## 3 Discussion  
Previous studies have reported that TCDD elicited NAFLD-like pathologies, dysregulated bile acid metabolism and gut microbiome dysbiosis [9; 11; 12; 28; 30]. This study further elucidated the shifts in the gut microbiota associated with TCDD treatment using shotgun metagenomic sequencing. We show that TCDD dose-dependently shifted the gut microbiota composition by enriching for Lactobacillus species, consistent with hepatic disruption of host and microbial bile acid metabolism. In addition, TCDD enriched for genes involved in mevalonate dependent isoprenoid precursor biosynthesis and menaquinone biosynthesis, crucial for microbial cell growth and survival. Over-representation of these microbial associated pathways were also identified in human cirrhosis stool metagenomics datasets. TCDD-elicited gut dysbiosis is in agreement with observed effects in published in vivo studies following treatment with endogenous (i.e., FICZ) and exogenous (i.e., TCDD and TCDF) AhR agonists [8; 9; 11; 28; 29; 30]. More specifically, we observed an increased Firmicutes/Bacteroides ratio with dose-dependent increases in Lactobacillus species [28; 30]. Lactobacillus species are often associated with NAFLD and with increased abundances in patients with diabetes and liver fibrosis [45]. Probiotic Lactobacillus species, including _L. reuteri_ supplementation, have also been reported to alleviate NAFLD pathologies by reducing steatosis [46], fibrosis [47], insulin resistance [48] and serum cholesterol levels [49]. However, Lactobacillus species supplementation may also exacerbate fibrosis [50]. In humans and mice, _L. reuteri_ supplementation can modulate the gut microbiota and alter bile acid metabolism. _L. reuteri_ enrichment also approached comparable levels compared to samples from humans and mice administered probiotic supplementation [51; 52]. We observed a species-specific increase of _L. reuteri_ with a concurrent decrease in _L. murinus_ suggesting shifts in Lactobacillus composition at the species and/or strain levels. Further, decreased abundance of _L. murinus_ has been reported in human NAFLD [53]. Other taxa enriched following treatment included _Turicbacter sanguinis_, an anaerobic expansitive bacillus commonly found in animals, including humans [54]. Interestingly, _T. sanguinis_ has been shown to deconjugate bile acids and metabolize serotonin affecting lipid and steroid metabolism [54; 55]. Quantitative trait locus analysis correlated _T. sanguinis_ abundance with cholic acid levels and expression of the intestinal bile acid transporter _Slc10a2_[54]. Both cholic acid levels and _Slc10a2_ expression are dose-dependently increased by TCDD [9]. Consequently, the dose-dependent taxonomic shift in Lactobacillus and Turicbacter species known to deconjugate conjugated bile acids is consistent with increased levels of secondary bile acids following TCDD treatment. Some host relevant intestinal health and homeostatic effects can be attributed to Lactobacillus species mediated by bile salt hydrolases (BSHs), which are responsible for deconjugation reactions, the gateway step for conversion of conjugated primary bile acid to secondary bile acids [56]. A majority of Lactobacillus species possess BSHs, often containing multiple different gene copies within their genome, some with different bile acid substrate preferences [33; 38]. However, the presence of _bsh_ sequences does not simply infer bile acid tolerance as growth inhibition and reduced fitness is also possible depending on the conjugated or deconjugated bile acids present and/or BSH specificity [33; 38; 57]. For example, _L. gasseri bsh_ knockout mutants exhibit increased fitness compared to wild type strains [38]. Interestingly, L. gasseri _bsh_ sequences were not identified despite increased _L. gasseri_ abundance following TCDD treatment. Our _bsh_ analysis also found TCDD enriched Lactobacillus-associated sequences that may impart bile acid tolerance. For example, the _bsh_ sequence enriched by TCDD annotated to _L.