Explore The Neurotoxic Impact Of Caenorhabditis Elegans By GABA_A Receptors With Methyl Tert-butyl Ether Exposure

As an additive,Methyl Tert-Butyl Ether(MTBE)is widely used in chemical production and pharmaceutical manufacturing.However,its toxicity in biological organisms is rarely studied.Caenorhabditis elegans is widely distributed in the soil,with strong adaptability,complete genome sequence,easy to cultivate in the laboratory,and sensitive to the external environment,so it is widely used as a model organism in environmental toxicology research.In this study,Caenorhabditis elegans was exposed to 0-100 mg/L MTBE solution with the aim to study the chronic(1d)neurotoxic(10 d)effects of MTBE on Caenorhabditis elegans at physiological,biochemical and molecular levels.(1)At the level of physiological indicators,the body length,body bending frequency,head swing frequency and exploration behavior of Caenorhabditis elegans were all inhibited by MTBE to varying degrees,and had a certain dose-effect relationship.The suppression caused by chronic exposure is more significant than acute exposure.Compared with other indicators,the nematode's body bending frequency is a more sensitive indicator.Under the acute exposure of 10 mg/L MTBE and 1 mg/L MTBE chronic exposure,the nematode's body bending frequency was significantly lower than that of the control group(p<0.05).Sensitivity of exploratory behavior was second.Under chronic exposure of MTBE,compared with the control group,a dose of 10 mg/L could significantly inhibit the exploratory behavior of nematodes when food was induced(p<0.05).Body bending frequency and exploratory behavior indicators can be used as screening indicators for the sensitivity of MTBE to the physiological level of C.elegans neurotoxic effects.(2)At the level of biochemical indicators,MTBE interferes with Reactive Oxygen Species(ROS),extracellular signal-regulated kinase(ERK)and phosphorylated extracellular signal-regulated kinase(Phosphorylated extracellular signal-regulated kinase,p-ERK).Compared with the control group,acute exposure to 100 mg/L MTBE caused a 35.47%increase in ROS,and chronic exposure to 0.01 mg/L MTBE increased the ROS level in C.elegans by 33.38%.Compared with the control group,acute exposure of 100 mg/L MTBE increased the ERK enzyme content by 10.27%,and chronic exposure of 0.1 mg/L MTBE increased the ERK enzyme of C.elegans by 11.49%.In addition,compared with the control group,acute exposure of 10 mg/L MTBE increased p-ERK enzyme by 32.54%,and chronic exposure of 0.01 mg/L MTBE increased p-ERK enzyme in C.elegans.44.53%.In addition,after stimulation with y-aminobutyric acid A(GABAA)receptor agonist(muscimol),changes in ROS and physiological indicators mean that the GABAA receptor is impaired.Compared with chronic exposure,Acute exposure causes less damage to C.elegans GABAA receptors.(3)Since chronic exposure of MTBE has a greater impact on C.elegans,qRT-PCR was used to analyze the changes in mRNA levels of MTBE after exposure to C.elegans for 10 days.The results showed that the expression of sod-1 and unc-47 genes increased significantly,and the expression of unc-49 genes decreased significantly.Combined with the results of Pearson correlation analysis,the mutant strains Caenorhabditis elegans CB382 and CB407 were used to further examine the effect of the unc-49 gene on the nematode.Compared with the wild type,the CB382 mutant strain nematode had no significant difference in ERK enzyme content The content of p-ERK enzyme increased significantly.Under the chronic exposure of MTBE,the transgenic strain Caenorhabditis elegans EG1653 labeled with green fluorescent protein,0 mg/L treatment fluid,was significantly richer in ventral fluorescence than dorsal fluorescence.At 100 mg/L MTBE,the fluorescence intensity of the dorsal and ventral sides of Caenorhabditis elegans was significantly reduced compared with the control group.In summary,under acute exposure conditions,when the MTBE concentration is 10-100 mg/L,C.elegans body length,head swing frequency,body bending frequency,and exploratory behavior have different degrees of negative effects.At the same time,when the MTBE concentration is 10-100 mg/L,the ROS content,ERK enzyme and p-ERK enzyme increase significantly.Under chronic exposure conditions,when the MTBE concentration is 0.1-100 mg/L,C.elegans body length,head swing frequency,body bending frequency,and exploratory behavior have different degrees of negative effects.At the same time,when the MTBE exposure concentration was 0.01-100 mg/L,the ROS content,ERK enzyme and p-ERK enzyme increased significantly,and the GABAA receptor was damaged.Pearson correlation analysis of chronic exposure indicators showed that the unc-49 gene was closely related to the physiological and biochemical indicators of C.elegans.Combined with the application of the mutant strain and the transgenic strain Caenorhabditis elegans,it was found that the unc-49 gene may regulate external interference through p-ERK enzyme and have a greater impact on the dorsal and ventral sides.Therefore,Caenorhabditis elegans can be used to evaluate the neurotoxic effects of MTBE.The research conclusions can provide toxicological reference for the ecological risk assessment and environmental management of MTBE pollution.