title: Gene expression analysis of CL-20-induced reversible neurotoxicity reveals GABA(A) receptors as potential targets in the earthworm Eisenia fetida.
abstract: The earthworm Eisenia fetida is one of the most used species in standardized soil ecotoxicity tests. End points 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 apply 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/cm(2) 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 days 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 shotgun oligo array. Statistical and bioinformatic analyses suggest that CL-20 initiated neurotoxicity by noncompetitively blocking the ligand-gated GABA(A) 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.
authors.affiliation: Environmental Services, SpecPro Inc., San Antonio, Texas 78216, United States. ping.gong@usace.army.mil
authors.forename: Ping
authors.initials: P
authors.lastname: Gong
authors.forename: Xin
authors.initials: X
authors.lastname: Guan
authors.forename: Mehdi
authors.initials: M
authors.lastname: Pirooznia
authors.forename: Chun
authors.initials: C
authors.lastname: Liang
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Perkins
mesh_terms: D000818:Animals; D001372:Aza Compounds / Q000633:toxicity*; D016000:Cluster Analysis; D005786:Gene Expression Regulation / Q000187:drug effects*; D006571:Heterocyclic Compounds / Q000633:toxicity*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000187:drug effects*; D009835:Oligochaeta / Q000187:drug effects* / Q000378:metabolism*; D040081:Protein Array Analysis; D011963:Receptors, GABA-A / Q000378:metabolism*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C482139:2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaaza-isowurtzitane; D001372:Aza Compounds; D006571:Heterocyclic Compounds; D009419:Nerve Tissue Proteins; D011963:Receptors, GABA-A