SQUAD bile acid profiling reveals cannabis-driven remodeling of the gut microbiome

SQUAD bile acid profiling reveals cannabis-driven remodeling of the gut microbiome — concise expert summary

Importance of the topic

Oral consumption of cannabis (edibles) is widespread and growing, and individual responses range from therapeutic benefit to adverse effects such as cannabinoid hyperemesis syndrome and psychiatric events. The gut microbiome and its metabolic outputs — particularly bile acids transformed by gut bacteria — are central regulators of inflammation, mucosal immunity and gut-brain signaling. Understanding how cannabis intake reshapes microbial communities and bile acid chemistry is therefore important for explaining variable host responses, identifying biomarkers of effect or harm, and designing interventions to mitigate adverse outcomes.

Study objectives and overview

This study tested whether repeated oral administration of a complex cannabis extract remodels the gut microbiome and alters bile acid composition in ways consistent with anti-inflammatory or otherwise biologically significant shifts. The design compared male and female C57BL/6J mice given cannabis extract (≤10 mg/kg) or vehicle every other day for two weeks (n = 12 per group), with fecal and brain samples analyzed for metabolomics and fecal microbiome composition by 16S rRNA sequencing. The SQUAD workflow combined targeted bile acid quantitation with untargeted metabolomics to link microbiome changes to functional metabolic readouts.

Methodology

- Animal model: Male and female C57BL/6J mice, cannabis extract dosing every other day for two weeks, fecal samples collected at study end; brain tissue also analyzed.

- Microbiome profiling: 16S rRNA sequencing to assess community composition, alpha and beta diversity, and taxon-level shifts by sex and treatment.

- Metabolomics: SQUAD workflow integrating targeted bile acid quantification and untargeted small-molecule metabolomics in matched fecal and brain samples to detect changes in primary, secondary, conjugated and sulfated bile acids.

Instrumentation used

Key analytical platforms employed in the study included:

• MoveKit BA (bile acid analytical kit/workflow) for targeted bile acid processing and quantitation.
• Thermo Fisher Orbitrap Tribrid Apex small molecule mass spectrometry platform for high-resolution targeted and untargeted metabolomics.
• Standard 16S rRNA sequencing workflows for microbiome taxonomic profiling.

Key results and discussion

- Sex-dependent microbiome and bile acid responses: Strong sex differences were evident in fecal bile acid profiles regardless of cannabis exposure. Cannabis effects were sex-specific.

- Female mice: Cannabis consumption reduced fecal microbial alpha diversity in females and shifted fecal bile acid composition toward increased conjugated (tauro-conjugates) and sulfated bile acids. This pattern suggests either enhanced microbial or host-mediated conjugation/sulfation or reduced microbial deconjugation and 7α-dehydroxylation activity, leading to decreased levels of hydrophobic (potentially cytotoxic) secondary bile acids.

- Taxonomic shifts in females: Cannabis increased the abundance of Ileibacterium and Streptococcaceae and increased members of the order Oscillospirales, while decreasing Dubosiella, Alistipes and the family Rikenellaceae. Changes in Alistipes and Rikenellaceae are notable given growing links of these taxa to neuroinflammation, anxiety/depression models and gut-brain signaling.

- Male mice: Bile acid trends were generally opposite to females, with a notable brain-specific increase in ursodeoxycholic acid (UDCA) reaching statistical significance in males. This highlights compartment-specific (fecal versus brain) and sex-dependent biochemical responses to the same cannabis exposure.

- Integration of microbiome and metabolome data supports a mechanistic link whereby cannabis-associated shifts in community composition correlate with altered bile acid transformation pathways, favoring increased conjugation/sulfation in females and divergent outcomes in males.

Practical implications and benefits of the method

- The combined targeted/untargeted SQUAD approach enables sensitive, comprehensive profiling of bile acid pools alongside broader metabolomic context, improving detection of biologically meaningful shifts induced by xenobiotics such as cannabis.

- Findings provide candidate metabolic signatures (e.g., increased sulfated and taurine-conjugated bile acids in feces) that could serve as biomarkers to stratify individuals by risk of adverse responses or to monitor therapeutic modulation of host–microbiome interactions.

- Identification of specific taxa altered by cannabis (e.g., Alistipes, Rikenellaceae) points to potential microbial targets for interventions aimed at modulating bile acid metabolism and downstream host effects, including neurobehavioral outcomes.

Limitations and interpretive caveats

- Short exposure duration and use of a complex cannabis extract limit mechanistic attribution to specific cannabinoids; dose translation to humans is not direct.

- Mouse models have species-specific bile acid and microbial ecology; therefore, translation to human physiology requires cautious validation in clinical cohorts.

- Fecal bile acid measures reflect pooled luminal output and do not directly quantify enterohepatic circulation, bile acid signaling at the intestinal epithelium, or serum/portal levels.

Future trends and potential applications

- Larger, longitudinal studies in rodents and humans to map dose-response and time-course relationships between cannabis ingestion, microbiome remodeling and bile acid dynamics.

- Mechanistic follow-up using metagenomics/metatranscriptomics to identify microbial enzymes (e.g., bile salt hydrolases, hydroxylases, sulfotransferases) driving the observed bile acid shifts and targeted microbial culturing or gnotobiotic models to test causality.

- Integration with host transcriptomics and immune profiling to connect bile acid changes to inflammatory signaling, epithelial barrier function and neuroimmune pathways relevant to anxiety, CHS and other cannabis-associated effects.

- Development of clinically actionable biomarkers for monitoring edible cannabis safety and personalized guidance, and exploration of microbiome-directed therapies (probiotics, prebiotics, enzyme inhibitors) to modulate bile acid pools.

Conclusion

This study demonstrates that repeated oral administration of a complex cannabis extract produces sex-dependent remodeling of the gut microbiome and corresponding shifts in bile acid composition in mice. In females, cannabis tended to increase fecal conjugated and sulfated bile acids and reduce microbial alpha diversity, consistent with reduced hydrophobic secondary bile acids and altered microbial bile acid metabolism. Males displayed distinct, sometimes opposite trends, including a brain-specific increase in UDCA. The SQUAD workflow combining targeted bile acid quantitation with untargeted metabolomics and 16S profiling is a powerful platform to link microbial community changes to functional metabolic consequences and to generate hypotheses about gut-brain interactions influenced by cannabis.

References

Reisdorph N., Amer B., Doenges K., Anderson N., Booher W., Lowry C., Bird S. S. SQUAD bile acid profiling reveals cannabis-driven remodeling of the gut microbiome. (Affiliations: University of Colorado Anschutz Medical Campus; Thermo Fisher Scientific; University of Colorado Boulder). Acknowledged support: ALSAM Foundation, Skaggs School of Pharmacy and Pharmaceutical Sciences, Institute of Cannabis Research. No competing financial interests declared.