Cytotoxic Effects And Mechanisms Of Nanoplastics And Heavy Metal Ions In Different Exposure Scenarios

Nanoplastics（NPs）are ubiquitous in our environment.The content of NPs in the environment is increasing due to the production and living needs.Existing studies have found the presence of NPs in microorganisms,plants,and even animals.NPs are likely to enter the human body through the food chain and cause immeasurable potential harm to the human body.Therefore,there is an urgent need for hazard assessment of NPs to determine the potential risks they pose to humans.In fact,NPs often do not exist alone in the environment,and the joint toxicity caused by the interaction between NPs and heavy metal pollutants has attracted much attention.To date,many studies have investigated the joint toxicity of simultaneous exposure of NPs and typical heavy metal pollutants in the environment.In the traditional percept,it is generally believed that"the dose determines the toxicity".However,exposure to toxic substances in the real environment is usually intermittent and repetitive.When an organism is exposed to multiple poisons simultaneously or sequentially,the time of action of the poisons should also be considered.Therefore,the sequential exposure effect hypothesis is generated:when an organism is exposed to two poisons sequentially,the toxic effects may be different if the order is reversed.Hence,when studying the joint toxicity of NPs and heavy metal pollutants,the differences in toxicity caused by sequential exposure should not be ignored.However,it is still a difficult point and a hot spot to quantitatively detect NPs in the environment.At present,the quantitative methods of NPs mainly include chromatography and spectroscopy,etc.In laboratory research,the quantitative method with metal doped NPs is an emerging detection and analysis method,which has the advantages of accurate quantification and good reproducibility.This method has been applied to quantify and trace NPs in aquatic organisms,plants and environmental substrates.Therefore,in this study,Pt/Pd was firstly mixed into polyacrylonitrile（PAN）by gradient emulsion polymerization.The polystyrene nanoparticles（PSNPs）were then prepared by polymerization reaction.We performed a series of characterization on the synthesized PSNPs to show the stability of PSNPs over time.The Zeta potentials of PSNPs（Pt）in ultra-pure water and RPMI-1640 medium were-39.64±3.87m V and-25.64±3.31 m V,respectively.Zeta potentials of PSNPs（Pd）were-44.48±6.96m V and-18.03±7.02 m V in ultrapure water and RPMI-1640 medium,respectively.PSNPs（Pt）and PSNPs（Pd）were raspberry-like core-shell structures in electron microscopy with sizes of 160.30±34.69 nm and 149.10±23.20 nm,respectively.The Pt element in PSNPs（Pt）is uniformly distributed in the particle,while the Pd element in PSNPs（Pd）is aggregated and distributed in the particle core.Stability test showed that the highest leaching rates of PSNPs（Pt）and PSNPs（Pd）over time were 16.52±0.32%and 1.76±0.12%,respectively.The above characterization results indicated that PSNPs（Pd）were more stable,and Pd element was more suitable as a tracer of PSNPs.Then,we used the synthesized PSNPs to investigate the effects of single,simultaneous and sequential exposure of PSNPs and cadmium ion(Cd²⁺)on the uptake and cytotoxicity of GES-1 cells.Compared with PSNPs alone exposure,the cell intake amount of PSNPs were slightly higher than PSNPs and Cd²⁺were simultaneously exposed,and that significantly higher in the Cd-PSNPs treatment than in the PSNPs-Cd treatment.The pathway of cellular internalization of PSNPs may include pinocytosis pathway,clathrin-mediated endocytosis pathway and caveolin-mediated endocytosis pathway.Compared with PSNPs alone exposure,PSNPs+Cd treatment promoted the pinocytosis pathway of cellular internalization of PSNPs.Compared with the PSNPs-Cd treatment,Cd-PSNPs treatment also promoted the pinocytosis pathway of PSNPs uptake.This suggests that the presence of Cd²⁺enhances the cellular internalization of PSNPs through pinocytosis pathway,either by simultaneous exposure with PSNPs or by preconditioning with Cd²⁺.The clathrin-mediated pathway has a similar effect.Conversely,there was no significant difference in the relative amount of PSNPs taken up by caveolin-mediated endocytosis pathway with different exposure patterns,suggesting that Cd²⁺may have no significant effect on cellular internalization of PSNPs by caveolin-mediated endocytosis pathway.Compared with Cd²⁺alone treatment,PSNPs+Cd treatment reduced cytotoxicity andoxidative stress damage.A similar phenomenon was observed in PSNPs-Cd treatment when compared with Cd-PSNPs treatment.This may be the result that PSNPs enhanced SOD and CAT activities,while Cd²⁺decreased SOD and CAT activities in cells.Furthermore,the difference in cytotoxicity may also be caused by the difference in cell intake.At the same time,PSNPs and Cd²⁺showed antagonistic effect on inducing cell cycle arrest.Compare with Cd²⁺alone exposure,Both PSNPs+Cd and PSNPs-Cd treatment reduced the apoptosis.In this study,the pathway of NPs internalization and exportation was quantitatively analyzedby metal doping method.Meanwhile,based on the sequence-effect hypothesis,we incorporated the exposure sequence into the joint toxicity study system of NPs and heavy metal pollutants.The biological effects and possible biological mechanisms of single exposure,simultaneous exposure and sequential exposure of PSNPs and Cd²⁺on GES-1 cells were comprehensively evaluated.It provided a theoretical basis for the establishing a comprehensive and systematic environmental risk assessment system of NPs in the future.Our research is of great significance to the environmental pollutant control,environmental protection and biological protection.