Intestinal Toxicity And Underlying Mechanisms Of Microplastics As Determined By The In Vitro Gastrointestinal Tract Model

Consumption of microplastics through food and drinking water represent an oral exposure pathway for human.The ingested microplastics tend to accumulate in the human intestine for a long time and pose potential health risks.Based on the current literature,it can be deduced that microplastics could induce intestinal barrier dysfunction and inflammation in the gut of both aquatic organisms and mice,exhibiting size-dependent toxicity.However,there is little knowledge of the influence of the gastrointestinal tract environment(p H,residence time,nutritional status,etc.)on the properties and potential intestinal toxicity of microplastics.Therefore,we employed a dynamic in vitro digestion assay to mimic the human ingestion of microplastics and assessed the toxicity of digested microplastics in the in vitro gastrointestinal tract model.Caco-2 cells,which are derived from colonic epithelial adenocarcinoma cells,can differentiate into a cell monolayer after being cultured in the transwell system,which can effectively simulate the intestinal epithelial barrier.The in vitro intestinal epithelial barrier model has been widely used to investigate the intestinal translocation and toxicity of pollutants.In this study,two different sizes of PS-MPs(100 nm and 5?m)were applied to determine size-dependent effects,intestinal toxicity,and mechanism of microplastics in the in vitro gastrointestinal tract model,which may provide new insights into the human health risk of microplastics.The detail research contents include1)the uptake and cytotoxicity analysis of two different sizes of PS-MPs in the Caco-2cells;2)the toxicity and mechanism of microplastics towards in vitro intestinal epithelial barrier model before and after in vitro digestion treatment;3)the combined toxicity of microplastics and arsenic towards in vitro intestinal epithelial barrier model before and after in vitro digestion treatment.(1)In view of the potential cytotoxicity of different sizes of microplastics,the uptake and cytotoxicity of 100 nm and 5?m PS-MPs were analyzed in the Caco-2 cells.Both sizes of PS-MPs could enter cells and be localized to the lysosomes,however,entry of 5?m PS-MPs was harder than 100 nm PS-MPs.Both sizes of PS-MPs did not significantly alter cell viability,plasma membrane integrity and fluidity,except for the up-regulation of intracellular ROS at 200?g/m L.However,100 nm PS-MPs(?20?g/m L)disrupted the mitochondrial membrane potential through accumulating in the lysosome and damaging the mitochondria.5?m PS-MPs at a concentration of 1?g/m L disrupted the mitochondrial membrane potential through escaping from the lysosome and further destroying the lysosome,leading to serious consequence compared with100 nm PS-MPs.Furthermore,both sizes of PS-MPs inhibited plasma membrane ATP-binding cassette(ABC)transporter activity and 100 nm PS-MPs could act as substrates of ABC transporter.(2)In view of the influence of gastrointestinal tract environment on the properties and potential intestinal toxicity of microplastics,an in vitro digestion assay an in vitro gastrointestinal tract simulation system and an in vitro Caco-2 monolayer model were built.Results showed that 20?g/m L 100 nm original PS-MPs significantly increased LDH release via inducing oxidative stress and damaged the Caco-2 monolayer,while 5?m original PS-MPs showed no significant toxic effect.Both sizes of PS-MPs did not significantly increase the gene expression and protein secretion levels of MCP-1 and IL-8.However,after in vitro digestion,20?g/m L 100 nm PS-MPs did not cause an increase in LDH release but significantly increased the gene expression and protein secretion levels of MCP-1 and IL-8.In vitro digestion eliminated the damage induced by the 20?g/m L 100 nm PS-MPs,but increased the proinflammatory effects.The change in toxicity was attributed to the formation and reunion of protein crowns on the100 nm microplastic surface during the digestion process.(3)In view of the joint toxicity of microplastics and other pollutants in the intestine,As(III)was selected as the representative co-existing pollutant.The study found that both sizes of PS-MPs significantly increased As-induced ROS generation in Caco-2cells,resulting in a synergy effect.Base on the in vitro Caco-2 monolayer model,the study found that As transport rate was not influenced by the original PS-MPs,but the digestion treatment reduced As transport rates from 28.5%to 12.5%and 13.2%for the100 nm and 5?m PS-MP groups,respectively.Compared with exposure to As alone,co-exposure of As and 20?g/m L 100 nm PS-MPs significantly increased the permeability of LY and LDH release,reduced the activity of Na~+-K~+-ATPase,suggesting deteriorative effects on the Caco-2 monolayer.However,the combined toxicities of PS-MPs and As were also decreased by digestive treatment.The in vitro digestion treatment decreased the intestinal bioavailability of As in the intestine when coexisting with PS-MPs,which was due to digestion treatment weakening the damage of the transport function of the Caco-2 monolayer model caused by PS-MPs.In summary,this study explored intestinal toxicity and underlying mechanism of microplastics by using in vitro gastrointestinal tract model,finding that both sizes of PS-MPs could enter Caco-2 cells,reduced the mitochondrial membrane potential and inhibited ABC transporter activity to varying degrees;In vitro digestion eliminated the damage induced by the 100 nm PS-MPs towards in vitro Caco-2 monolayer model,but produced an inflammatory response;Compared with exposure to As alone,co-exposure of As and 100 nm PS-MPs significantly increased the toxicity on the Caco-2 monolayer,while in vitro digestion eliminated the PS-MPs-induced toxicity sensitization effect.This study provides basic information for intestinal toxicity studies of microplastics by oral exposure.