Combined Toxicity Of Cadmium And Aflatoxin B₁

The contamination of aflatoxin B₁(AFB₁)and cadmium is a prominent problem in food and feed safety,and the effects and toxicological mechanism of the combined toxicity of AFB₁ and cadmium are still unclear.Five experiments were designed in this study.The combined toxicity of AFB₁ and CdCl₂ was systematically studied from the animal's level,cellular level and gene expression profile,respectively.The aim was to evaluate the combined toxic effects of AFB₁ and cadmium and to elucidate the toxicological mechanism of AFB₁ and cadmium.The research results are summarized as follows.Experiment 1: Evaluation of combined acute oral toxicity and toxic effects of AFB₁ and CdCl₂ in KM miceThe results revealed that the LD₅₀ of equal toxicity mixture for AFB₁ and CdCl₂ by oral route in male KM mice was 48.79 mg/kg and in female KM mice was 62.56mg/kg,regardless female or male KM mice was 55.27 mg/kg;the combined effect coefficient K was 1.27 for female mice,1.63 for male mice and 1.44 for mice(regardless of female or male).The combined toxicity effect of AFB₁ with CdCl₂ showed additive effect regardless of female or male KM mice.Blood test results showed that the indicators PDW,WBC,NEUT,LYMPH,MONO,EOS,BASO,NEUT%,EOS%,BASO%,HGB,MCV,MCH,MCHC,RDWCV,and RDWSD exceeded the reference range while the indicators RBC,HCT and PLT were below the reference range.Blood biochemical indicators exceeded the reference range as follows: CREA,UREA,PHOS,ALT,ALKP,TBIL,AMYL,and LIPA;the indicators GLU,CA and ALB were below the reference range.The results of organ coefficient(liver,heart,spleen,lung and kidney),and liver and kidney tissue sections all showed that regardless of male or female mice,the increase of mixed dose of AFB₁ and CdCl₂ resulted in higher level of toxicity with more severe damage the kidneys.Experiment 2: Study of single toxicity and combined toxicity of AFB₁ and CdCl₂ to LLC-PK1 cells based on factorial designThe aim of this study was to investigate firstly,the single toxic dose-effect relationship of AFB₁ and CdCl₂ on LLC-PK1 cells by MTT assay,and secondly the combined toxicity of AFB₁ and CdCl₂ on LLC-PK1 cells by using factorial design.The single toxic effects of AFB₁ or CdCl₂ on LLC-PK1 cells at 24 h and 48 h were time-and dose-dependent.IC_{(50,CdCl2,24h)}=14.526(10.758²0.981)?M,IC_{(50,CdCl2,48h)}=1.623(0.688².633)?M,IC_{(50,AFB1,24h)}=58.952(48.750⁷3.980)?M,IC_{(50,AFB1,48h)}= 6.399(1.549 10.775)?M.The results of factorial design experiment confirmed that the combined toxicity of AFB₁ and CdCl₂ on LLC-PK1 cells for 24 h and 48 h was synergistic.Experiment 3: Effect of AFB₁,CdCl₂ and AFB₁+CdCl₂ on cell damage and morphology of LLC-PK1 cellsThe aim of this study was to investigate the effects of AFB₁,CdCl₂ and AFB₁+CdCl₂ on the cell structural damage,oxidative damage and morphology of LLC-PK1 cells after 24 h treatment.The LDH value of the combined exposure group were significantly higher than the control group(P<0.05).Morphological observations by light microscopy and transmission electron microscopy both showed that the combined exposure group had greater structural damage to the cells than the single AFB₁ or CdCl₂ treatment group.The results showed that ATP,SOD and CAT values significantly decreased after AFB₁ and CdCl₂ combined exposure,and the value of MDA was significantly increased.Compared with the control group,there was significantly different(P<0.05);with the increase of the combined dose,the combined toxicity was enhanced.Under the light microscope,the cell atrophy increased with increase toxin concentration in the combined exposure group,and cells became smaller and lost adherence;a large number of floating dead cells and cell debris appeared in the culture solution.Under transmission electron microscopy,the nuclear chromatin concentration of most cells were distributed in the nucleus or moved to the nuclear membrane inside or side,more mitochondria with high electron density substances,and more autophagosomes appeared with increase toxin concentration in the combined exposure group.Experiment 4: The effect of AFB₁,CdCl₂,and AFB₁+CdCl₂ on apoptosis of LLC-PK1 cellsThe aim of this study was to investigate the effects of AFB₁,CdCl₂ and AFB₁+CdCl₂ on apoptosis of LLC-PK1 cells by AO-EB staining cell glass slides,DNA ladder assay and flow cytometry.The results showed that the total apoptotic rate,early apoptotic rate and late apoptotic rate of LLC-PK1 cells in AFB₁+CdCl₂ treatment group were higher than those of single CdCl₂ or AFB₁ treatment cells.Experiment 5: The effect of AFB₁,CdCl₂,and AFB₁+CdCl₂ on gene expression profiles of LLC-PK1 cellsBased on the toxicity evaluation,Agilent porcine expression profiling chip was used to detect changes of genome-wide expression profile of single AFB₁,CdCl₂,and AFB₁+CdCl₂ to treated LLC-PK1 cells for 24 hours.In the AFB₁+CdCl₂ group,there were 4306 genes(fold change>2)were up-regulated,accounting for 29.18%;3324genes(fold change>2)were down-regulated,accounting for 21.05%.The GO analysis selected 104 differential gene after AFB₁ and CdCl₂ combined treated,they related to the function of cell biological processes,cell molecular functions and cellular components.KEGG Pathway analysis found that AFB₁ and CdCl₂ combined treatment for 24 h compared with other experimental groups,the differentially expressed genes were mainly concentrated in PPAR signaling pathways,proteoglycans in cancer,etc.The pathways of up-regulation and down-regulation in AFB₁+CdCl₂ group were the highest and more obvious than in other experiments groups.Among the 20 selected pathways,there were 9 up-regulations and 11 down-regulations in AFB₁+CdCl₂ group.Cross-matching of all Pathway IDs in each experimental group revealed that AFB₁+CdCl₂ group also had synergistic effect in signal pathway distribution.The reliability of the microarray results was verified by Real-time PCR using 10 selected differentially expressed genes.Functional analysis of 10 verified genes revealed that they played an important role in tumorigenesis,mitochondrial damage and apoptosis.In summary,the combined toxicity effect of AFB₁ with CdCl₂ showed additive effect regardless of female or male KM mice,and the combined toxicity of AFB₁ and CdCl₂ on LLC-PK1 cells for 24 h and 48 h was synergistic,also the combined toxicity of AFB₁ and CdCl₂ on gene expression profiles for 24 h was synergistic.With the increase of the combined dose,the toxic effects on animals,cell viability,cell structure and oxidative damage,apoptosis and gene expression profiles were more and more obvious,and the toxicity of the combined exposure group was higher than that of single exposure group.Therefore,it is necessary to pay attention to the risks and hazards caused by the combination of cadmium and aflatoxin B₁ on human and animal health.