Long-term TCDD oral exposure changes gut microbial glycosylation patterns in female but not male C57BL/6 mice

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) at high doses damages the liver and disrupts gut microbiota in mice. We investigated whether very low TCDD doses also cause dysbiosis and if sex-specific differences exist. Male and female C57BL/6 mice received 1 µg TCDD/kg body weight initially and maintenance doses biweekly for 12 weeks; controls received DMSO/corn oil. Fecal pellets were collected throughout, and cecal, liver, and gut samples at euthanasia. Mice did not suffer weight loss, and the liver weight to body weight ratio remained normal in all groups. Barrier genes’ expression was unaffected in all groups. 16S sequencing of fecal pellets revealed an increase in α-diversity over the 12-week treatment period, regardless of TCDD exposure or sex. No significant differences in α-diversity were observed between control and TCDD-treated groups. Analysis of β-diversity indicated differences in microbiota composition between male and female mice, as well as over time. However, probably due to low sample numbers, we could not detect significant differences using UniFrac or other ß-diversity metrics. However, our flow cytometry-based analysis tool, FlowSoFine™, identified significantly distinct microbial signatures in TCDD-treated female mice compared to control females. We hypothesized that this could be due to the formation of bacterial clusters or to different glycosylation patterns, both undetectable by sequencing. We therefore stained the cecal material with a panel of seven fluorescent lectins, which detect typical bacterial sugar structures, i.e., glycans. Significant changes were detected for concanavalin A, soybean agglutinin, and Dolichos biflorus agglutinin binding in the gut microbiota in female but not in male mice. We isolated bacteria by fluorescence-activated cell sorting (FACS), which differed between TCDD-treated and control female mice. 16S sequencing of these bacteria revealed a high frequency of Lachnospiraceae and reduced amount of Lactobacillaceae, CAG-508, and Oscillospiraceae in the TCDD-exposed samples. Glycoconjugates govern biofilm formation, infectious behavior of bacteria, and host immune responses, but little is known about diet or xenobiotic induced changes in glycosylation. We conclude that assessing glycosylation parameters in studies of dysbiosis is relevant, as well as stratifying for sex-specific differences.