Ecotoxicological Effects And Mechanism Of Single And Combined Pollution Of Manganese And Fluoride

Manganese and fluorine are considered as important pollution indicators of shallow groundwater pollution.Manganese is one of the essential elements of the human body,but excessive manganese can produce neurotoxicity.The biological toxicity of fluorine and its toxicological mechanisms have not been yet clear.In the context of global climate change,especially under the influence of temperature,the ecotoxicological effects of pollutants need to be explored urgently.Caenorhabditis elegans,Escherichia coli,and animal cells are widely used as model organisms in the field of environmental biology and as indicator organisms for toxicology models.Molecular biomarkers can directly reflect the characteristics of the interaction between external physical/chemical factors and biological molecules.Cytokines can characterize the toxicity of exogenous chemical substances as a systemic stress indicator,and the stress protein Hsps has the significant stress response.In this study,C.elegans,E.coli and animal cell were combined with in vivo and in vitro experiments to explore toxicities of manganese and fluorine under temperature influence,and to reveal the manganese and fluorine environmental stress response mechanism and their joint toxicity patterns.Hsp90 and Hsp70 as biomarkers of manganese and fluorine contaminations were also explored based on the nematode endpoint determination system,the mutant function and protein expressions.In this study,E.coli was used as a living single cell organism and a transitional model of three model organisms in the toxicity test of this paper.Low concentrations of Mn stress showed an inhibitory effect on the growth of E.coli.Hsp70,EOTAXIN,IL-8,EGF,and RANTES expressions showed the good linear relationship with Mn exposure concentrations,showing their characteristics of biomarkers as Mn toxicity response.Hsp70,IL-1b,RANTES,IL-1ra,MIP-1b and Hsp90 showed the good linear relationship with fluoride exposure concentration.The 24-hour median lethal LC₅₀ of C.elegans of C.elegans at 20 ^oC as normal culture temperature was 131.75 mM,and the LC₅₀ of fluoride for 24 hours was 101.16mM.The temporal dynamics of Mn stress showed that JT6130 mutant and BC10067mutant had higher tolerance to short-term acute toxicity.hsp90 and hsp70 showed a regulatory function on Mn exposure under the heat influence.Similar to the performance under Mn stress,hsp90 showed a negative regulatory effect under fluorosis.Thermal effects could stimulate the behavioral response,but the fluoride stress could depress this effect,which indicatd the joint exposures pattern of heat and fluoride was antagonism.hsp70 was significantly involved in aging regulation of C.elegans under Mn and Fluorine stress,and showed the positive role in the signal response of aging pathway.As a representativestress protein,C.elegans Hsp70 was significantly affected by cold and thermal shock,and its expression increased with the increase of temperature impact strength.At 20?,the expression level of Hsp70 in adult C.elegans increased with the increase of Mn exposure concentration,further verifying the positive regulation of hsp70 in the stress response of Mn.Under the combination of temperature and Mn stress,the expression level of Hsp70 increased with the increase of the stress concentration within a certain range,which showed the synergistic effect of the combined effect of temperature and Mn on the molecular level.Similar to the expression effect of Mn exposure,the expression level of Hsp70 in the wild-type C.elegans under fluorine increased with the increase of exposure concentration,which was consistent with the decrease of resistance in hsp70-deficient mutant BC10067under fluorine exposure.The results showed that hsp70 was positively regulated by stress response to fluoride in C.elegans,and the temperature shock amplified this induction effect.Hsp70 had biomarker characteristics for Mn and fluorine contamination.The expression of Hsp90 showed the opposite characteristics to Hsp70,which is that the expression of Hsp90 in C.elegans decreased with the increase of thermal shock,decreased with the increase of Mn or fluoride exposure.Cold shock enhanced the inhibition of Hsp90 in C.elegans,suggesting that hsp90 may enhance the regulation of Mn stress response under chemical exposure.Hsp90 showed the biomarker characteristics under low temperature,which further proved that hsp90/daf-21 played a negative regulatory role in the signal transduction of Mn stress response.Moderate thermal effects decreased the inhibitory effect of fluorine on Hsp90 expression,whereas both cold and ultimate thermal shock potentiated this inhibitory effect,indicating the complexity and abundance of hsp90 under the influence of temperature on the regulatory effect of fluoride stress.That and hsp90-deficient mutant with increased resistance to fluorine toxicity double suggested the negative role of hsp90 of C.elegans in stress responsive signal transduction under fluoride exposure.Combined toxicity test results of manganese and fluorine showed that the low concentration of manganese and the low concentration of fluorine had a weak joint toxic effect.The low concentration of fluorine and high concentration of manganese show a strong synergistic effect,and the higher concentration of fluorine had a synergistic effect with manganese toxicity.Under the combined stress of Mn and F,the head thrash rate,reproductive rate and life span of C.elegans were significantly reduced,showing the synergetic effect of compound pollution of the two chemicals.Compound stress of low and medium concentrations showed cetain positive synergistic effect on the induced expression of Hsp70 in C.elegans,while the combined effect of medium and high concentrations of manganese and fluorine showed a significant negative synergistic effect on the induced expression of Hsp90 in C.elegans.In conclusion,Hsp70 and Hsp90,as stress proteins,played an important role in the regulation of stress responses of Mn and fluoride in vivo.The toxicology model of E.coli-cell?C.elegans and the combination of endpoints with molecular-behavior-reproduction-longevity are a good indicator system to assess the toxicity of Manganese and fluorine.