Cadmium (Cd) is a toxic metal known to accumulate in and have adverse effects on the olfactory systems of fish. The objective of this project was to investigate the effect of cadmium on zebrafish larvae, specifically the effects on the olfactory system at cellular and functional levels. Zebrafish larvae (72 hours post fertilization) were exposed to sublethal concentrations of cadmium (0.5, 1, 5, and 10 µM) for 96 h. Whole-body cadmium accumulation during this exposure period as quantified using GFAAS (graphite furnace atomic absorption spectroscopy) was found to increase with both exposure length and concentration. Using a transgenic strain of hsp70/eGFP (heat shock protein 70/enhanced green fluorescent protein reporter gene) zebrafish, dose-dependent induction of the heat shock response was observed in the olfactory epithelium. Expression of hsp70/eGFP in the olfactory epithelium was a highly sensitive biomarker for the effects of cadmium in the olfactory system with a lowest observed effects concentration (LOEC) of 0.5 µM Cd. Strong induction of the transgenic reporter gene correlated closely with cell death (LOEC of 5 µM Cd) and histological alterations (LOEC of 1 µM Cd) in the olfactory epithelium of zebrafish larvae following cadmium exposure. Additionally, loss of sensory cilia from the surface of the olfactory epithelium was observed in larvae exposed to 5 and 10 µM Cd. Furthermore, behaviour tests to assess olfactory function revealed sensory deficits, likely due in part to the cadmium-induced degeneration of the olfactory epithelium (p<0.05 for 1 µM; p<0.001 for 5 and 10 µM Cd). <p>To determine if cadmium was entering the cells of the olfactory epithelium by acting as a calcium (Ca) antagonist, zebrafish larvae were co-exposed to 1, 5, or 10 µM Cd with 1 or 5 mM Ca for 96 h. Whole-body cadmium accumulation as quantified using ICP-MS (inductively coupled plasma mass spectrometry) was decreased in larvae co-exposed to cadmium and calcium. Additionally, induction of the heat shock response was reduced in the presence of increasing calcium co-treatment. These ameliorating effects of calcium were further revealed in cell death and histological analyses of the olfactory epithelium. Also, larvae co-exposed to cadmium and calcium exhibited greater olfactory sensory function compared to larvae exposed to cadmium only. Significant increases in aversion response were observed in larvae exposed to 5 µM Cd with 1 and 5 mM Ca (p<0.05). These results indicate that cadmium gains entry to the olfactory epithelium via calcium uptake systems, wherein it causes damage to the olfactory system and can lead to sensory impairment.

Cadmium (Cd) is a toxic metal known to accumulate in and have adverse effects on the olfactory systems of fish. The objective of this project was to investigate the effect of cadmium on zebrafish larvae, specifically the effects on the olfactory system at cellular and functional levels. Zebrafish larvae (72 hours post fertilization) were exposed to sublethal concentrations of cadmium (0.5, 1, 5, and 10 µM) for 96 h. Whole-body cadmium accumulation during this exposure period as quantified using GFAAS (graphite furnace atomic absorption spectroscopy) was found to increase with both exposure length and concentration. Using a transgenic strain of hsp70/eGFP (heat shock protein 70/enhanced green fluorescent protein reporter gene) zebrafish, dose-dependent induction of the heat shock response was observed in the olfactory epithelium. Expression of hsp70/eGFP in the olfactory epithelium was a highly sensitive biomarker for the effects of cadmium in the olfactory system with a lowest observed effects concentration (LOEC) of 0.5 µM Cd. Strong induction of the transgenic reporter gene correlated closely with cell death (LOEC of 5 µM Cd) and histological alterations (LOEC of 1 µM Cd) in the olfactory epithelium of zebrafish larvae following cadmium exposure. Additionally, loss of sensory cilia from the surface of the olfactory epithelium was observed in larvae exposed to 5 and 10 µM Cd. Furthermore, behaviour tests to assess olfactory function revealed sensory deficits, likely due in part to the cadmium-induced degeneration of the olfactory epithelium (p<0.05 for 1 µM; p<0.001 for 5 and 10 µM Cd). <p>To determine if cadmium was entering the cells of the olfactory epithelium by acting as a calcium (Ca) antagonist, zebrafish larvae were co-exposed to 1, 5, or 10 µM Cd with 1 or 5 mM Ca for 96 h. Whole-body cadmium accumulation as quantified using ICP-MS (inductively coupled plasma mass spectrometry) was decreased in larvae co-exposed to cadmium and calcium. Additionally, induction of the heat shock response was reduced in the presence of increasing calcium co-treatment. These ameliorating effects of calcium were further revealed in cell death and histological analyses of the olfactory epithelium. Also, larvae co-exposed to cadmium and calcium exhibited greater olfactory sensory function compared to larvae exposed to cadmium only. Significant increases in aversion response were observed in larvae exposed to 5 µM Cd with 1 and 5 mM Ca (p<0.05). These results indicate that cadmium gains entry to the olfactory epithelium via calcium uptake systems, wherein it causes damage to the olfactory system and can lead to sensory impairment.

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Mercury (Hg) compounds are some of the most toxic compounds of any heavy metal on earth. Due to long-range transport from point sources Hg can be found world-wide in air, soil, water, and living organisms. Mercury compounds can cause a number of adverse effects, with the unborn fetus, infants, and children being most susceptible. Zebrafish (Danio rerio) are an excellent vertebrate model system for toxicological studies, including developmental effects. The overall objective of this research was to investigate the effects of inorganic forms of Hg in zebrafish larvae. Unique accumulation patterns were observed using synchrotron X-ray fluorescence imaging after zebrafish were exposed to one of four Hg compounds (i.e. mercuric chloride, mercury bis-L-cysteineate, methylmercury chloride, methylmercury L-cysteineate). Specifically, we noted chemical form dependant and tissue-specific Hg accumulation including the sensory cells of the olfactory epithelia and the neuromasts. Phenylthiourea (PTU) is commonly used to block zebrafish melanogenesis to generate transparent larvae to aid with enhanced visualization of immunohistochemical and vital stains. It was determined that PTU dramatically alters Hg toxicity through chemical interaction with Hg so that further studies were conducted in the absence of PTU. To investigate the effects of Hg on primary neurons, the immunohistochemistry protocol using anti-acetylated tubulin was performed and the results demonstrated that mercuric chloride damages primary neurons particularly in the olfactory pits. To study potential detoxification of Hg in zebrafish we examined the efficacy of two sequestration agents, dimercaptosuccinic acid and alpha lipoic acid, as well as endogenous selenium. The levels of Hg were not significantly lower following treatment with either sequestration agent under the conditions used in this research. Previous work examining the antagonistic relationship between Hg and selenium has been conducted by dosing animals with both Hg and selenium (Se). We discovered a mixed chalcogenide of the general form HgSxSe(1-x) forming in vivo following exposure to mercuric chloride without the addition of selenium. Indeed the selenium may have been remobilized from natural stores in the pigment spots. The research presented herein demonstrates that the target tissues for Hg depend strongly on chemical form. In particular inorganic Hg can accumulate in a number of important tissues including sensory systems. The formation of insoluble and non-toxic HgSxSe(1-x) in zebrafish larvae suggests that endogenous selenium may play critical roles in modulating toxicity.