Cytotoxicity Detection Of Emerging Contaminants Based On Electrochemical Method And Mechanism Study

The environmental pollution and eco-toxicity of emerging contaminants(ECs)have become one of the major environmental problems in the world.Antibiotics,bisphenols,perfluorinated compounds and their substitutes are three types of ECs.These pollutants themselves or their intermediate products have been frequently detected in environmental media,which are potentially harmful to human health and the ecological environment.Therefore,it is of great significance to study their cytotoxicity and action mechanism.However,the conventional in vitro toxicity testing methods have the disadvantages of long cycle,high cost and some of the stains are toxic.The emerging cell electrochemical method is developed based on electrochemistry and cell biology,which has the advantages of no labeling,simplicity,sensitivity,and speed.It is an effective means to evaluate the toxicity of ECs.At present,most of the cells used in electrochemical detection are human cancer cells.In contrast,fish cells can more directly reflect the toxic effects of pollutants on aquatic organisms.However,the electrochemical signals of fish cells are weak.Therefore,it is necessary to prepare modified electrodes with good electrocatalytic properties to improve the detection sensitivity.In summary,carbon nanotubes,graphene and carbon nanohorn were used to prepare three composite modified electrodes with high electrocatalytic activities.The electrodes were characterized by the scanning electron microscopy(SEM),transmission electron microscopy(TEM),X-ray diffraction(XRD),Raman spectroscopy(RAM),X-ray energy dispersive spectroscopy(EDS)and cyclic voltammetry(CV).Using ctenopharyngodon idellus kidney(CIK)cells as model cells,the cell electrochemical methods based on different nano-modified electrodes were established to study the toxicity of antibiotics,bisphenols(BPs),perfluorinated compounds(PFCs)and their substitutes.The results were compared with those of the traditional MTT assay.Furthermore,combined with the indicators of oxidative stress and apoptosis,the mechanism of cytotoxicity was deeply revealed.The main findings are as follows:1.The f-MWCNTs/BCP/GCE was prepared by using functional multi-walled carbon nanotubes(f-MWCNTs)and bromocresol purple(BCP)to modify the glass carbon electrode(GCE).The characterization results showed that the electrode had a large electroactive surface area(0.149 cm²)and good electron transfer performance.The electrode showed good electrocatalytic activity to CIK cells.Combined with high performance liquid chromatography analysis(HPLC),it was confirmed that the electrochemical signal was attributed to guanine and xanthine in cells.On the basis of optimizing the detection conditions,a cell electrochemical method based on f-MWCNTs/BCP/GCE was established to evaluate the toxicity of antibiotics(tetracyclines,quinolones and sulfonamides).The results showed that the six antibiotics all had significant inhibition effects on the growth of CIK cells,and the cytotoxicity had a linear relationship with the logarithm concentration.The half inhibitory concentration(IC₅₀)values of tetracycline(TC),oxytetracycline(OTC),ciprofloxacin(CIP),Ofloxacin(OFX),sulfasoxazole(SOX),sulfamethoxazole(SMZ)on CIK cells were 184.51,229.09,354.98,380.19,660.69 and 831.51?M,respectively.Tetracycline antibiotics are more toxic to CIK cells than quinolones and sulfonamides.The toxicity order was consistent with that of the traditional methyl thiazolyl tetrazolium(MTT)assay.The IC₅₀ values of antibiotics measured by electrochemistry were lower than those of the MTT assay,demonstrating that the electrochemical method was reliable and more sensitive.2.The MWCNTs-COOH/RhB/AuNPs/SPE was developed by using carboxylated multi-walled carbon nanotubes(MWCNTs-COOH),rhodamine B(Rh B)and gold nanoparticles(Au NPs)to modify screen printing electrodes(SPE).The electrode can be used for micro cell detection.The characterization results showed there is a synergistic effect among MWCNTs-COOH,Au NPs and Rh B,which can significantly increase the electron transfer rate and the electroactive surface area(2.21times that of the SPE).The modified electrode exhibited excellent electrocatalytic activity to CIK cells.On the basis of optimizing the detection conditions,a cell electrochemical method based on MWCNTs-COOH/RhB/AuNPs/SPE was established to evaluate the cytotoxicity of BPs.The five BPs all had significant inhibition effects on the growth CIK cells,and the cytotoxicity had a linear relationship with the logarithm concentration.The IC₅₀ values of bisphenol AF(BPAF),bisphenol B(BPB),bisphenol A(BPA),bisphenol F(BPF)and bisphenol S(BPS)on CIK cells of 48 h were 21.33,72.44,112.20,158.49,and 173.78?M,respectively.BPAF and BPB were more toxic to CIK cells than BPA,indicating that they are not environmentally friendly alternatives for BPA.Compared with the MTT method,the cell electrochemical method was more sensitive and rapid.3.The f-CNH/GR/Au NPs/SPE was fabricated by using functional single-walled carbon nanohorns(f-CNH)and gold nanoparticle-loaded graphene(GR/Au NPs)to modify SPE.The characterization results showed that the combination of GR/Au NPs and f-CNH can effectively solve the problem of easy agglomeration between GR sheets.The electroactive surface area was increased by 2.37 times that of SPE,which greatly promoted the electron transfer rate.The electrode showed excellent electrocatalytic activity toward CIK cells.On the basis of optimizing the detection conditions,a simple and rapid cell electrochemical method based on f-CNH/GR/Au NPs/SPE was established to evaluate the cytotoxicity of PFCs and their alternatives.The five PFCs and their alternatives had significant inhibition effects on the growth of CIK cells,and their cytotoxicity had a linear relationship with the logarithm concentration.The IC₅₀ values of 48 h of HFPO-TA,HFPO-DA,PFOS,PFOA and 6:2 FTCA on CIK cells were 88.53,192.84,252.99,506.22,and 627.55?M,respectively.HFPO-TA and HFPO-DA were more toxic to CIK cells than PFOA and PFOS,indicating that they are not environmentally friendly alternatives for PFCs.The toxicity order obtained by the cell electrochemical method was consistent with that of the MTT assay.Howerer,the IC₅₀ values obtained by the former method were lower,indicating that its sensitivity is higher.4.Correlation analysis of the cytotoxicity data of the three ECs measured by electrochemical methods with oxidative stress and apoptosis parameters showed that there was a significant correlation between the cytotoxicity and oxidative stress and apoptosis indicators.Cytotoxicity was positively correlated with intracellular reactive oxygen species(ROS)and DNA damage tail moment(TM).Cytotoxicity was negatively correlated with glutathione(GSH)content,superoxide dismutase(SOD)activity,and mitochondrial membrane potential(MMP)red-green light ratio.Intracellular ROS was significantly negatively correlated with GSH,SOD,and MMP,and significantly positively correlated with TM.These results were consistent with the metabolic mechanism of cell oxidative stress damage and apoptosis caused by the rise of intracellular ROS.The three ECs can cause the increase of intracellular ROS content,which promotes the imbalance of the intracellular antioxidant system,the decrease of GSH content,and the decrease of SOD enzyme activity,which in turn lead to the decrease of intracellular mitochondrial membrane potential and the damage of DNA,and ultimately leads to cell apoptosis.However,the toxic mechanisms of those ECs are also different.Antibiotics can interact with GSH and the basic amino acids that constitute SOD enzymes,resulting in a decrease in the activity of GSH and SOD enzymes,and aggravating the oxidative stress damage of CIK cells.Additionally,antibiotics can cause DNA damage by affecting DNA synthesis in cells.Bisphenols can produce more ROS in cells,aggravate cell oxidative stress damage,and cause DNA damage.The weak acidity of perfluorinated compounds and their substitutes makes them interact with the basic amino acids that constitute SOD enzymes,resulting in a decrease in the activity of SOD enzymes,and aggravate cell oxidative stress damage by interacting with GSH.Furthermore,they can reduce the mitochondrial membrane potential by acting on mitochondria,thereby aggravating cell apoptosis.