Grantee Research Project Results

EPA is supporting the development of technologies to better identify and monitor sources of pollution and protect water quality.  Microarrays are a promising new technology; however, so far they have mainly been employed to identify the mechanisms of toxicity.  The aim of this work was to show that Ceriodaphnia dubia microarrays can be a practical technology for identifying, characterizing, and monitoring environmental impact.  This work showed that microarrays can provide information on degree of environmental impact and on the identity and effects of multiple contaminants in complex mixtures.

Three reference chemicals (diazinon, chlorpyrifos, and copper) whose toxic effects on C. dubia have been well defined by conventional testing were used to generate gene expression signatures.  Exposure to each chemical was tested individually or as a binary mixture of diazinon with chlorpyrifos or copper over a range of concentrations.  We measured lethality and gene expression at each concentration with three independent repeats for each concentration of the three individual toxins.  Gene expression data was generated by isolating RNA from both toxin exposed and control unexposed C. dubia populations.  The RNA was fluorescently labeled and hybridized to a 10,000-element spotted cDNA array developed in previous work.  Microarrays were scanned using a GenePix 4000B to quantify Cy3 and Cy5 fluorescence. Scanned data were imported into Acuity4.0 (Molecular Devices), Lowess normalized, and screened for significance using a t-test.  From the results of these analyses, probes with p < 0.05 and a greater than 2.0-fold change in either direction was considered differentially expressed.

We hoped to show that it is feasible to use microarrays.  They can provide information on degree of environmental impact and on the identity and effects of multiple contaminants in complex mixtures.  We identified different expression signatures at different levels of toxin concentration—likely corresponding to specific toxicity responses at low dose, and more general stress and cell death responses at higher doses where acute lethality became apparent.  We found that, as expected, diazinon and chlorpyrifos expression effects were more similar to each other than to the effects of copper as measured using hierarchical clustering.  Additionally, the lethality and gene expression signatures resulting from binary mixtures of the three chemicals were examined and compared to the signatures of the individual components.  Importantly, we found that the expression signature for binary mixtures had significant and identifiable similarity to the signatures for each of the component toxins.

By the end of Phase II work, we will have commercially available environmental toxicity testing microarrays that will enable practical and powerful assessment characterization and monitoring of environmental contaminants in applications ranging from Toxicity Identification Evaluation (TIE) studies to new chemical registration to Whole Effluent Toxicity (WET) testing.  Microarrays will be able to identify synergistic toxicities, identifying conditions with ecologically harmful effects even though individual contaminant concentrations do not exceed critical thresholds.