Dietary riboflavin deficiency induces genomic instability of esophageal squamous cells that is associated with gut microbiota dysbiosis in rats

Abstract

Scope: Epidemiologic evidence suggests that riboflavin (RBF) deficiency is a specific nutritional predisposition for esophageal cancer. The aim of this study is to investigate the potential roles of gut microbiota in esophageal tumorigenesis caused by the RBF deficiency. Methods: Male F344 rats were subcutaneously injected with the chemical carcinogen N-nitrosomethylbenzylamine (NMBA, 0.35 mg kg⁻¹). Rats were assigned to 4 groups, denoted as R₆ (normal RBF, 6 mg kg⁻¹), R₆N (normal RBF combined with NMBA), R₆N → R₀N (normal RBF conversion to RBF-deficiency), and R₀N → R₆N (RBF-deficiency conversion to normal RBF). Bacterial communities were analyzed based on high-throughput 16S rRNA gene sequencing. Oxidative DNA damage and double-strand break markers were studied by immunohistochemistry. Results: The R₆N → R₀N diet enhanced the incidence of esophageal intraepithelial neoplasia (EIN, 40 weeks 66.7% vs. 25 weeks 16.7%, P < 0.05). RBF deficiency and replenishment modulated the gut microbiota composition. The gut microbiota (e.g. Caulobacteraceae, Sphingomonas and Bradyrhizobium) affected xenobiotic biodegradation and the genomic instability of the host. Furthermore, the RBF deficiency aggravated oxidative DNA damage and DNA double-strand breaks (immunohistochemistry) in the esophageal epithelium, whereas the RBF replenishment had the opposite effect (P < 0.05, respectively). Conclusions: RBF deficiency promotes NMBA-induced esophageal tumorigenesis, which is associated with gut microbiota-associated genomic instability, and offers new insights into the role of RBF deficiency in esophageal carcinogenesis.