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Gut Microbiota Profiling of Aflatoxin B1-Induced Rats Treated with Lactobacillus casei Shirota.

ABSTRACT: Aflatoxin B1 (AFB1) is a ubiquitous carcinogenic food contaminant. Gut microbiota is of vital importance for the host's health, regrettably, limited studies have reported the effects of xenobiotic toxins towards gut microbiota. Thus, the present study aims to investigate the interactions between AFB1 and the gut microbiota. Besides, an AFB1-binding microorganism, Lactobacillus casei Shirota (Lcs) was tested on its ability to ameliorate the changes on gut microbiota induced by AFB1. The fecal contents of three groups of rats included an untreated control group, an AFB1 group, as well as an Lcs + AFB1 group, were analyzed. Using the MiSeq platform, the PCR products of 16S rDNA gene extracted from the feces were subjected to next-generation sequencing. The alpha diversity index (Shannon) showed that the richness of communities increased significantly in the Lcs + AFB1 group compared to the control and AFB1 groups. Meanwhile, beta diversity indices demonstrated that AFB1 group significantly deviated from the control and Lcs + AFB1 groups. AFB1-exposed rats were especially high in Alloprevotella spp. abundance. Such alteration in the bacterial composition might give an insight on the interactions of AFB1 towards gut microbiota and how Lcs plays its role in detoxification of AFB1.

SUBMITTER: Liew WP

PROVIDER: S-EPMC6357033 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Project description:Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1's carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT's) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the rat transcriptome contains many previously unidentified, AFB1-responsive exons and transcripts supporting RNA-Seq's capabilities to provide new insights into AFB1-mediated gene expression leading to hepatocellular carcinoma.

Project description:UnlabelledStress-induced abdominal dysfunction is an attractive target for probiotics. To investigate the effects of the probiotic Lactobacillus casei strain Shirota on abdominal dysfunction, a double-blind, placebo-controlled trial was conducted with healthy medical students undertaking an authorized nationwide examination for academic advancement. For 8 weeks, until the day before the examination, 23 and 24 subjects consumed an L. casei strain Shirota-fermented milk and a placebo milk daily, respectively. In addition to assessments of abdominal symptoms, psychophysical state, and salivary stress markers, gene expression changes in peripheral blood leukocytes and composition of the gut microbiota were analyzed using DNA microarray analysis and 16S rRNA gene amplicon sequence analysis, respectively, before and after the intervention. Stress-induced increases in a visual analog scale measuring feelings of stress, the total score of abdominal dysfunction, and the number of genes with changes in expression of more than 2-fold in leukocytes were significantly suppressed in the L. casei strain Shirota group compared with those in the placebo group. A significant increase in salivary cortisol levels before the examination was observed only in the placebo group. The administration of L. casei strain Shirota, but not placebo, significantly reduced gastrointestinal symptoms. Moreover, 16S rRNA gene amplicon sequencing demonstrated that the L. casei strain Shirota group had significantly higher numbers of species, a marker of the alpha-diversity index, in their gut microbiota and a significantly lower percentage of Bacteroidaceae than the placebo group. Our findings indicate that the daily consumption of probiotics, such as L. casei strain Shirota, preserves the diversity of the gut microbiota and may relieve stress-associated responses of abdominal dysfunction in healthy subjects exposed to stressful situations.ImportanceA novel clinical trial was conducted with healthy medical students under examination stress conditions. It was demonstrated that the daily consumption of lactic acid bacteria provided health benefits to prevent the onset of stress-associated abdominal symptoms and a good change of gut microbiota in healthy medical students.

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Gut Microbiota Profiling of Aflatoxin B1-Induced Rats Treated with Lactobacillus casei Shirota.

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