Metals Toxicity as Seen from The Rainbow Trout Transcriptome

Although lethal toxicity of cationic metals in fish has been well-studied, the molecular determinants of how toxicity varies across metal, exposure concentration, and exposure duration are not well understood. Therefore, we compared transcriptome changes in gill and liver of juvenile rainbow trout (Oncorhynchus mykiss) exposed to copper (Cu), cadmium (Cd), or zinc (Zn) to control laboratory waters, 17, or 50% of available metal-specific 96-hour LC50 concentrations for 48 and 96 hours (N=6 within each of 36 sample groups). From 190 of 216 fish, 15,305,931 ± 25,483 trimmed Illumina reads/sample were aligned to an Omyk_RefSeq template consisting of 79,244 transcripts. Expression levels compared to controls were normalized with DESeq2, and a combined set of 4664 differentially expressed genes (DEGs) were identified (i.e., ≥2-fold change, corrected P ≤0.05 & 95% confidence). The number of DEGs differed by metal (P<0.05); Cu (3751), Zn (1047), and Cd (24), with only a single DEG shared by all 3 metals. While Cu primarily affected gene expression in the liver, Cd and Zn effects were concentrated within gill tissue. The total number of DEGs increased with exposure duration (P<0.05), but not exposure concentration (P=0.864). Principal components analysis showed that PC1 accounted for 63.6% of the variation in the expression data and was associated with tissue differences, with the influence of metal being secondary. Gene Ontology (GO) enrichment analyses were performed for all up and down regulated DEG sets (corrected P≤0.05, FDR ≤ 0.05). A total of 1484 GOs were enriched for 18 DEG sets. Interestingly, the single shared transcript had 76 GO annotations plus 3 enzyme codes related to transferring phosphorous-containing groups. Altered expression of DEGs coding for enzymes occurred in 112 KEGG pathways. Six of the pathways were affected by 10 or more treatment conditions. Most notably, expression of genes for multiple enzymes in purine and thiamine metabolism pathways were variously changed for 15 and 13 of the DEG sets, respectively, which spanned all metals, durations, concentrations, and tissues. The unique gene expression patterns across metals and tissues suggests that each metal may bind tissue sites differently during exposure; however, there appears to be some convergence among impacted downstream biochemical pathways that warrants further investigation.

Impact/Purpose

Metals occur in surface waters naturally but may be elevated as a result of various human and non-human activities. Aquatic life, including Federally Threatened and Endangered Species (T&E Species) may be exposed and experience ill effects. EPA regularly conducts ecological risk assessments to determine if its decisions incorporate an understanding of the sensitivity of T&E Species to metals exposure. However, because toxicity information will generally be limited for Listed species, new approaches are needed to help the agency use existing toxicity information for its assessments. Using advanced genomics tools and focusing their initial work on a Salmonid species, the rainbow trout, the authors have conducted an analysis to determine if the commonly occurring and toxic metals, cadmium, zine, and copper, exert their toxicity through similar or unique molecular processes. Understanding the mode of action in this way provides a stronger basis for a more informed use of existing toxicity data. The authors found that although the three metals appear to differently bind to biological receptor sites, a common biochemical pathway was affected by all three metals. Such knowledge indicates that the extrapolation of metals toxicity data has a defined biological basis. Additionally, this information provides a point of focus for their future work in which multiple species will be exposed to zinc, so as to more fully evaluate the biological basis for metals toxicity data extrapolation during risk assessments focused on T&E Species. The project aims to enhance the scientific basis for ecological risk assessment work at EPA and beyond in order to more efficiently accomplish the mission to protect environmental health.

Citation

Reichman, J., R. Hockett, H. Ren, A. Fisher, James Markwiese, AND M. Jankowski. Metals Toxicity as Seen from The Rainbow Trout Transcriptome. SETAC North America, 41st Annual Meeting, Virtual, OR, November 15 - 19, 2020.