Zebrafish larvae were exposed to IMX-101 at 0, 3.8, 30.2 and 58.2 mg/L (measured concentrations). Danio rerio larvae were exposed in static nonrenewal acute 96-h bioassays. Exposure chambers were 300-ml high-form lipless beakers with a test solution volume of 250 ml. Ten larval fish per beaker were exposed to IMX-101 where all treatments included 4 exposure replicates. Larval zebrafish were fed newly hatched Artemia nauplii two hours before the initiation of exposures and at the 48-h timepoint. Surviving fish were enumerated daily and any fish found to be deceased were promptly removed from exposure chambers.
Growth protocol
Exposure chambers were 300-ml high-form lipless beakers with a test solution volume of 250 ml. Ten larval fish per beaker were exposed in the IMX-101 exposures where all treatments included 4 exposure replicates. Larval zebrafish were fed newly hatched Artemia nauplii two hours before the initiation of exposures and at the 48-h timepoint.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from 4 whole fish/replicate for all transcript expression experiments. All samples were flash frozen upon collection and stored at -80 °C until RNA extraction. Samples were homogenized in lysis buffer with a FAST Prep-24 instrument (MP Biomedicals, Santa Ana, CA) before RNA isolation with RNeasy kits (Qiagen, Valencia, CA). Total RNA concentrations were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop technologies, Wilmington, DE, USA). The integrity of total RNA was assessed on an Agilent 2100 Bioanalyzer (Palo Alto, CA). Criteria for acceptable RNA integrity included a RNA integrity number >7.0 from the Agilent 2100 Bioanalyzer and a 260/280 spectrophotometric reading >2.0.
Label
Cy3
Label protocol
The Agilent Low-Input Quick Amp, One Color Microarray Hybridization protocol (Agilent Technologies) was utilized following manufacturer’s recommendations where 600 ng of total RNA served as the starting material for each biological replicate.
Hybridization protocol
All samples were hybridized to Agilent 8 x 60K, zebrafish microarrays (AMADID #36575). Microarray hybridization experimental designs for IMX-101 exposures were completely randomized (using a random number generator). Exposure concentrations were selected to represent both sublethal concentrations and concentrations beyond the threshold of lethality to facilitate identification of mechanisms of toxicity. The IMX-101 experiment included the measured exposure concentrations: 3.8, 30.2 and 85.2 mg/L (measured concentrations) each including 4 replicates, in addition to 4 control (0 mg/L) exposure replicates. Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the custom-designed Agilent test array for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed at room temperature with GE Wash Buffer 1 (Agilent) and with 37°C GE Wash buffer 2 (Agilent).
Scan protocol
An Agilent Surescan Microarray Scanner (G2505 C, Agilent Technologies Inc.) was used to scan microarrays at 2 μm resolution.
Data processing
Data were extracted from microarray images using Agilent Feature Extraction software (Agilent Technologies). Analysis of internal control spikes indicated that signal data was within the linear range of detection. Microarray data were normalized to the 75th percentile within each array followed by median scaling among all exposures using GeneSpring Software version GX14.5 (Agilent Technologies). GeneSpring was additionally used to conduct statistical analyses including gene set enrichment analysis (GSEA) and ANOVA-based differential transcript expression analyses. GSEA was conducted for IMX-101. The “50 hallmark gene sets” developed and curated by the Broad Institute (Liberzon et al 2015) were used to provide exploratory screening for biological processes affected by the IMX-101 exposure. Algorithm parameters included a minimum of 15 matching genes within a gene set, a maximum number of 100 permutations and a false discovery rate q-value cut off ≤ 0.3. In order to rank the importance of significant gene sets identified by GSEA, composite scores were developed using four criteria derived from the GSEA output where significant p-value (p = 0.05), significant q-value (p = 0.3), an enrichment score > |0.5|, and a normalized enrichment score > |1.5| each received scores of 0 or 1 depending if the criteria were met. Additionally, one-way ANOVA was conducted to assess the effects on transcript expression relative to controls. In the one-way ANOVA tests a p-value cutoff of 0.005 was utilized to identify significantly affected transcripts followed by post hoc pairwise tests comparing expression for each dose-level relative to controls by moderated t-test (p = 0.05) and a 1.5 fold change cut-off.
