Metabolomics Analysis Of Mice Kidney With Cadmium Exposure And Hep3B Cells With Aflatoxin B1 Exposure

Cadmium and aflatoxin B1(AFB1),two of the most common environmental carcinogens,have been classified as class I human carcinogens by the International Agency for Research on Cancer(IARC).The intake of the two carcinogens is extremely harmful to human and animals.The investigation of metabolic changes of tissues or cells induced by carcinogens can panoramically reveal the effects of carcinogens on metabolic pathways and help us gain insights into toxicological mechanism of carcinogens.Moreover,it will be helpful for finding the potential drug targets which can be used to prevent and treat toxic effects or disease caused by carcinogens,as well as finding biomarkers for evaluation of the toxicological effects.In the present study,non-targeted metabolomics studies of mice kidney with cadmium exposure and Hep3 B cells with AFB1 exposure were performed to explore the toxicological mechanisms of cadmium and AFB1 and their effects on metabolic pathways of mice kidney and Hep3 B cells.Therefore,we could provide potential drug targets and experimental basis to prevent and treat damage caused by cadmium and AFB1.Firstly,we used mice as a model to explore the accumulation and excretion of cadmium.Forty-eight mice were randomly assigned to three groups,ie,high cadmiumtreated groups(100 ppm),low cadmium-treated groups(25 ppm),and control group.There are sixteen mice in each group.Mice in cadmium-treated groups drank water with cadmium dissolved in and mice in control group drank purified water.At the 4th and 8th week following cadmium exposure,eight mice in each group were transferred to metabolic cages to collect their urine,feces,livers and kidneys after executions.We used ICP-MS to determine the concentration of cadmium in urine,feces,liver and kidney of mice.The results showed that the concentration of cadmium in feces was much higher than that in urine after cadmium exposure,indicating that cadmium in mice was excreted mostly with feces.At the 4th week and the 8th week,the concentration of cadmium in feces of the 25 ppm and 100 ppm groups was about 3680～9089 times higher than that in urine.Therefore,compared to measuring cadmium in urine traditionally,detecting cadmium in animals or human feces by ICP-MS will be a high-sensitive method of screening cadmium exposure.In addition,our results indicated that cadmium accumulated in mice liver and kidney predominantly.The concentration of cadmium in mice kidney was approximately twice higher than that in mice liver,which indicated that kidney was the first main target organ of cadmium.And the accumulation of cadmium in mice liver and kidney will cause inflammation,oxidative stress and damage to them.Secondly,we applied the ultra-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry(UPLC-QTOF-MS/MS)to characterize the metabolite profiling of all mice kidney tissues following cadmium exposure.Each sample was analyzed using four modes,i.e.,reversed-phase liquid chromatography(RPLC)separation combined with positive ion mode of mass spectrometry(RPLC-POS),RPLC separation combined with negative ion mode of mass spectrometry(RPLC-NEG),hydrophilic interaction chromatography(HILIC)separation combined with positive ion mode of mass spectrometry(HILIC-POS),and HILIC separation combined with negative ion mode of mass spectrometry(HILIC-NEG).Acquired data were processed by Progenesis QI and metabolite identification was achieved by searching the database Metlin and HMDB.Multivariate statistical analysis,including principal component analysis(PCA),partial least square discriminant analysis(PLS-DA),orthogonal partial least square discriminant analysis(OPLS-DA)and single-factor analysis of variance(One-way ANOVA)was performed using EZinfo software.Differential metabolites between groups were filtered according to the following criteria,ie,ANOVA(p)< 0.05,variable importance for the projection(VIP)> 1,and fold change(FC)≥ 1.5or ≤ 0.67,respectively.Our results showed that cadmium intake could cause comprehensive metabolic disorder in mice.In total,1334,1040,934 and 756 metabolites were putatively identified by RPLC-POS,RPLC-NEG,HILIC-POS and HILIC-NEG,respectively.In total,2287 metabolites were obtained by combining all metabolites identified using the four methods.According to the criteria of ANOVA(p)< 0.05,FC ≥ 1.5or ≤ 0.67 and VIP>1,184 and324 differential metabolites were characterized in the 4th and 8th week,respectively.Pathway analysis by Metabo Analyst 5.0 indicated that multiple key metabolic pathways were changed significantly,including histidine metabolism,aminoacyl-t RNA synthesis,purine metabolism,cysteine and methionine metabolism,glycerol phospholipid metabolism,amino sugar and nucleotide sugar metabolism and alanine,aspartic acid and glutamate metabolism.The findings of alterations of amino sugar and nucleotide sugar metabolism provided novel insights into toxicity and related mechanisms of cadmium exposure.Multiple metabolites changed in each metabolic pathway,and the trends of many metabolites were consistent with those reported in the literature,indicating that our data have good coverage and our results are reliable.Several differential metabolites could be used for evaluating the early renal damage caused by cadmium exposure,including 3-methyl-L-histidine,L-tryptophan and uric acid.Finally,a non-targeted metabolomics analysis of Hep3 B cells was performed using the same UPLC-QTOF-MS/MS technique as described above.The cells were treated with16 μM and 32 μM AFB1 for 6 consecutive days,and 0.1%DMSO treated cells as control.The results showed that the metabolic profile of Hep3 B cells changed significantly following AFB1 exposure.In total,1227,665,825 and 840 metabolites were identified by RPLC-POS,RPLC-NEG,HILIC-POS and HILIC-NEG,respectively.And 2679 metabolites were identified when combining all metabolites from the four methods.According to the criteria of ANOVA(p)< 0.05,FC ≥ 1.5or ≤ 0.67 and VIP>1,392 differential metabolites were identified in four modes.The differential metabolites were introduced into the Metabo Analyst 5.0 for enrichment analysis and construction of pathways.Our data indicated that AFB1 exposure induced significant metabolic reprogramming,including significantly decreased purine metabolism,hexosamine pathway and sialylation,fatty acid oxidation,fatty acid synthesis,TCA cycle,remarkably increased pyrimidine metabolism,glycerophospholipid metabolism,and perturbed glutathione metabolism,and amino acid metabolism.The findings of alterations of purine and pyrimidine metabolism and the decrease of hexosamine pathways and sialylation provided novel insights into toxicity and related mechanisms of AFB1.In addition,we tested cell viability for 6 consecutive days and examined cell apoptosis on the sixth day.The results showed that AFB1 significantly inhibited the proliferation of Hep3 B cells by promoting their apoptosis.In conclusion,we comprehensively analyzed the cadmium-induced metabonomic changes in mice kidney and AFB1-induced metabonomic changes in Hep3 B cells using UPLC-QTOF-MS/MS-based untargeted metabolomics technique.Our data discovered that key metabolic pathways significantly altered following cadmium and AFB1 exposure,which provided potential drug targets and evidence for preventing and treating carcinogen-induced damage.Kinds of key differential metabolites were identified and quantified,which can be used to evaluate the damage of cadmium and AFB1 and to screen cadmium and AFB1 exposure in early stage.In addition,we greatly expanded the metabolome coverage by combining the orthogonal HILIC-MS and RPLC-MS in both positive and negative ionization modes,which benefits the discovery of more differential metabolites.Therefore,this approach can be widely used in various non-targeted metabolomics analysis.