Gene Expression Analysis of CL-20-Induced Reversible Neurotoxicity Reveals GABA_AReceptors as Potential Targets in the EarthwormEisenia 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 eco-toxicologically relevant but provide little mechanistic insight into toxicity pathways, especially at the molecular level. Here we applied a toxicogenomic approach to investigate the mode of action underlying the reversible neurotoxicity of hexanitrohexaazaisowurtzitane (CL-20), a cyclic nitroamine explosives compound. We developed an E. fetida-specific shotgun microarray targeting 15119 unique E. fetida transcripts. Using this array we profiled gene expression in E. fetida in response to exposure to CL-20. Eighteen earthworms were exposed for 6 days to 0.2 μg/cm2 of CL-20 on filter paper, half of which were allowed to recover in a clean environment for 7 days. Nine vehicle control earthworms were sacrificed at day 6 and 13, separately. Electrophysiological measurements indicated that the conduction velocity of earthworm medial giant nerve fiber decreased significantly after 6-day exposure to CL-20, but was restored after 7 days of recovery. Total RNA was isolated from the four treatment groups including 6-day control, 6-day exposed, 13-day control and 13-day exposed (i.e. 6-day exposure followed by 7-day recovery), and was hybridized to the 15K shot-gun oligo array. Statistical and bioinformatic analyses suggest that CL-20 initiated neurotoxicity by non-competitively blocking the ligand-gated GABAA receptor ion channel, leading to altered expression of genes involved in GABAergic, cholinergic, and Agrin-MuSK pathways. In the recovery phase, expression of affected genes returned to normality, possibly as a result of autophagy and CL-20 dissociation/metabolism. This study provides significant insights into potential mechanisms of CL-20-induced neurotoxicity and the recovery of earthworms from transient neurotoxicity stress.

Comparative toxicogenomics of three insensitive munitions constituents 2,4-dinitroanisole, nitroguanidine and nitrotriazolone in the soil nematode Caenorhabditis elegans

“…One hundred ng of total RNA was first reverse-transcribed into cDNA, followed by cDNA labeling using Low Input Quick Amp Labeling Kits (Agilent, Palo Alto, CA) in the presence of cyanine 3-CTP dye. The labeled cDNA was hybridized to the C. elegans -specific 44 K-olig array (one sample/array) at 65 °C for 17 h using Agilent’s Gene Expression Hybridization Kits [ 39 ]. A total of 48 samples (3 chemicals × 4 treatments per chemical × 4 replicates per treatment) were randomly assigned and hybridized to six 8 × 60 K-array slides containing 44 K oligonucleotide probes per array.…”

Cerebral ganglionic variations and movement behaviors of Lumbricus terrestris on exposure to neurotoxin

“…There may be a neuronal membrane depolarization in a reversible manner and an increased level of free calcium which could be either or due to its release from the organelle or inhibition on its reuptake. 42 The oxidant/antioxidant status of the worms did show significant changes beyond the concentration of 7.0 mg/kg bw of ACR leading toan imbalance is exacerbating loss of ionic imbalance. It is assumed that the oxidative stress and membrane potential have a role in the sensory modulation of locomotion movements in the annelids.…”

Biological Activity of Hexaazaisowurtzitane Derivatives

“…Among them, CL-20 (1) is the most wellknown representative and extensive studies have confirmed its xenobiotic nature (Figure 1). Toxicology studies investigating the effects of high doses of CL-20 on animal organisms have revealed that its negative impact, mediated through various biotransformation pathways, is mostly reversible [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. Interestingly, current genetic research methods demonstrate the reversibility of the neurotoxic effects of compound 1 on Eisenia fetida [11].…”