## 3 Discussion
Simvastatin, which is primarily excreted in the feces [69], has been reported to reduce bacterial growth by directly inhibiting bacterial HMG-CoA synthesis while PPIs inhibit Streptococcus species growth [70; 71; 72]. These microbiome-drug interactions highlight off target effects that should be considered when investigating novel NAFLD treatments, such as new drug development and/or probiotic interventions. In addition to increased mevalonate-dependent isoprenoid biosynthesis genes in cirrhotic patients, menaquinone biosynthesis gene abundance was also increased. This suggests taxa with the ability to produce menaquinone may have a competitive advantage when intestinal environmental conditions shift during disease progression. In cirrhosis patients, _E. coli_ and _B. vulgaris_ were associated with genes providing a majority of the menaquinone biosynthesis capacity. These species are also increased in human NAFLD [73]. Similar to the results in mice exposed to TCDD, _L. reuteri_ was associated with several menaquinone biosynthesis genes and only detected in decompensated cirrhosis patients but lacked the complete pathway (Table S10). In cirrhosis patients, it is unclear whether _L. reuteri_ is participating in menaquinone metabolism and/or benefiting from increased abundance of species, like _E. coli_ that are capable of producing menaquinone. This study was designed to account for factors affecting gut microbiota analysis, including cage effects, coprophagia, and circadian rhythms [74]. Significant shifts in taxa were observed in Lactobacillus species. However, the small group size (\(n=3\)) following adjustment for multiple testing lacked sufficient power to confirm more subtle shifts, such as the two fold enrichment of _Lachospiraceae A4_, an abundant community member associated with _bsh_ sequences. Samples were also collected within the same Zeitgeber period to account for possible variations in relative microbiota levels due to circadian rhythm/diurnal regulation. In fact, _L. reuteri_ is one gut microbiome member demonstrating changes in relative abundance in human samples due to circadian/diurnal regulation [75]. TCDD disrupted diurnal regulation of hepatic gene expression, including bile acid biosynthesis genes, which may contribute to _L. reuteri_ enrichment [76]. The same dose range (sesame oil vehicle and 0.01-30 \(\mathrm{\SIUnitSymbolMicro g}/\mathrm{kg}\) TCDD) and dosing regimen (every 4 days for 28 days) has been used in previous studies to examine TCDD-elicited effects in mice relevant to NAFLD D development and progression [76; 77; 78; 79; 11; 12; 13; 14; 15; 30]. This model recapitulates similar NAFLD progression and pathologies with hepatocyte vacuolization at doses as low as 0.3 \(\mathrm{\SIUnitSymbolMicro g}/\mathrm{kg}\) TCDD. In addition, immune cell infiltration (steatohepatitisis) was observed at doses as low 3 \(\mathrm{\SIUnitSymbolMicro g}/\mathrm{kg}\) TCDD with collagen deposition (fibrosis) reported at 30 \(\mathrm{\SIUnitSymbolMicro g}/\mathrm{kg}\) TCDD [9]. Using this study design, TCDD also disrupted the bile acid metabolism and enteronehepatic circulation with increased hepatic and serum total bile acids and secondary bile acid DCA [9]. Likewise, increased serum bile acid levels, including DCA, have been reported in patients with steatohepatitisis and fibrosis [79; 80; 81; 82]. Furthermore, NAFLD patients with increased bile acid levels have increased levels of bacterial genes from the _bai_ operon associated with 7\(\mathrm{\SIUnitSymbolMicro c}/\mathrm{dehydroxylation}\) of bile acids leading to the production of DCA from cholic acid [83]. Although the consequences of TCDD-elicited immune system effects on the gut microbiome were not assessed in this study, it is most likely a factor impacting _L. reuteri_ enrichment. TCDD causes macrophage and dendritic cell migration out of the _lamina propria_ with increased accumulation in the liver, possibly exacerbating hepatic inflammation and affecting intestinal immune responses [14]. The ability of _L.