Daphnia magna Ecotoxicogenomics Provides Mechanistic Insights into Metal Toxicity

Abstract

Toxicogenomics has provided innovative approaches to chemical screening, risk assessment, and predictive toxicology. If applied to ecotoxicology, genomics tools could greatly enhance the ability to understand the modes of toxicity in environmentally relevant organisms. Daphnia magna, a small aquatic crustacean, is considered a "keystone" species in ecological food webs and is an indicator species for toxicant exposure. Our objective was to demonstrate the potential utility of gene expression profiling in ecotoxicology by identifying novel biomarkers and uncovering potential modes of action in D. magna. Using a custom D. magna cDNA microarray, we identified distinct expression profiles in response to sublethal copper, cadmium, and zinc exposures and discovered specific biomarkers of exposure including two probable metallothioneins, and a ferritin mRNA with a functional IRE. The gene expression patterns support known mechanisms of metal toxicity and reveal novel modes of action including zinc inhibition of chitinase activity. By integrating gene expression profiling into an environmentally important organism, this study provides experimental support for the utility of ecotoxicogenomics.

Gene Expression Profiling in Daphnia magna Part I: Concentration-Dependent Profiles Provide Support for the No Observed Transcriptional Effect Level

“…The complete list of differentially expressed genes organized into their functional categories is shown in Table S3 in the Supporting Information. We also included data from our previous study at the 1/10 LC50 for comparison (6). The log2 ratio averaged over all six microarray hybridizations is also shown so expression levels can be compared across the different treatments.…”

“…The results of the q-RT-PCR analysis compared to microarray ratios are shown in Table S4 in the Supporting Information. We previously carried out confirmatory q-RT-PCR for several genes at one concentration including monooxygenase, IMPase and GST (6); in the present study, we assayed the differential expression of these genes across different concentrations. For the majority of these genes, the q-RT-PCR results confirm the differential expression determined by microarray analysis thereby providing support for their use as biomarkers of exposure.…”

“…We therefore expanded upon our initial study, which examined gene expression profiles for the metal toxicants, Cu, Cd, and Zn, in the ecotoxicology model organism, Daphnia magna (6), to include a range of biologically relevant concentrations. This study reported the gene expression patterns for Cu, Cd, and Zn at the 1/10 LC50, representing a sublethal concentration that resulted in chronic effects to reproduction.…”

Gene Expression Profiling in Daphnia magna, Part II: Validation of a Copper Specific Gene Expression Signature with Effluent from Two Copper Mines in California

“…We selected candidate biomarkers that were both associated with Cu or general metal exposure from our previous microarray studies (6,10) and differentially expressed in the five mine samples containing high levels of Cu. These include biomarkers predicative of general metal exposure, metallothionein (MT) (a) DV437799, monoxygenase DV437798, slit homologue DV437805, cellulase DV437797, and preamylase ES408209, and those specific for Cu exposure, inositol monophosphatase (IMPase) DV437806, lectin DV437813, and ES408276.…”

“…Because three exposures were performed for each metal, and RNA from each exposure was hybridized to two different microarrays, there were six hybridizations for each exposure condition. Details related to the construction of the D. magna microarray and the microarray hybridization procedure have been described previously (6) and are available in the Supporting Information. Information about experimental design, raw signal intensity values, and other MAIME compliant data are available at the Gene Expression Omnibus (GEO) (located at http://www.ncbi.nlm.nih.gov/geo) with the accession numbers GSE7666 (Walker Mine data) and GSE7667 (Greenhorn Mine data).…”