Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Toxicogenomic effects of sub-lethal exposures of organophosphates on mouse liver cells [mRNA Expression]

ABSTRACT: In order to examine the long term effects of the OPs, murine liver cells (BNL CL.2, ATCCÂ® TIB-73) have been exposed to sub-lethal doses of three OPs: diisopropylfluorophosphate (DFP) representative of sarin and soman, O,S-diethyl methylphosphonothioate (OSD) serving as a simulant for VX, and paraoxon as an example of OP insecticides. Dosing levels of these compounds was set at 20% of the IV LD50 for each, with a 4 hour exposure time. Gene arrays and physiological tests were run at three time points following exposure; 2 hours, 2 days, and 2 weeks. The physiological results showed little to no effect upon exposure to sub-lethal dose of OPs. Gene expression and microRNA (miRNA) profiles using GeneChip Mouse Gene 1.0 ST arrays and miRNA arrays (Affymetrix, Santa Clara, CA) found that the OPs did alter expression of genes related to several systems previously implicated in OP exposure with no long term effects. In addition, a significant number of sRNA/snRNA and ribosomal RNA were found to be affected suggesting the need for further study of the changes in these regulators. Three biological replicates of liver cells were exposed to three representative OPs and then harvested at three time points (2 hours, 72 hours, and two weeks) following exposure. These time points were designed for a determination of both early and late effects as well as identification of persistent, chronic changes. The RNAs from the collected cells were extracted and processed on commercial microarrays to determine the gene expression patterns and miRNA profiles associated with response to exposure to the OPs.

ORGANISM(S): Mus musculus

SUBMITTER: Baochuan Lin

PROVIDER: E-GEOD-78222 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The principal toxicity of acute organophosphate (OP) pesticides poisoning is the disruption of neurotransmission through inhibition of acetylcholinesterase (AChE). However, other mechanisms leading to persistent effects and neurodegeneration remain controversial and difficult to detect. Because Caenorhabditis elegans is relatively resistant to OP lethalityM-bM-^@M-^Tparticularly through the inhibition of AChEM-bM-^@M-^Tstudies in this nematode provide an opportunity to observe alterations in global gene expression following OP exposure that cannot be readily observed in less resistant organisms. We exposed cultures of worms in axenic, defined medium to dichlorvos under three exposure protocols. In the first, worms were exposed continuously throughout the experiment. In the second and third, the worms were exposed for either 2 or 8 h, the dichlorvos was washed out of the culture, and the worms were allowed to recover. We then analyzed gene expression using whole genome microarrays from RNA sampled at multiple time points throughout the exposure. The worms showed a time-dependent response that progressively worsened. Early in the exposure, the predominant effect was on metabolic processes, while at later times, an immune-like response and cellular repair mechanisms dominated the expression pattern. Following removal of dichlorvos, the gene expression in the worms appeared to relatively, rapidly return to steady-state levels. The changes in gene expression observed in the worms following exposure to dichlorvos point towards two potential mechanisms of toxicity: inhibition of AChE and mitochondrial disruption. Synchronized cultures of worms at the L3/L4 molt were treated with dichlorvos (0.6 M-BM-5M or 15 M-BM-5M) or maintained as a controls according to one of three protocols. In the first protocol, the set of flasks was incubated without interruption for the duration of the experiment with flasks being harvested at 2, 8, 14, 20, and 26 h. The other two sets were incubated for 2 or 8 h, at which time the worms were centrifuged out of the exposure medium, washed 3 times with a modified CeHR medium (Washout Buffer), resuspended in fresh CeHR medium without dichlorvos, and returned to the incubator. Flasks were harvested at 6 h intervals following the washout through 26 h. Flasks of worms were also harvested prior to beginning the exposure as the 0 h controls. Total RNA was isolated from the frozen harvested worms then analyzed using Affymetrix GeneChips. A total of 148 microarrays were processed (47 per exposure protocol with 4 replicates) however only 146 were included in final analysis as 2 falied to pass QC criteria.

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Toxicogenomic effects of sub-lethal exposures of organophosphates on mouse liver cells [mRNA Expression]

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