ResultsDeveloping an Aquatic Toxicity Biomarker using Hemoglobin Gene Expression Bonnie Chang Abstract Current toxicity protocol involving acute and chronic toxicity assays provide little information about the mechanism of toxicity and are limited in their sensitivity. The use of gene expression as a biomarker for environmental toxicity is a recent concept. By looking at the expression of specific genes, it may be possible to see the effects and mechanisms of toxicity at sub-lethal levels. In this experiment, we studied the expression of the hemoglobin gene in Daphnia magna after metal exposure. Hemoglobin RNA was extracted from Daphnia magna exposed to different concentration levels of copper, cadmium, and lead. Two exposure levels derived from acute toxicity assays were used for each metal: the NOEC (no observable effect concentration), and the midpoint between the NOEC and LC50. Expression levels of hemoglobin were compared using northern analysis. It was found that the daphnia hemoglobin gene was greatly expressed following lead exposure. There was also a difference in expression between the two lead concentrations. Little to no expression of the hemoglobin gene was expressed following copper and cadmium exposure. The results indicate that the hemoglobin gene may be specific to lead toxicity. More importantly, the difference in expression indicates that hemoglobin gene expression may serve as a biomarker for lead toxicity upon further studies. The analysis suggests that changes in gene expression may be a more biologically relevant predictor of environmental impact because changes were detected at sub-lethal levels, making it a more sensitive test.Introduction Economic growth does not come without sacrifice. Over the past two centuries, the increasing need for minerals in industry has created some of the worst hazardous waste areas in the United States (van Geen and Luoma 1999). Although metals are naturally occurring chemicals, human influences have altered their distribution in the environment (van Geen and Luoma 1999; Eaton 1979). By extracting metals from ores, manipulating their chemical speciation in industry, and dumping them into the environment in altered forms, humans have disrupted the natural processes that govern the fate of metals (Waalkes 1995). Despite our extensive knowledge concerning the deleterious effects of metal contamination on aquatic ecosystems, few reliable methods for assessing the level of this contamination exist. Direct measurement of the concentration of metals requires prior knowledge of the contaminants present and can be confounded by the speciation of the metals in the ecosystem, which can drastically alter their toxicity. Acute and chronic toxicity assays, which target a specific organism within the ecosystem, do not reveal the toxicant responsible for the contamination and also provide little information about the mechanism of toxicity (Chapman 2000). A more sensitive test is necessary to accurately assess the ecological impacts of metal contamination. Changes in gene expression are sensitive indicators of stress to an organism (Far and Dunn 1999). Measuring these changes after toxicant exposure may provide information about the sub-lethal effects of a toxicant to an organism. There is also the possibility of linking changes in gene expression to specific toxicants (Nuwaysir 1999). In a recent study by Custodia et al. using Caenorhabditis elegans, the xenobiotic response to varying concentrations of a vertebrate steroid hormone was correlated with gene expression changes (Poynton 2003, pers comm; Custodia 2001). The objective of this research is to develop and validate a new method to assess the effects of toxicants to an ecosystem by measuring the changes in hemoglobin gene expression in a common water toxicity bioassay organism, Daphnia magna. Daphnia are often used as indicators of contamination (USEPA 1993); therefore, effects on this organism can be used to predict ecosystem health. Hemoglobin levels in daphnia are affected by metal exposure. Previous studies have found a complex response pattern of hemoglobin content after exposure to copper,cadmium, and lead (Dave 1984; Berglind 1985; Berglind et al. 1985). This research will further elaborate on these studies by measuring the gene expression of hemoglobin in daphnia after exposure to copper, cadmium, and lead. This research will address the hypothesis that the hemoglobin gene in Daphnia magna is differentially expressed at different levels of metal exposure. These results will serve as a benchmark for further studies on gene expression in daphnia using cDNA microarrays. Further studies may include different genes (such as the metallothioneins) and a wider array of metals. Once gene expression profiles are established, it may become possible to use gene expression as an accurate bio-indicator of the ecological impacts of metal contaminants. Methods This study was done in Chris Vulpe's lab at the University of California-Berkeley. Daphnia magna was cultured according to standard USEPA protocol (1993). The metals involved in this study were copper, cadmium, and lead. These metals were chosen based on their relative concentrations in the environment and their toxicity to aquatic life (Crosby 1998). Concentrations of copper, cadmium, and lead were made using CuSO4, CdSO4, and PbSO4 solutions, respectively. The amount of sulfate added were negligible compared to the amount that existed in the media. There were four components to the experiment. (1) Test concentrations for copper cadmium and lead were established with an acute toxicity test. LC50’s were determined in order to establish lethal endpoints. Two sub-lethal concentrations below the LC50 were used in this study: the NOEC (no observable effect concentrations), and the 1/2 LC50 (midpoint between the NOEC and LC50). (2) Daphnia magna were exposed for 24 hours to the NOEC and 1/2 LC50 of each metal. (3) RNA immediately extracted from the exposed daphnia. (4) Hemoglobin levels were determined by northern analysis. The results were visually analyzed due to the lack of an easily quantifiable technique. A difference in gene expression was shown by a difference in band intensity (brightness and length). An acute toxicity assay was used to find the NOEC and 1/2 LC50. The test was conducted using a procedure similar to USEPA Whole Effluent Toxicity (WET) protocol (1993). Acute toxicity
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