Using synchrotron x-ray fluorescence mapping, we have examined the uptake and localization of organic mercury in zebrafish larvae. Strikingly, the greatest accumulation of methyl and ethyl mercury compounds was highly localized in the rapidly dividing lens epithelium, with lower levels going to brain, optic nerve, and various other organs. The data suggest that the reported impairment of visual processes by mercury may arise not only from previously reported neurological effects, but also from direct effects on the ocular tissue. This novel approach is a powerful tool for directly investigating the molecular toxicology of heavy metals, and should be equally applicable to the study of a wide range of elements in developing embryos.methylmercury ͉ thimerosal ͉ x-ray fluorescence mapping ͉ eye lens T oxic organic mercury compounds worry many communities worldwide, yet the detailed mechanisms underlying their transport and toxicity remain uncertain (1). These neurotoxic compounds are particularly insidious due to the latency in onset of toxic symptoms and have caused several devastating masspoisonings of humans. Adults are affected, but exposure in utero has resulted in severe consequences such as microcephaly, cerebropalsy, seizures, and mental retardation. Methylmercury (MeHg) compounds are actively transported across cell membranes (2) although not, as originally thought, by molecular mimicry of methionine (3). Beyond this, however, our knowledge of the mechanisms underlying organic mercury (Hg) toxicity is fragmentary. MeHg-induced changes in cellular Ca 2ϩ have been shown to be important (4-7), as has oxidative stress (4), and glutamate metabolism (8). In both yeast and human cells, overexpression of the ubiquitin-targeting enzyme Cdc34 confers protection against the cytotoxic effects of MeHg, which leads to the suggestion that an unknown protein containing a signal for ubiquitination by Cdc34 is involved in the development of the cytotoxic effects of MeHg (9-11).Zebrafish (Danio rerio) are common model organisms for the study of embryonic development, and have found increasing use in vertebrate toxicology (12). While embryonic and larval zebrafish previously have been used to study the toxic effects of MeHg exposure at the whole body (13,14) and at the molecular level (15), there are no available data on MeHg uptake and accumulation with respect to different tissues and organs. Such information is critical to properly integrate information on tissue and cell specific impacts of Hg with uptake and accumulation of the metal at the whole animal level. We present herein a novel approach to the investigation of heavy-metal toxicity using synchrotron x-ray fluorescence imaging (16) to directly image metal localization within zebrafish larvae and to observe remarkable differential accumulation of organic Hg using this technique. Strikingly, we find that both methyl and ethyl Hg derivatives are concentrated to the greatest degree in lens epithelium with lower levels present in the brain, optic nerve, and other organs....

The toxic effects of cadmium and other heavy metals have been well established, and many of these and other environmental pollutants are known to be embryotoxic or teratogenic. However, it has proven difficult to identify individual cells that respond to toxicants among the wide range of cell populations in an intact animal, particularly during early development when cells are continually changing their molecular and physiologic characteristics as they differentiate. Here we report the establishment of an in vivo system that uses hsp70 gene activation as a measure of cadmium toxicity in living early larvae of transgenic zebrafish carrying a stably integrated hsp70-enhanced green fluorescent protein (eGFP) reporter gene. We demonstrate that eGFP expression in this strain of fish acts as an accurate and reproducible indicator of cell-specific induction of hsp70 gene expression. Furthermore, the transgene responds in a dose-dependent manner at concentrations similar to those observed for morphologic indicators of early-life-stage toxicity and is sensitive enough to detect cadmium at doses below the median combined adverse effect concentration and the median lethal concentration. The stable nature of this transgenic line should allow for extremely rapid and reproducible toxicologic profiling of embryos and larvae throughout development.

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In the present study, we show that the stress-inducible hsp70 gene in zebrafish is strongly and specifically expressed during normal lens formation from 28 to 38 hours post-fertilization, and is subsequently downregulated by 2 days of age. Only weak constitutive hsp70 mRNA signal was sporadically observed in other embryonic tissues. Similarly, transgenic fish carrying a 1.5 kb fragment of the hsp70 promoter linked to eGFP exhibited fluorescence only in the lens. In contrast, both the endogenous hsp70 gene and the transgene were strongly expressed throughout the embryo following heat shock at the same developmental stages.

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