Oral exposure to benzalkonium chlorides in male and female mice reveals sex-dependent alteration of the gut microbiome and bile acid profile

Benzalkonium chlorides (BACs) are commonly used disinfectants in a variety of consumer and food-processing settings, and the COVID-19 pandemic has led to increased usage of BACs. The prevalence of BACs raises the concern that BAC exposure could disrupt the gastrointestinal microbiota, thus interfering with the beneficial functions of the microbes. We hypothesize that BAC exposure can alter the gut microbiome diversity and composition, which will disrupt bile acid homeostasis along the gut-liver axis. In this study, male and female mice were exposed orally to d7-C12- and d7-C16-BACs at 120 µg/g/day for one week. UPLC-MS/MS analysis of liver, blood, and fecal samples of BAC-treated mice demonstrated the absorption and metabolism of BACs. Both parent BACs and their metabolites were detected in all exposed samples. Additionally, 16S rRNA sequencing was carried out on the bacterial DNA isolated from the cecum intestinal content. For female mice, and to a lesser extent in males, we found that treatment with either d7-C12- or d7-C16-BAC led to decreased alpha diversity and differential composition of gut bacteria with notably decreased actinobacteria phylum. Lastly, through a targeted bile acid quantitation analysis, we observed decreases in secondary bile acids in BAC-treated mice, which was more pronounced in the female mice. This finding is supported by decreases in bacteria known to metabolize primary bile acids into secondary bile acids, such as the families of Ruminococcaceae and Lachnospiraceae. Together, these data signify the potential impact of BACs on human health through disturbance of the gut microbiome and gut-liver interactions.

Methods
Total DNA was extracted from the cecum of untreated and treated mice using E.Z.N.A. DNA Stool Kit (Omega Bio-tek, Inc., Norcross, GA) according to the manufacturer’s protocol. The concentration of DNA was determined via Qubit 2.0 Fluorometer (Life Technologies/Thermo Fisher Scientific, Grand Island, NY). The integrity and quality of DNA samples were confirmed by the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA). The V4 region of the 16S rRNA gene was amplified and sequenced using HiSeq. 2500 platform (250-bp paired-end) (Novogene, Beijing, China) (n = 4-6).