Study On The Toxicity Of Arsenic In Vivo And In Vitro Based On Hsp Response In C.Elegans

Heavy metals are ubiquitous pollutants worldwide, while herbicides are some of themost prevalently used agrochemicals and became more pollutant in environment. Theenvironmental factors such as temperatures and other chemicals could affect the toxicity ofcontaminations. Arsenic (As), an element of wide distribution and multiple purposes, isknown to be a toxic environmental pollutant and carcinogen. Environmental factors such astemperature could affect the toxicology of As stress to organisms. Nematodes are frequentlyused for toxicological studies in various exposures, and becomes a useful animal model forthe study of the ecotoxicological relevance of chemical-Caenorhabditis elegans, a free-livingsoil nematode, is an excellent model organism because of its short lifespan, ease ofmanipulation, and low cost. Because of its high quality and inexpensive advantage, C. eleganis widely used as a model organism in the field of life sciences. Both nematode dormancy(dauer) state and stress protein (Hsps) expression have the characteristic of outstanding stressresponse and could be environmental ecotoxicological biomarkers in potential. induced andbiochemical level responses. Classical heat shock responses in C. elegans were describedmore than three decade ago.In this study, we chose nematode C. Caenorhabditis elegans as the model organism invivo and mammal cell line in vitro to study toxicological effects of arsenic. We used theending-points in C. elegans including24-h median lethal concentration (LC50), head thrash,reproductive rates/brood size to observe the effects of temperature (cold shock and heat shock)on of As stress to adults and dauer larval nematodes, and detecting mortality and Hsp90protein of nematode expression under Joint effects of As and pesticides stresses. Bothnematode dormancy (dauer) state and Hsps expression have the characteristic of outstandingstress response and could be environmental ecotoxicological biomarkers in potential.24-hLC50of As on wild type adults at20°C was130. mg·L-1through Probit analysis. Cold shockenhanced resistance of nematodes under arsenic stress, and high temperature significantlyreduced nematode tolerance to arsenic exposure. The head thrash and brood size declinedunder the increasing As extends.We also used C. elegans to investigate24h acute toxicities and joint toxicity of arsenicwith glyphosate, dichlorvos and cooper. Results showed that the24h-LC50for glyphosate toC. elegans were18.5mg·L-1. Glyphosate showed a high toxic effect on the reproduction to C.elegans. Results of equivalent toxicity design indicated that the joint toxicity of arsenic andglyphosate on C. elegans followed synergic especially when the concentration of As is high.Lower concentration glyphosate had a strong depression on the nematodes head thrash. Jointtoxicity of As and glyphosate at low concentration showed synergic effects on head thrash andreproductive rate of C. elegans. However, high concentrations of As and glyphosate had aantagonistic effects on the reproductive rate of C. elegans. Compared to As, Cu as a classicalheavy metal especially at low concentrations had a different toxic effect on C. elegans. The joint toxicity of As and Cu on C. elegans showed to be synergic. Cu and low concentration ofAs made a significant decrease of reproductive rate in C. elegans, while at high concentration,the joint toxicity of As and Cu showed a antagonistic effects on reproductive rate of C.elegans.We observed temperature played an important role in determining LC50values of As. Weexamined the differences in lethal toxicity induced by As exposure in dauer larvae and adultsdaf-21mutant at various temperatures. Hsp90is an essential gene in C. elegans, and the geneencoding Hsp90was identified as daf-21which is involved in C. elegans dauer formation.The lethal values in daf-21adults increased with temperatures increasing which indicates thatdauer larval had strong resistance to combined heat and As effects. It also implicated thespecial role of daf-21in dauer formation and maintenance. We further confirm using ELISAto assay the changes in Hsp90expression under the combined effects of heat and As. In thepresent study, we integrated the application of Western blot and ELISA assay method. Therewere differences of Hsp90expressions by Western blots and ELISA in C. elegans underdifferent concentrations of arsenic stress. The consistency in the results of protein expressionobtained from both methods confirms that temperature and pollutants has joint effect onchange of heat shock proteins expression in the nematode, which could be effective detectedand characterized by ELISA. Our results showed decreased Hsp90accumulation followingexposure of C. elegans to As stress, which indicates daf-21regulatory function in the pathwayof heat stress and possible role in As toxic response pathway in C. elegans. The significantdecreases in Hsp90levels observed in wild type adults exposed to As at higher temperaturessuggested that Hsp90could be a biomarker for heavy As pollution.For As toxicity study in vitro, PC12cell line (rat pheochromo-cytoma cell line) has beenselected as a mode cell line for evaluating toxicological effects of As. The value (47?M) of24h median lethal concentration (LC50) of NaAsO2for PC12cells has been obtained byMTT assay. The cytotoxicity of As has been systematically studied by exposure of PC12cells to relatively low concentration (5and20?M) of As. We found that As is able tosignificantly decrease cell proliferation, generate strong genotoxicity and increase ROS(reactive oxygen species) level of PC12cells. The experimental results demonstrate that lowconcentration of (e.g.,5?M, about10%of its LC50) As can generate high cytotoxicitytowards PC12cell line.