Research ReportAcute copper exposure induces oxidative stress and cell death in lateral line hair cells of zebrafish larvae
Introduction
The death of hair cells induced by a variety of environmental factors has been extensively documented. The effects of acoustic trauma, aminoglycoside antibiotics and, more recently the anti-cancer drugs cisplatin and other platinum derivatives, have been used to study hair cell death and there is an accumulating body of literature on the mechanisms and clinical outcomes of damage induced by these agents (Cheng et al., 2005, Henderson et al., 2006, Rizzi and Hirose, 2007, Selimoglu, 2007). Hair cells in the mammalian ear are highly sensitive to oxidative stress induced by aminoglycosides and cisplatin, but loading them with antioxidant scavengers prior to exposure can effectively protect the cells (Priuska and Schacht, 1995, Clerici et al., 1996, Hirose et al., 1997, Ton and Parng, 2005). Both necrotic and apoptotic pathways of cell death have been documented in the mammalian inner ear after toxic or mechanical damage (Yang et al., 2004, Cheng et al., 2005, Rybak and Whitworth, 2005, Eshraghi and Van de Water, 2006, Bohne et al., 2007).
Recently, dissolved copper as CuSO4 in low micromolar concentrations has been reported to be toxic to hair cells, a finding first analyzed in lateral line neuromasts of zebrafish larvae (Hernández et al., 2006, Linbo et al., 2006). This sensory system is extremely sensitive to copper concentrations that do not affect the survival of the fish. Copper generates a dose-dependent toxicity in neuromasts, producing death of hair cells followed by proliferation and regeneration of these cells, or permanent damage if concentrations are higher than 50 μM (Hernández et al., 2006).
The lateral line system in zebrafish offers a unique experimental setting to study cellular and molecular events during hair cell death and regeneration, mainly because of the accessibility of neuromasts for examination and direct application of different compounds. Its utility has also been proven in chemical screens for assessing the effect of different ototoxic and otoprotective compounds (Ton and Parng, 2005, Owens et al., 2008). In zebrafish larvae, the lateral line is composed of neuromasts located at stereotypical positions on the head and the body. Neuromasts contain discrete hair cell clusters and two described populations of accessory cells: mantle cells which cover the apical surface of hair cells forming a single cell-wide sheet, and supporting cells forming a basal cell layer projecting apical processes that intredigitate with hair cells (Balak et al., 1990, Williams and Holder, 2000). The transparency of larvae and accessibility of neuromasts allows for live hair cell labeling with fluorescent markers of mechanotransduction such as FM1-43 (Gale et al., 2001, Meyers et al., 2003, Corey et al., 2004) and other membrane and nuclear stains (Murakami et al., 2003, Santos et al., 2006).
Copper is an essential micronutrient but misregulation of intracellular levels can become toxic to many cell types. In fishes this metal can accumulate when excess is found in the water though dietary sources are tightly regulated (Clearwater et al., 2002). One of the effects of copper toxicity in zebrafish is oxidative damage in gills and the liver as well as changes in the expression of genes involved in cellular respiration (Craig et al., 2007). In lateral line neuromasts of zebrafish larvae, copper concentrations beginning from 0.15 μM, can significantly diminish hair cell number in a matter of minutes (Linbo et al., 2006). Short treatments (2 h) with as low as 1 μM CuSO4 can completely eliminate all hair cells in neuromasts and higher concentrations differently affect accessory cells and the regeneration capacity of this sensory system (Hernández et al., 2006, Hernández et al., 2007).
Several methods have been used to characterize hair cell and accessory cell damage, including staining of hair cells with vital dyes (Daspei, Di-Asp and FM1-43), transgenic lines that label neuromasts with GFP, Acridine Orange, immunofluorescence, light microscopy and scanning electron microscopy. These methods were useful to demonstrate that toxicity produced by these copper concentrations is restricted to neuromasts, hair cells being the most sensitive cell type, and to characterize the temporal and concentration-dependent aspects of hair cell disappearance. However, the occurrence of specific cell death pathways and the molecular events associated with copper exposure in hair cells remain unclear.
In the present work we sought to provide insights on processes occurring during copper-induced toxicity in hair cells using the zebrafish model. As a first approximation we developed a simple method to identify dying hair cells in lateral line neuromasts in vivo, using confocal microscopy and fluorescent dyes to monitor hair cell morphology during exposure. Hair cells were stained with two dyes, one for membrane labeling (Bodipy TR C5-ceramide) and a second with DNA binding properties (Sytogreen 24). In this way we could visualize HC shape and the fate of nuclear material during copper exposure. We analyzed the effects of 1 and 10 μM CuSO4, copper concentrations previously used to study hair cell regeneration (Hernández et al., 2007), and we compared the outcomes that these two conditions produce in terms of the death pathways followed by HCs. To characterize further events occurring in hair cells exposed to copper we used transmission electron microscopy to observe dead cells in neuromasts. DNA fragmentation and ROS production was analyzed using methods such as TUNEL, antioxidant otoprotection and dichlorodihydrofluorescein labeling.
Our results show that copper exposure elicits necrotic hair cell death in neuromasts, an effect that is partially prevented by antioxidants. In addition, we detect significant DNA damage and an increase in ROS levels in copper-exposed hair cells.
Section snippets
Hair cell death in neuromasts recognized by confocal microscopy and fluorescent vital dye staining
Hair cell (HC) labeling was performed in 3-day-old larvae (72 hpf), by incubating them in a mix of Bodipy TR C5-ceramide and Sytogreen 24 in fish medium, at concentrations that result in only HC staining (see Experimental procedures). Labeled neuromasts in untreated fish were monitored over time and no morphological changes were observed 15 h after staining, indicating that these dyes produce no toxic effects on HCs. Specific labeling of HCs was confirmed using the SqET4 transgenic line, which
Discussion
Lateral line neuromasts in zebrafish larvae have emerged as a very useful model to study hair cell ototoxicity and regeneration. Recent studies have validated this model showing that lateral line hair cells are sensitive to aminoglycosides and cisplatin (Song et al., 1995, Williams and Holder, 2000, Murakami et al., 2003, Harris et al., 2003, Ton and Parng, 2005, Santos et al., 2006, Owens et al., 2007, Ou et al., 2007) demonstrating a functional parallel with hair cells found in the mammalian
Maintenance of zebrafish, copper application and image acquisition
Our own breeding colony of the Tübingen wild-type strain of zebrafish (Danio rerio) was maintained at 28.5 °C on a 14-h light/10-h dark cycle (Westerfield, 1994). The transgenic strains SqET4 and SqET20 (previously known as ET4 and ET20) were obtained from Vladimir Korzh (Parinov et al., 2004). All embryos were collected by natural spawning, staged according to Kimmel et al. (1995), and were raised at 28 °C in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.3 mM MgSO4, and 0.1% methylene
Acknowledgments
We are especially indebted to Julieta González and Nancy Olea for discussions and help with electron microscopy. We thank Vladimir Korzh and Hernán López-Schier for transgenic fish lines and valuable reagents. Catalina Lafourcade and Florencio Espinoza provided technical help. This work was supported by the Millennium Science Initiative, Grant number P06-039F and by FONDECYT, Grant number 1070867.
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