Exposure to Microcystin-LR Promotes Colorectal Cancer Progression by Altering Gut Microbiota and Associated Metabolites in APCmin/+ Mice

Abstract

Microcystins (MCs), toxins generated by cyanobacteria, feature microcystin-LR (MC-LR) as one of the most prevalent and toxic variants in aquatic environments. MC-LR not only causes environmental problems but also presents a substantial risk to human health. This study aimed to investigate the impact of MC-LR on APC^min/+ mice, considered as an ideal animal model for intestinal tumors. We administered 40 µg/kg MC-LR to mice by gavage for 8 weeks, followed by histopathological examination, microbial diversity and metabolomics analysis. The mice exposed to MC-LR exhibited a significant promotion in colorectal cancer progression and impaired intestinal barrier function in the APC^min/+ mice compared with the control. Gut microbial dysbiosis was observed in the MC-LR-exposed mice, manifesting a notable alteration in the structure of the gut microbiota. This included the enrichment of Marvinbryantia, Gordonibacter and Family_XIII_AD3011_group and reductions in Faecalibaculum and Lachnoclostridium. Metabolomics analysis revealed increased bile acid (BA) metabolites in the intestinal contents of the mice exposed to MC-LR, particularly taurocholic acid (TCA), alpha-muricholic acid (α-MCA), 3-dehydrocholic acid (3-DHCA), 7-ketodeoxycholic acid (7-KDCA) and 12-ketodeoxycholic acid (12-KDCA). Moreover, we found that Marvinbryantia and Family_XIII_AD3011_group showed the strongest positive correlation with taurocholic acid (TCA) in the mice exposed to MC-LR. These findings provide new insights into the roles and mechanisms of MC-LR in susceptible populations, providing a basis for guiding values of MC-LR in drinking water.

Keywords: Apcmin/+ mice; bile acid; colorectal cancer; gut microbiota; microcystin-LR.

Figure 1

MC-LR promoted the colorectal tumor progression and impaired intestinal barrier function in APC^min/+ mice. (A) The workflow diagram of mice experiment. (B) Changes in body weight of mice from week 0 to week 8. (C) Representative colon images of the colorectum. (D) Tumor number, tumor load and length of colorectum in control and MC-LR-exposed groups. (E) Images of H&E-stained areas in the adenoma and adenocarcinoma areas of the control and MC-LR-exposed groups (scale bars: 500 μm). (F) The percentage of adenoma area and adenocarcinoma area of mice colorectum. (G) Representative images of PAS staining (scale bars: 100 μm). (H) The number of colon goblet cells was evaluated by PAS staining. * p < 0.05, *** p < 0.001.

Figure 2

MC-LR exposure did not induce changes in the alpha diversity of the intestinal microbial community. (A) Coverage and Shannon curves in the gut microbial community. (B) Alpha diversity was compared between control and MC-LR-exposed groups using Sobs, Simpson and Chao index. NS, not significant.

Figure 3

Exposure to MC-LR altered the structure of the gut microbiota. (A) Relative abundance of microbial taxa at phylum levels in MC-LR-exposed group and the control group. (B) Relative abundance of microbial taxa at genus levels in MC-LR-exposed group and the control group. (C) PCoA plots based on the Bray–Curtis distance matrix indicate beta diversity in MC-LR-exposed group and the control group. (D) LEfSe cladogram displays the dominant bacteria in the three enterotype subgroups (phylum-to-genus level). Only taxa with an LDA score >  2 are presented. (E) The LDA score of the discriminative microbial taxa (genus-to-species level) between MC-LR-exposed and control groups. Only taxa with an LDA score >  2 are presented. (F–H) Comparisons of the relative abundance of Marvinbryantia, Gordonibacter, Family_XIII_AD3011_group, Faecalibaculum, Lachnoclostridium, Bifidobacterium and Turicibacter in MC-LR-exposed and control groups. * p < 0.05, ** p < 0.01.

Figure 4

MC-LR exposure aggravated microbial-induced dysregulation of BA metabolism. (A) PLS-DA together with OPLS-DA score plots comparing metabolic profile between control and MC-LR-exposed groups. (B) KEGG pathway classification: detection and annotation of metabolites. The x-axis stands for second-grade items of the KEGG pathway, while the y-axis stands for the number of identified metabolites. (C) Heat map of the significantly differential metabolites in MC-LR-exposed and control group (p < 0.05, VIP >1). (D) Relative abundance of differential BAs in MC-LR-exposed and control groups. (E) Correlation of differentially altered microbes with metabolites after MC-LR exposure analyzed using Spearman’s correlation. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Mechanisms by which MC-LR promotes CRC progression. Figure was created with elements from BioRender.com, accessed on 25 April 2024.