated (top) and benzamil treated (bottom) fish following treatment with either 0 (left) or 60 (right) μM cadmium chloride. Due to reduced hair cell numbers in cdh23 mutants data is normalized to the 0 cadmium chloride control for each treatment group. Data are displayed as mean ± standard deviation. *p < 0.05, ****p < 0.0001 by Two-Way ANOVA and Šídák multiple comparisons test. Black stars above the error bars denote significant differences in cdh23 mutants or benzamil treated fish as compared to their respective wild-type sibling and DMSO control groups treated with the same dose of cadmium chloride. Gray stars below the error bars denote significant differences in cdh23 mutants or benzamil treated fish at that cadmium chloride dose compared to the 0 cadmium chloride control group of the same mutant or drug condition. n = 10 fish for all groups.

Genetic mutants resistant to neomycin-induced hair cell death due to impaired mechanotransduction activity are also resistant to cadmium-induced hair cell death. (A)ift88^tz288 mutants, which have previously been shown to have reduced mechanotransduction activity (Kindt et al., 2012; Stawicki et al., 2016), are partially resistant to cadmium-induced hair cell death. (B) Representative images of the IO4 Neuromast in ift88 wild-type siblings (top) and ift88 mutant fish (bottom) following treatment with either 0 (left) or 60 (right) μM cadmium chloride. (C)cc2d2a^w38 mutants, which have previously been shown to have normal mechanotransduction activity (Owens et al., 2008; Stawicki et al., 2016), are not resistant to cadmium-induced hair cell death. (D) Representative images of the IO4 Neuromast in cc2d2a wild-type siblings (top) and cc2d2a mutant fish (bottom) following treatment with either 0 (left) or 60 (right) μM cadmium chloride. Due to reduced hair cell number in ift88 mutants data is normalized to the 0 cadmium chloride control for each treatment group. Data are displayed as mean ± standard deviation. *p < 0.05, ***p < 0.001, ****p < 0.0001 by Two-Way ANOVA and Šídák multiple comparisons test. For the ift88 mutant experiment n = 10 fish for wild-type sibling 15 and 120 μM cadmium chloride and mutant 0, 15, and 30 μM cadmium chloride, n = 9 for wild-type sibling 0 μM cadmium chloride and n = 8 for wild-type sibling 30 and 60 μM cadmium chloride and mutant 60 and 120 μM cadmium chloride. For the cc2d2a experiment n = 10 fish for all groups.

Zinc shows limited protection against cadmium-induced hair cell death. Zinc sulfate failed to reduce hair cell death when administered (A) alongside cadmium chloride and only showed a slight reduction at the highest dose tested when administered (B) 1 h before and during cadmium chloride treatment. This protection was not seen when tested against a range of cadmium chloride doses (C). Data are displayed as mean ± standard deviation. ***p < 0.001 as compared to the 0 zinc sulfate control by Two-Way ANOVA and Šídák multiple comparisons test. n = 10 fish for all groups. (D) Representative images of the IO4 Neuromast in control (top) and zinc sulfate treated (bottom) fish following treatment with either 0 (left) or 60 (right) μM cadmium chloride.

Copper fails to protect against cadmium-induced hair cell death. (A) At doses of 0.5 μM and higher copper sulfate caused significant hair cell death on its own. ****p < 0.0001 by Two-Way ANOVA and Šídák multiple comparisons test as compared to the 0-copper sulfate control. Using copper sulfate in concentrations up to 1 μM failed to protect against hair cell death in response to 30 μM of cadmium chloride. (B) The 0.25 μM dose of copper sulfate failed to show a significant reduction in hair cell death at all cadmium chloride doses tested. Data are displayed as mean ± standard deviation. n = 10 fish for all groups. (C) Representative images of the O2 Neuromast in control (top) and copper sulfate treated (bottom) fish following treatment with either 0 (left) or 60 (right) μM cadmium chloride.