Comparative effects of cadmium, zinc, arsenic and chromium on olfactory-mediated neurobehavior and gene expression in larval zebrafish (Danio rerio)

Abstract

Studies have shown that olfactory-mediated behaviors that are critical to survival can be disrupted by exposure to certain metals. Aquatic Superfund sites often contain elevated levels of various metals, yet few have been characterized for their potential to cause olfactory toxicity. A larval zebrafish behavioral assay was developed to characterize concentration-response curves for zinc (Zn), hexavalent chromium (Cr), and arsenate (As) olfaction inhibition. Cadmium (Cd), an established olfactory toxicant, was used as a positive control. As expected, following a 24-hour exposure to Cd, we observed a reduced response to taurocholic acid (TCA), a behaviourally-active odorant, thus validating the behavioral assay. Zn exposure also decreased TCA response (IC50: 34.9 µg/L and 69.0 µg/L respectively). Behavioral responses to another odorant, L-cysteine, revealed that Zn was potentially of specific toxicity to ciliated olfactory sensory neurons.No significant changes in olfactory responses were observed from Cr and As exposures, even at exposures far greater than observed environmental concentrations.Exposures to binary mixtures of Cd and Zn indicated that Zn had a protective effect against Cd toxicity at low concentrations, but this effect disappeared at higher Zn concentrations. Quantitative analysis of whole larvae gene expression of 8 genes 4 important in mitigating metal induced oxidative stress, confirmed an antioxidant response was evoked by Cd, but not for the other metals, suggesting that oxidative stress was not a primary mechanism of Zn induced olfactory dysfunction. In summary, our study identified that Zn inhibits olfaction at environmentally-relevant concentrations, and that it may reduce Cd when present in mixtures. Our study extends the utility of zebrafish as a model species to assess olfactory toxicity in fish.5 Abbreviations: Nrf2: nuclear factor, erythroid 2-like 2, omp: olfactory marker protein b, dpf: days post fertilization, EM: E3 embryo medium, SRP: superfund research project, Zn: zinc, As: arsenic, Cr: chromium, Cd: cadmium, TCA: taurocholic acid, OE: olfactory epithelium, OSN: olfactory sensory neuron, OR: olfactory receptor, MOR: major olfactory receptor, TRPC2: Transient Receptor Potential Cation Channel Subfamily C Member 2, OB: olfactory bulb, hmox1a: heme oxygenase 1a, gstp: glutathione s-transferase pi, gclc:glutamate-cysteine ligase, catalytic subunit, nqo1: NAD(P)H dehydrogenase, quinone 1, prdx1: peroxiredoxin 1, gpx1a: glutathione peroxidase 1a, sod1: superoxide dismutase 1, soluble, sod2: superoxide dismutase 2, mitochondrial, hsp70: heat shock cognate 70-kd protein, tandem duplicate 3, actb1: actin, beta 1, gapdh: glyceraldehyde-3-phosphate dehydrogenase, hprt1: hypoxanthine phosphoribosyltransferase 1, gadd45bb: growth arrest and DNA-damage-inducible, beta b

Mechanotransduction Activity Facilitates Hair Cell Toxicity Caused by the Heavy Metal Cadmium

“…Previous experiments have shown that cadmium enters other cell types through zinc transporters (Dalton et al, 2005;Fujishiro et al, 2009), and that zinc can block cadmium uptake (Barbier et al, 2004;Girijashanker et al, 2008). Zinc has also previously been shown to protect against impaired behavioral responses to odorants caused by cadmium in larval zebrafish (Heffern et al, 2018) and to protect against impaired auditory responses caused by cadmium in rats (Agirdir et al, 2002). To test whether zinc could likewise protect against cadmiuminduced hair cell death in zebrafish, we co-treated fish with 30 µM of cadmium chloride and varying doses of zinc sulfate, based on doses that had previously been shown to protect against cadmium-induced defects in olfactory behavior in larval zebrafish (Heffern et al, 2018), with and without a 1-h zinc sulfate pretreatment.…”

“…Zinc has also previously been shown to protect against impaired behavioral responses to odorants caused by cadmium in larval zebrafish (Heffern et al, 2018) and to protect against impaired auditory responses caused by cadmium in rats (Agirdir et al, 2002). To test whether zinc could likewise protect against cadmiuminduced hair cell death in zebrafish, we co-treated fish with 30 µM of cadmium chloride and varying doses of zinc sulfate, based on doses that had previously been shown to protect against cadmium-induced defects in olfactory behavior in larval zebrafish (Heffern et al, 2018), with and without a 1-h zinc sulfate pretreatment. We found no significant reduction in hair cell death in response to cotreatment alone ( Figure 4A), and only a slight reduction at the highest zinc sulfate dose tested 1,364 µM, which is equivalent to 88 µg/L zinc, in response to the 1-h pretreatment combined with cotreatment ( Figure 4B).…”

Mechanotransduction activity facilitates hair cell toxicity caused by the heavy metal cadmium

“…Zinc has also previously been shown to protect against impaired behavioral responses 191 to odorants caused by cadmium in larval zebrafish (Heffern et al, 2018) and to protect 192 against impaired auditory responses caused by cadmium in rats (Agirdir et al, 2002). 193…”

“…To test whether zinc could likewise protect against cadmium-induced hair cell death in 194 zebrafish, we co-treated fish with 30 µM of cadmium and varying doses of zinc, based 195 on doses that had previously been shown to protect against cadmium-induced defects 196 in olfactory behavior in larval zebrafish (Heffern et al, 2018), with and without a one-197 hour zinc pretreatment. We found no significant reduction in hair cell death in response 198 to cotreatment alone ( Figure 4A), and only a slight reduction at the highest zinc dose, 199 88 µg/L, in response to the one-hour pretreatment combined with cotreatment ( Figure 200 4B).…”

“…Copper, in particular, kills hair cells at doses considerably lower than cadmium. The zinc 271 doses we used were based on previous work showing that 22 µg/L zinc could protect 272 against cadmium-induced olfactory damage whereas lower and higher doses could not 273 (Heffern et al, 2018). We failed to see protection at 22 µg/L of zinc.…”