Integrated analyses of the microbiome, metabolome, and spatial transcriptomics reveal sex-dependent bladder vulnerability to chronic inorganic arsenic.

Inorganic arsenic (iAs) is a widespread environmental carcinogen; however, the mechanisms by which chronic low-dose exposure promotes bladder carcinogenesis in a sex-dependent manner remain insufficiently characterized. Analyses of patient bladder tumors revealed arsenic concentrations consistent with chronic environmental exposure, even when urinary arsenic levels were within current guideline limits. To replicate physiologically relevant exposure, male and female mice received iAs in drinking water for 12 months. This exposure induced urothelial hyperplasia, hemorrhage, inflammation, early neoplastic changes in the bladder, and dose-dependent lung injury. Arsenic speciation demonstrated significant sex differences: males accumulated higher levels of monomethylated arsenic (MMA), while females exhibited increased dimethylarsinic acid (DMA) and greater methylation capacity. Arsenic exposure reduced protective commensal populations and enriched stress-tolerant, pro-inflammatory taxa across the gut, urinary, and bladder microbiomes. In males, proliferative and DNA methylation signatures were predominant, whereas in females, oxidative, endocrine, and redox stress signatures were more pronounced. Spatial transcriptomics identified sex-specific activation of carcinogenic and immune-stromal pathways in the bladder epithelium, with increased populations of cancer-epithelial cells and macrophages. Integrated analyses of the microbiome, metabolome, and spatial transcriptome indicated that chronic arsenic exposure reprograms the gut-bladder axis in both dose- and sex-dependent manners. These findings define a mechanistic, sex-dependent gut-bladder axis through which chronic low-dose arsenic toxicity promotes premalignant bladder pathology.