Attributing Effects of Aqueous C 60 Nano-Aggregates to Tetrahydrofuran Decomposition Products in Larval Zebrafish by Assessment of Gene Expression

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Abstract
C(60) is a highly insoluble nanoparticle that can form colloidal suspended aggregates in water, which may lead to environmental exposure in aquatic organisms. Previous research has indicated toxicity from C(60) aggregate; however, effects could be because of tetrahydrofuran (THF) vehicle used to prepare aggregates. Our goal was to investigate changes in survival and gene expression in larval zebrafish Danio rerio after exposure to aggregates of C(60) prepared by two methods: a) stirring and sonication of C(60) in water (C(60)-water); and b) suspension of C(60) in THF followed by rotovaping, resuspension in water, and sparging with nitrogen gas (THF-C(60)). Survival of larval zebrafish was reduced in THF-C(60) and THF-water but not in C(60)-water. The greatest differences in gene expression were observed in fish exposed to THF-C(60) and most (182) of these genes were similarly expressed in fish exposed to THF-water. Significant up-regulation (3- to 7-fold) of genes involved in controlling oxidative damage was observed after exposure to THF-C(60) and THF-water. Analyses of THF-C(60) and THF-water by gas chromatography-mass spectrometry did not detect THF but found THF oxidation products gamma-butyrolactone and tetrahydro-2-furanol. Toxicity of gamma-butyrolactone (72-hr lethal concentration predicted to kill 50% was 47 ppm) indicated effects in THF treatments can result from gamma-butyrolactone toxicity. This research is the first to link toxic effects directly to a THF degradation product (gamma-butyrolactone) rather than to C(60) and may explain toxicity attributed to C(60) in other investigations. The present work was first presented at the meeting "Overcoming Obstacles to Effective Research Design in Nanotoxicology" held 24-26 April 2006 in Cambridge, Massachusetts, USA.