Dietary Exposure Risk Assessment Of Cadmium In Wheat And Wheat Flour In Shandong Province And Brain Damage Of Mice Induced By Cadmium Combined With Microplastics Exposure

Objective Cadmium(Cd)is a heavy metal that is widely used in industrial production.With the acceleration of industrialization and urbanization,Cd could pollute soil by various routes such as industrial and agricultural activities including the production and use of fertilizers and pesticides and plastic films,industrial waste discharge and sewage irrigation and atmospheric settlement,which further increase the Cd content in food crops.The diet is the main route of Cd exposure for non-smoking and non-occupational populations.Wheat is the staple food of peoples in Shandong Province.We detected the level of Cd in wheat and wheat flour in Shandong Province,characterizing its exposure of Cd and evaluating the health risks.This results could provide basic data for the prevention and control of health damage caused by Cd.As a matter of fact,real environment cantain a mixture of pollutants.Microplastics(Mps)is regared as the plastic particle size below 5 mm,which came from the fragmentation and decomposition of waste plastics under the physical and chemical action.It also derived from direct addition in the production process of industrial,agricultural and daily washing and care supplies.Apart from its toxicity,Mps has the ability to enrich other environmental compounds due to its small particle size,large specific surface area,small steric hindrance and low crystallinity.Therefore,the possibility of combined contamination of Cd and Mps exists in the real environment.Therefore,it is of great scientific significance to reveal the toxic effects and mechanism of action caused by combined exposure to heavy metals.In this study,we analyzed the Cd content of wheat and wheat flour in Shandong Province and evaluated the risk of Cd exposure to wheat flour;in addition,we further observed the damage effects on mouse brain under subchronic exposure to different doses of Cd and combined Mps,and the results of this study may provide a new perspective for the study of the combined effects of Mps and heavy metals pollution and health risks.Methods Cd dietary exposure risk assessment in Shandong Province:A total of 1789 samples of wheat and wheat flour were collected from farmers’ markets,shops,supermarkets and farmers of 16 cities in Shandong Province,and their Cd contents were detected by inductively coupled plasma mass spectrometry Spectrometry(ICP-MS).Based on the consumption of wheat flour of Shandong Province,the risk assessment of Cd exposure of residents in Shandong Province were carried.Neurotoxicity induced by Cd and Cd combined with microplastics exposure in mice:Male SPF C57/6J mice were randomly divided into control group(Con),2 mg/kg Cd group(Cd-L),5 mg/kg Cd group(Cd-H),10 mg/kg Mps group,2 mg/kg Cd+10 mg/kg Mps group(Mps+Cd-L)and 5 mg/kg Cd+10 mg/kg Mps group(Mps+Cd-H),with 11 in each group.The animals were acclimatized and fed for 7 days,and the subjects were given successively by gavage for 90 days.Before the end of experiment,the neurobehavioural performances of mice were evaluated by open field experiments and new object recognition test respectively.Subsequently,five mice in each group were randomly selected and anesthetized with 5%chloral hydrate,followed by cardiacal perfusion fixation with 4%paraformaldehyde,the brains were taken out and post-fixed for 48 hours,dehydrated with 30%sucrose solution.The frozen brain slices at thickness of 30μm were prepared on a frozen microtome,and immunohistochemical staining was performed to observe microglial and astrocytes morphology,and degeneration of neuron.The remaining of mice in each groups were sacrificed and the brain was quickly harvested,and tissue homogenates are prepared with RIPA lysate,the Western blotting was employed and detected the expressions of the target proteins to evaluated the inflammatory state of brain and neuronal damage.ResultsCd dietary exposure risk assessment in Shandong Province:The data showed that Cd content in wheat was significantly higher than that in wheat flour(Z=-12.50,P<0.001).There was regional differences in Cd content in wheat and wheat flour.The estimated monthly Cd intake(EMI)for the average wheat flour consumption level of people was 1.18μg/kg BW,accounting for 4.72%of provisional tolerable monthly intake(PTMI).The EMI of people with high wheat flour consumption was 2.87 μg/kg BW,accounting for 11.47%of PTMI.Neurotoxicity study in mice exposed to cadmium combined with microplastics:The body weight of mice in all groups showed an increasing trend during the experiment,but the total weight gain of animals in the 5 mg/kg.bw Cd and Mps combined exposure group was lower than that of the control group,and the difference was statistically significant(P<0.01).There was no significant difference in the brain weight of animals in each experimental group compared with the control group(P>0.05),but the brain coefficient of the combined 5 mg/kg.bw Cd and Mps exposure group was significantly higher than that of the control group(P<0.05).The recognition index of mice in the Mps,Cd-H,Mps+Cd-H were all significantly lower than the control group(P<0.05);the residence time of mice in the central area of the open field was significantly shorter in the Mps group and Mps+Cd-L,the differences were statistically significant compared with the control group(P<0.05).Mps+Cd-L and Mps +CdH group mice had significantly shorter exercise distance compared with the control group(P<0.05).Compared with the control group,microglia in the brains of all experimental groups were significantly activated,with the most obvious changes in the Mps+Cd-H.Western blot showed that the trend of Iba-1 expression in each group was consistent with the immunohistochemical results;astrocytes in the brains of mice in each treatment group were also activated,and Western blot results showed that the expression of GFAP was increased in all treatment groups,but the difference was not statistically significant(P>0.05).NF-κB phosphorylation activation and p-NF-κB protein expression were significantly higher in the Cd-H,Mps,Mps+Cd-L,and Mps+Cd-H groups than in the control group(P<0.05).Compared with the control group,the hippocampal neurons in all experimental groups showed different degrees of degenerative changes such as loosening of cell arrangement,decrease in density and a little vacuolization;Western blot results showed that the expression of neuronal marker protein NeuN in the brains of mice in all experimental groups showed a tendency to decrease,and the expression of NeuN in the brains of Cd-H,Mps-treated,Mps+Cd-L and Mps+Cd-H-treated groups was reduced,and the difference was statistically significant(P<0.05).Conclusions1 The average consumer population in Shandong Province had a low risk of cadmium exposure through wheat flour,but the risk of dietary cadmium exposure of high-consumption people in wheat flour in cities with high cadmium content still needs further attention.2.Experimental doses of cadmium and microplastics can induce cognitive impairment and depression in mice,accompanied by inflammatory response of the brain and neuronal damage,Mps exposure enhances Cd neurotoxicity.