Study On The Combined Hepatotoxicity Of Microplastics And Cadmium And Its Mechanism Based On Gut Microbiota

Microplastics(MPs)are plastic fragments or particles less than 5 mm in diameter.With the massive use and improper disposal of plastic products in recent decades,researchers have found traces of microplastics in marine,freshwater,soil,and atmospheric systems.Studies have shown that microplastics can enter and accumulate in plants and animals in terrestrial and marine ecosystems and enter human food systems,including food production,food processing,and food consumption.A growing number of studies have shown that microplastics can enter the human body through the food chain,drinking water,food containers or packaging,and it is urgent to assess their safety risks to humans.Due to their small particle size,large specific surface area,and complex surface characteristics,MPs can exogenously accumulate other types of persistent toxic substances,such as heavy metals,through electrostatic adsorption,intermolecular interactions,and biofilm formation.Cadmium(Cd)is a representative heavy metal contaminant that has been detected in rice,vegetables and seafood,and is readily accumulated in the body through dietary intake.Cadmium and its compounds are widely used as pigments and stabilisers in plastics and can be endogenously contained or exogenously adsorbed on the surface of MPs.The combined toxicity of MPs upon adsorption of the cadmium and the potential pathogenic mechanisms are scientific questions that need to be addressed in the current food safety toxicological studies.However,existing studies on the toxicity of MPs and their adsorbed heavy metals are mostly focused on aquatic organisms,and studies on the toxic effects and mechanisms in terrestrial mammals are still lacking.In this thesis,the in vitro adsorption behaviour of Cd on MPs was investigated by simulating the natural environment,including theoretical monolayer saturation adsorption,adsorption fitting model,factors affecting adsorption,and changes of MPs before and after adsorption;then,the individual and combined exposure toxicity of MPs and Cd to the liver and colon were evaluated in an adult male mouse model,and the possible mechanism of enhanced toxicity was explored based on impaired intestinal barrier and inflammatory response to oxidative stress.Finally,we investigated the effects of single and combined exposure to MPs and Cd on the structural homeostasis of gut microbiota by using 16 S r RNA high-throughput sequencing,and verified the key role of gut microbial structural disruption in the colonic and hepatic toxic damage by fecal microbiota transplantation experiments.The contents of each chapter are organized as follows.Chapter 1: The current status of research on MPs and Cd is reviewed,including definitions,possible exposure pathways,known biotoxicity,adsorption behaviors and patterns,and an overview of the research ideas and specific contents of this thesis is provided.Chapter 2: In order to study the adsorption behavior of Cd on MPs under in vitro conditions and to identify the factors that may affect the adsorption process,commercially available polystyrene microplastics and cadmium chloride reagents were used in this chapter,and in vitro co-incubation was performed in a simulated environment based on batch experiments.The adsorption kinetics and isothermal curves of Cd on MPs were determined by inductively coupled plasma mass spectrometry(ICP-MS)and fitted to the data model.It was found that the adsorption of Cd on the surface of MPs gradually increased with the decrease of particle size,the increase of metal ion concentration,and the increase of incubation time,and the saturation adsorption value existed.In addition,the in vitro adsorption behavior of MPs and Cd could be well fitted and predicted by the pseudo-second-order kinetic model and the Langmuir isotherm model.The morphological changes of the MPs before and after the adsorption were observed by scanning electron microscope(SEM),and then the Cd content on the surface of the MPs before and after adsorption was subsequently visualized by using energy dispersive spectrometer(EDS).The results showed that the MPs exhibited poor dispersion,spherical stacking,slight degradation and aging of the surface layer after Cd adsorption.Based on the experimental conditions and results in this chapter,subsequent oral exposure animal experiments were conducted.Chapter 3: In order to investigate the similarities and differences in the biotoxicity of MPs and Cd single and compound/combined exposure,this chapter establishes an8-week oral exposure mouse model by free drinking water poisoning with MPs alone,Cd/MPs compound,Cd alone,and Cd+MPs mixture,and a series of phenological indicators,biochemical indicators of serum and liver,indicators of oxidative stress and inflammation,histopathological changes of liver and colon,expression levels of related genes,Cd accumulation levels in liver and colon tissues of mice were analyzed and measured after exposure.The results showed that exposure to Cd/MPs compound and Cd+MPs mixture caused more severe colon and liver injury than Cd and MPs single exposure,including weight loss,colon shortening,increased histopathologic injury of colon and liver,increased ROS levels in colon and liver,abnormal levels of oxidative stress-related biochemical indicators,and upregulation of inflammatory factor protein and gene expression in liver and colon tissues,etc.Possible mechanisms include superimposed effects at the level of oxidative stress and increased Cd accumulation due to impaired intestinal barrier,resulting in more Cd entering the systemic circulation to reach the liver and cause damage.Chapter 4: Considering the key role of gut microbiota in the development of several diseases,in this chapter,the changes of gut microbiota in mice after MPs and Cd single exposure and compound/combined exposure were analyzed by using highthroughput sequencing of 16 S r RNA gut microbiota.The results showed that MPs adsorbed with the heavy metal Cd induced a more pronounced disruption of gut microbial homeostasis in the mouse intestine compared to the single MPs exposure group.Cd exposure had a strong disruptive effect on the gut microbial homeostasis,and the combined exposure of MPs and Cd enhanced this effect.This was manifested by a decrease in the gut microbiota diversity index(α-diversity and β-diversity),deviation of the microbiota structure from the control group mice(principal component analysis and principal coordinate analysis),changes in the genus of the constituent gut microbiota(at the level of phylum,class,order,family),changes in the dominant genus(linear discriminant analysis effect size and linear discriminant analysis),abnormal abundance of specific genera(Lactobacillus,Clostridia,Helicobacter,Alistipes,Prevotellaceae,Parasutterella,Akkermansia,Faecalibaculum,Coriobacteriaceae,etc.),and altered metabolic pathways(PICRUSt2-based prediction of KEGG metabolic pathway).To verify the key mediating role of gut microbiota in MPs and Cd-induced enterohepatic injury,the verification of toxicogenic mechanisms based on gut microbiota dysbiosis was performed in this section by fecal microbiota transplantation experiments.After transplantation of MPs-and Cd-affected fecal microbiota from donor mice,various degrees of colonic and hepatic injury were observed in the recipient mice,including weight loss,colon shortening,increased histopathologic damage in the colon and liver,and abnormalities in oxidative stress and inflammationrelated biochemical indices.In conclusion,MPs can adsorb heavy metal Cd in the in vitro environment and media,and the physicochemical properties and biotoxicity of MPs are altered after adsorption,and the compound/combined exposure can cause more severe colon and liver injury and imbalance of intestinal microbial structure homeostasis in adult male mice.Possible mechanisms of enhanced toxicity include superimposed effects at the level of oxidative stress,increased accumulation of Cd in tissues due to impaired intestinal barrier,increased abundance of harmful bacteria in the intestinal flora and decreased abundance of beneficial bacteria.