Multi-Omics Reveal Additive Cytotoxicity Effects of Aflatoxin B1 and Aflatoxin M1 toward Intestinal NCM460 Cells

Abstract

Aflatoxin B1 (AFB1) is a common crop contaminant, while aflatoxin M1 (AFM1) is implicated in milk safety. Humans are likely to be simultaneously exposed to AFB1 and AFM1; however, studies on the combined interactive effects of AFB1 and AFM1 are lacking. To fill this knowledge gap, transcriptomic, proteomic, and microRNA (miRNA)-sequencing approaches were used to investigate the toxic mechanisms underpinning combined AFB1 and AFM1 actions in vitro. Exposure to AFB1 (1.25-20 μM) and AFM1 (5-20 μM) for 48 h significantly decreased cell viability in the intestinal cell line, NCM460. Multi-omics analyses demonstrated that additive toxic effects were induced by combined AFB1 (2.5 μM) and AFM1 (2.5 μM) in NCM460 cells and were associated with p53 signaling pathway, a common pathway enriched by differentially expressed mRNAs/proteins/miRNAs. Specifically, based on p53 signaling, cross-omics showed that AFB1 and AFM1 reduced NCM460 cell viability via the hsa-miR-628-3p- and hsa-miR-217-5p-mediated regulation of cell surface death receptor (FAS), and also the hsa-miR-11-y-mediated regulation of cyclin dependent kinase 2 (CDK2). We provide new insights on biomarkers which reflect the cytotoxic effects of combined AFB1 and AFM1 toxicity.

Keywords: additive effects; aflatoxin B1; aflatoxin M1; multi-omics analyses.

Figure 1

Concentration-response curves and combination index (CI) in NCM460 cells exposed to individual and combined AFM1 and AFB1 at different concentrations for 48 h. (A) NCM460 cell viability. Compared with the control group, ** p < 0.01, *** p < 0.001. (B) Combination index (CI) represents the interactive effects of AFM1 and AFB1 at different concentrations. The dotted line indicates additivity, the area under the dotted line synergism, and the area above of the dotted line antagonism. Data are mean values ± standard deviation (SD). Results of 3 independent experiments with 5 replicates. AFM1, aflatoxin M1. AFB1, aflatoxin B1.

Figure 2

Transcriptome analysis of NCM460 cells induced by individual and combined AFM1 and AFB1 treatments for 48 h. (A–C) Volcano plots showing the number of differentially expressed genes (DEGs) in AFM1/CTL, AFB1/CTL, and AFM1 + AFB1/CTL groups. (D) Hierarchical DEGs clustering in different group in NCM460 cells. (E) Venn diagram analysis of AFM1, AFB1, and AFM1 + AFB1 groups. CTL represents the cells with cell medium treatment, AFM1 = 2.5 μM, AFB1 = 2.5 μM, and M1_B1 = AFM1 (2.5 μM) + AFB1 (2.5 μM).

Figure 3

The enrichment and validation of transcriptome. (A) Compared with control group, Gene Ontology (GO) terms enriched by the DEGs in AFM1, AFB1, and AFM1 + AFB1, and the significance of GO terms were evaluated by corrected p value. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched by 2083 unique genes in the AFM1 + AFB1 treatment group. (C) Differentially expressed gene (DEG) in the AFM1 + AFB1/CTL treatment group validation by qRT-PCR. DEGs and their validation by qRT-PCR were analyzed between CTL and AFM1 + AFB1 treatment. Data from qRT-PCR are mean values ± SD. Results of 3 independent experiments with 3 replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

The enrichment and validation of transcriptome. (A) Compared with control group, Gene Ontology (GO) terms enriched by the DEGs in AFM1, AFB1, and AFM1 + AFB1, and the significance of GO terms were evaluated by corrected p value. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched by 2083 unique genes in the AFM1 + AFB1 treatment group. (C) Differentially expressed gene (DEG) in the AFM1 + AFB1/CTL treatment group validation by qRT-PCR. DEGs and their validation by qRT-PCR were analyzed between CTL and AFM1 + AFB1 treatment. Data from qRT-PCR are mean values ± SD. Results of 3 independent experiments with 3 replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Proteomic analysis of NCM460 cells exposed to individual and combined AFM1 and AFB1 at 2.5 μM for 48 h. To reduce the proteomic sample variability, three replicates of harvested NCM460 cells were pooled into one sample. Each treatment was run in three replicates. (A) Differentially expressed proteins (DEPs) in AFM1, AFB1, and AFM1 + AFB1 treatments. (B) Venn analysis of AFM1, AFB1, and AFM1 + AFB1 groups. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of 98 unique DEPs in the AFM1 + AFB1 group. (D) Validation of paxillin (PXN) production by western blotting. The results of western blotting are expressed as mean ± SD of three independent experiments. * p < 0.05.CTL represents the cells with cell medium treatment, AFM1 = 2.5 μM, AFB1 = 2.5 μM, and M1_B1 = AFM1 (2.5 μM) + AFB1 (2.5 μM).

Figure 5

Cross-omics analysis of transcriptomic and proteomic data in individual and combined AFM1 and AFB1 treatment. (A–C) Comparison of changes in differentially expressed genes (DEGs) and cognate differentially expressed proteins (DEPs) abundance. Quadrant b and h are those correlations that were significant for transcripts only, quadrant d and f are those correlations that were significant for proteins only, quadrant a, c, g and i are those correlations that were significant for both transcripts and proteins, and quadrant e are those that were not significant in either of the two data sets. (D) Venn diagram analysis of DEGs and DEPs in quadrants c and g of AFM1, AFB1, and AFM1 + AFB1 groups. (E) Protein–protein interaction (PPI) analysis of 49 key proteins related to cell viability in AFM1 + AFB1 group. Different colors represent different pathways. Node size represents connectivity strength. AFM1 = 2.5 μM, AFB1 = 2.5 μM, and M1_B1 = AFM1 (2.5 μM) + AFB1 (2.5 μM).

Figure 5

Cross-omics analysis of transcriptomic and proteomic data in individual and combined AFM1 and AFB1 treatment. (A–C) Comparison of changes in differentially expressed genes (DEGs) and cognate differentially expressed proteins (DEPs) abundance. Quadrant b and h are those correlations that were significant for transcripts only, quadrant d and f are those correlations that were significant for proteins only, quadrant a, c, g and i are those correlations that were significant for both transcripts and proteins, and quadrant e are those that were not significant in either of the two data sets. (D) Venn diagram analysis of DEGs and DEPs in quadrants c and g of AFM1, AFB1, and AFM1 + AFB1 groups. (E) Protein–protein interaction (PPI) analysis of 49 key proteins related to cell viability in AFM1 + AFB1 group. Different colors represent different pathways. Node size represents connectivity strength. AFM1 = 2.5 μM, AFB1 = 2.5 μM, and M1_B1 = AFM1 (2.5 μM) + AFB1 (2.5 μM).

Figure 6

MiRNA analysis in NCM460 cells exposed to individual and combined AFM1 and AFB1 treatments for 48 h. To reduce the miRNA-sequencing sample variability, three replicates of harvested NCM460 cells were pooled into one sample. A total of three samples were prepared from each treatment. (A) The number of differentially expressed miRNAs (DEmiRNAs) in AFM1, AFB1, and their combination treatments. (B) The top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched by DEmiRNAs targets. (C) A Sankey diagram of the key miRNA–mRNA network related to NCM460 cell viability.

Figure 7

Summary of key miRNA/genes/proteins involved in NCM460 cytotoxicity induced by AFM1 + AFB1 treatment based on multi-omics profiling. Black represents differentially expressed genes (DEGs) and differentially expressed proteins (DEPs). Blue represents differentially expressed miRNAs (DEmiRNAs).