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Microarray analysis of CL-20 reversible neurotoxicity in the earthworm Eisenia fetida

ABSTRACT: The earthworm Eisenia fetida is one of the most used species in standardized soil ecotoxicity tests. Endpoints such as survival, growth and reproduction are ecologically relevant but provide little mechanistic insight into the toxicity pathways, especially at the molecular level. To better understand toxicological modes of action and to facilitate the development of molecular biomarkers, we have obtained 30,245 unique EST sequences from E. fetida and have designed a novel microarray with 15,119 60-mer oligonucleotide probes. These probes target the unique non-redundant EST sequences identified in E. fetida. Using this array we have profiled gene expression of E. fetida after exposure to CL-20, a cage cyclic nitramine previously found exhibiting reversible neurotoxicity to worms. Worms were exposed for 6 days to CL-20. Half of the exposed worms were allowed to recover in a clean environment for 7 days. Electrophysiological analysis showed that the conduction velocity of worm medial giant nerve fiber was significantly decreased after 6-d exposure to CL-20, and that giant nerve fiber function was restored at the end of the 7-d recovery period. Total RNA samples isolated from four treatment groups (6 replicates per group), i.e., 6-d control, 6-d exposed, 13-d control and 6-d exposed with 7-d recovery, were analyzed using the new 15K oligo array. Bioinformatics and statistical analyses have identified specific neurological pathways affected by CL-20 and recovery of these pathways after CL-20 removal. These results provide significant insights on the CL-20 toxic mode of action and how earthworms can recover from chemical stressors.

ORGANISM(S): Eisenia fetida

PROVIDER: GSE18536 | GEO | 2009/10/14

SECONDARY ACCESSION(S): PRJNA121335

REPOSITORIES: GEO

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Project description:The earthworm Eisenia fetida is one of the most used species in standardized soil ecotoxicity tests. Endpoints such as survival, growth and reproduction are ecologically relevant but provide little mechanistic insight into the toxicity pathways, especially at the molecular level. To better understand toxicological modes of action and to facilitate the development of molecular biomarkers, we have obtained 30,245 unique EST sequences from E. fetida and have designed a novel microarray with 15,119 60-mer oligonucleotide probes. These probes target the unique non-redundant EST sequences identified in E. fetida. Using this array we have profiled gene expression of E. fetida after exposure to CL-20, a cage cyclic nitramine previously found exhibiting reversible neurotoxicity to worms. Worms were exposed for 6 days to CL-20. Half of the exposed worms were allowed to recover in a clean environment for 7 days. Electrophysiological analysis showed that the conduction velocity of worm medial giant nerve fiber was significantly decreased after 6-d exposure to CL-20, and that giant nerve fiber function was restored at the end of the 7-d recovery period. Total RNA samples isolated from four treatment groups (6 replicates per group), i.e., 6-d control, 6-d exposed, 13-d control and 6-d exposed with 7-d recovery, were analyzed using the new 15K oligo array. Bioinformatics and statistical analyses have identified specific neurological pathways affected by CL-20 and recovery of these pathways after CL-20 removal. These results provide significant insights on the CL-20 toxic mode of action and how earthworms can recover from chemical stressors. Adult earthworms (E. fetida) were exposed on filter paper to CL-20 (0.2 ug/cm2) for 6 days with or without 7-day recovery (4 treatment groups in total). Each treatment group had 9 replicate worms, six of which were used for gene expression analysis. Worms were measured for their medial giant nerve fiber conduction velocity using a non-invasive electrophysiological technique immediately before takedown. At the termination of the 6-d or 13-d experiment, worms were snap-frozen and fixed in RNAlater-ICE. Total RNA was isolated from the fixed worms. A total of 24 worm RNA samples were hybridized to three 8x15K custom-designed Agilent oligo arrays using Agilentâs one-color Low RNA Input Linear Amplification Kit. The array contains 15,208 non-redundant 60-mer probes, each targeting a unique E. fetida transcript. After hybridization and scanning, gene expression data were acquired using GenePix Pro 6.0.

Project description:We utilized a candidate gene approach using custom microarray constructed for our study species Drosophila montana and D. virilis to identify genes with modulated expression patterns under low temperature conditions. The flies were exposed to four different treatments (+5°C for 6 days, 0°C for 1 hour, two periods of recovery after cold stress and a control treatment). The aim of the study was to identify potential cold-responsive genes and to investigate differences in gene expression between the species. Microarray analysis revealed altogether 31 out of 219 genes on the array to show expression changes during different stages of cold stress. Among the potential stress tolerance genes detected earlier in D. melanogaster, hsr-omega was upregulated in both species during cold acclimation, expression changes in other genes being treatment- and species specific. Our microarray study clearly showed that different stages of cold response elicit changes at least in genes involved in heat shock response, circadian rhythm and metabolism. Cold- induced gene expression was investigated comparing four different treatments to the control treatment: 1) Cold acclimation: 14 days in control conditions, then 6 days at +5°C 2) Cold hardening: 20 days in control conditions, then1 hour at 0°C 3) 15-min recovery from chill coma: the 20-day-old flies were exposed to -6°C for 16 hours, after which they were let to recover for 15 minutes 4) 1-hour recovery from chill coma: the 20-day-old flies were exposed to -6°C for 16 hours, after which they were let to recover for 1 hour. In control treatment flies were kept for 20 days at 19°C. All the flies were 20-days old at the time of sample collection, and the light:dark cycle was 22 hours of light and 2 hours of dark. Because of the limited space on the array plate, the recovery samples were collected only for D. montana. All the samples were collected 5-6 hours after the lights had been turned on in the chamber and the flies were immediately immersed in liquid nitrogen, after which they were stored at -84ºC. Three pools of ten flies were collected from each treatment group.

Project description:Motivation: Monitoring, assessment and prediction of environmental risks that chemicals pose demand rapid and accurate diagnostic assays. A variety of toxicological effects have been associated with explosive compounds TNT, RDX and HMX. One important goal of microarray experiments is to discover novel biomarker genes for quantitative phenotypic prediction. We have developed an earthworm microarray containing 15,208 unique oligo probes. Our objective was to identify biomarker genes that can be used to quantitatively predict earthworm tissue residues of the explosives compounds that they were exposed to and took in from the HMX-spiked soil. Results: We collected a large microarray gene expression and earthworm tissue residue dataset. First, differentially expressed genes were identified for each exposure duration (4, 14 and 28 days). These genes were used in multivariate regression modeling for HMX residue prediction. Eighteen different regression models were tested and compared. The best performing model was able to achieve very high prediction accuracies with R2 values of 0.715, 0.728 and 0.822 for 4 days, 14 days and 28 days exposures, separately. Conclusions: This study demonstrated that multivariate regression coupled with high throughput microarray gene expression was a promising approach to quantitative phenotypic prediction. Adult earthworms (Eisenia fetida) were exposed in soil spiked with HMX (0, 8, 16, 32, 64, or 128 mg/kg) for 4, 14 or 28 days. Each treatment originally had 10 replicate worms with all 10 worms survived at the end of exposure. Total RNA was isolated from the surviving worms. A total of 120 worm RNA samples were hybridized to a custom-designed oligo array using AgilentM-bM-^@M-^Ys one-color Low RNA Input Linear Amplification Kit. The array contains 15,208 non-redundant 60-mer probes, each targeting a unique E. fetida transcript. After hybridization and scanning, gene expression data were acquired using AgilentM-bM-^@M-^Ys Feature Extraction Software (v.9.1.3). The 120-array dataset consists of three exposure groups (4 day, 14 day and 28 day) with each group having the following 8 treatments: day 0 control, blank control, solvent control, 8, 16, 32, 64, and 128 mg HMX/g soil. Each treatment had 5 biological replicates (i.e., five individual worms).

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Microarray analysis of CL-20 reversible neurotoxicity in the earthworm Eisenia fetida

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