Research On The Toxicity And Mechanism Of Three Types Of Surfactants At The Molecular And Cellular Levels

Surfactants,which possess some useful properties,have been widely used in personal care,household cleaning and detergents products and other aspects.With the development of related industries,the worldwide production and consumption of surfactants are growing rapidly.Residual surfactants after extensive use are able to accumulate in environment,which can affect waters,soils and even the ecosystem.Surfactants can enter the human body through the enrichment of food chain or directly contact,and distributed throughout the body with the blood.Among these,as a persistent organic pollutant,perfluoro-surfactants have attracted worldwide attention because of its potential toxicity and bioaccumulation in the body.The toxicity induced by surfactants has been reported at animal,cell and molecular levels.However,the toxicity during transport process about surfactants has rarely reported before.Meanwhile,since the use of perfluorooctane sulfonate has been banned,fewer studies are reported about the toxicity of substitutes.For full evaluate and elucidate the toxicity of surfactants during transport process,this work investigated the toxicity and mechanisms of surfactants in the process during transport process from the level of cell and molecule.In chapter one,the characteristics and current situation of environmental surfactants pollution as well as the research development of surfactants toxicity were simply introduced.The induction mechanism of oxidative stress,oxidative damage and the related regulated mechanism were overviewed.Through reviewing this knowledge,the research developments and the current scientific problems in the toxicology filed were concluded and experimental protocols were set for these problems.In chapter two,targeted transport proteins human serum albumin(HSA)and bovine hemoglobin(BHb),the interaction mechanism about different surfactants to HSA and BHb were investigated and compared at the molecular level.(1)The interaction mechanism about anionic surfactant sodium dodecyl sulfate(SDS),cationic surfactant cetyltrimethyl ammonium bromide(CTAB)to HSA was investigated and compared at the molecular level.Results showed that SDS and CTAB bound to HSA mainly by electrostatic force and affected the structure and function of HSA.With the increase of CTAB concentrations,the skeleton of HSA loosed apparently,the content of a-helix decreased from 51.6%to 36.8%and β-sheet increased from 7.5%to 14.9%.Whereas,with the addition of SDS,the skeleton of HSA did not changed obviously,the content of a-helix only decreased 3.2%and the β-sheet increased 2.9%.The influence to the structure of HSA caused by the cationic surfactant CTAB was stronger than the anionic surfactant SDS.When SDS was added,the esterase activity of HSA was inhibited significantly which means the catalytic function of HSA was affected.While,CTAB made esterase activity first increased then decreased.The inhibition caused by CTAB was weaker than that of SDS at high concentrations.Combined with the results of molecular simulation,since SDS bound to HSA in the esterase active center and interacted with key amino acid residues directly,the esterase activity of HSA was inhibited.While,CTAB did not bind to HSA in this region.The effect on the structure of HSA inhibited the esterase activity indirectly at high concentrations.(2)The interaction mechanism about perfluoro-surfactants perfluorooctane sulfonate(PFOS),perfluorohexane sulfonate(PFHS)and perfluorobutane sulfonate(PFBS)that contain different carbon chains to HSA was investigated and compared at the molecular level.Results showed that all three molecules bound to HSA by electrostatic and hydrophobic forces,in which the electrostatic force was dominant.After exposure to all three molecules,the skeleton and secondary structure of HSA did not changed significantly.With the addition of PFOS,the content of a-helix increased 1.8%,the content of p-sheet decreased 3.8%.With the addition of PFHS,the content of a-helix decreased 2.5%and β-sheet increased 1.5%.Whereas,PFBS did not cause clearly effect on the secondary structure of HSA.Molecular simulation results showed that all three molecules bound to HSA in the esterase active center,which prevented the substrate into the esterase activity center,leading the inhibition to the esterase activity of HSA.At the highest concentration,the esterase activity of HSA decreased about 70%,60%and 50%under the exposure of PFOS,PFHS and PFBS,respectively.These indicated that the influence on the structure and function of HSA was strengthened with the lengthening of carbon chain.(3)The interaction mechanism about perfluoro-surfactants PFOS,PFHS and PFBS to BHb was investigated and compared at the molecular level.Results showed that all three molecules bound to BHb by electrostatic and hydrophobic forces,in which the electrostatic force was dominant.With the addition of all three molecules,the microenvironment of tryptophan(Trp)and tyrosine(Tyr)residues of BHb did not altered clearly.Combined with the molecular simulation results,because of binding to BHb with the key Trp37 residue,the effect caused by PFOS and PFHS was stronger than that of PFBS which did not bound to BHb in this critical region,in accordance with the changes of fluorescence spectrum.With the increase of PFOS,PFHS and PFBS concentrations,the skeleton of BHb did not significantly changed and the content of α-helix increased 3.81%,2.62%and 1.90%,respectively.The addition of PFOS inhibited the ability of BHb to bind oxygen and caused the formation of hemichrome,which affected the function of transporting oxygen.Whereas,the addition of PFHS and PFBS did not impact the function significantly.These suggested that the influence on the structure and function of BHb was weakened with the shortening of carbon chain.In chapter three,the toxicity of different surfactants on erythrocytes and leukocytes were evaluated and analyzed at cell level.(1)The toxicity of perfluoro-surfactants PFOS,PFHS and PFBS on erythrocytes and leukocytes were evaluated and analyzed by spectroscopy and cell experiments.Results showed that the structure of erythrocytes did not alter obviously with the addition of PFOS,PFHS and PFBS.While the physiological function of erythrocytes was changed.After 4h exposure,with the addition of all three molecules,the total antioxidant capacity of blood slightly increased to relieve oxidative damage induced by oxidative stress.However,at the highest concentration,the antioxidant system of the blood was insufficient to cope with the oxidative stress.Excessive ROS induced oxidative damage,resulting in a decrease in the total antioxidant capacity.After 24h exposure,the leukocytes viability was affected,the proportion of leukocytes apoptosis changed and reactive oxygen species(ROS)in leukocytes increased.These indicate that all three molecules induced oxidative stress during the transportation and were toxic to blood cells.The toxicity increased with the lengthening of carbon chain.(2)The toxicity of SDS and CTAB on erythrocytes and leukocytes were evaluated and analyzed by spectroscopy and cell experiments.Results show that,at low concentrations,the physiological function of erythrocytes were changed with the addition of SDS and CTAB.After 1.0×10-5 mol/L and 5×10-6 mol/L respectively,SDS and CTAB led apparent hemolysis,which destroyed the structure of erythrocytes.After 4h exposure,with the addition of two molecules,the total antioxidant capacity of blood and biomarkers of oxidative stress increased to alleviate oxidative damage induced by oxidative stress.However,at the highest concentration,the antioxidant system of the blood was insufficient to deal with the oxidative stress.Excessive ROS induced oxidative damage,resulting in a decrease in related indicators.24h exposure made more significant toxic effects,leading to an obvious decline in leukocytes viability,changing the proportion of leukocytes apoptosis.Meanwhile.ROS in leukocytes increased.These indicate that all three molecules induced oxidative stress during the transportation and were toxic to blood cells.CTAB showed a stronger toxic effect than SDS.In chapter four,based on the results of of each part of the study,targeted HSA and blood cells and choosed PFOS as a representative of perfluoro-surfactants,we compared similarities and differences about toxicity that induced by three types of surfactants.From the molecular level,at low dosage,all three molecules did not changed the skeleton and secondary structure of HSA significantly,which showed a certain similarity.In contrast,after 1 ×10-5 mol/L,CTAB could change skeleton and secondary structure of HSA apparently under high dosage,while SDS and PFOS had no significant effect on the skeleton and secondary structure of HSA.Due to the binding to HSA in the esterase active center,the esterase activity of HSA was significantly inhibited when SDS and PFOS were added.With the increase of concentrations,the inhibition induced by SDS was significantly stronger than that of PFOS.While,CTAB made esterase activity first increased then decreased.Though CTAB did not bind to HSA in this region.The effect on the structure of HSA inhibited the esterase activity indirectly at high concentrations.The toxicity of three surfactants to the structure of HSA followed by anionic surfactant CTAB,cationic surfactant SDS,perfluoro-surfactants PFOS.Owing to the different location of combination,the effects on the esterase activity of HSA shows exactly an opposite result.From the cell level,after 4h exposure,the toxicity of three surfactants to the physiological function of erythrocytes at low concentrations followed by anionic surfactant CTAB,cationic surfactant SDS,perfluoro-surfactants PFOS.At high concentrations,SDS and CTAB led apparent hemolysis,which destroyed the structure of erythrocyte.While,PFOS did not influence the structure of erythrocytes.When SDS and CTAB were added,the total antioxidant capacity of blood and biomarkers of oxidative stress increased to improve the resistance to oxidative stress.PFOS only made the total antioxidative capacity of blood rise slightly.However,at the highest concentration,the antioxidant system was insufficient to deal with the oxidative stress.Excessive ROS induced oxidative damage,resulting in a decrease in related indicators.After 24 hours of exposure,all three molecules showed a certain toxic effect on leukocytes,leading significant changes on the proportion of leukocytes apoptosis,Meanwhile.ROS in leukocytes increased.SDS and CTAB caused an obvious decline in leukocytes viability,and the effect of CTAB was significantly stronger than that of SDS,whereas PFOS did not affect leukocytes viability apparently.In summary,the toxicity of three surfactants to blood cells followed by anionic surfactant CTAB,cationic surfactant SDS,perfluoro-surfactants PFOS.In chapter five,every part of this thesis was concluded.The innovation points were also summarized.Subsequently,the development direction and future research in this filed were outlooked according to the shortage of the research protocols.