The human gut microbiota is the totality of the microorganisms that colonizes our gut and many studies have demonstrated its involvement in human health and in the development of many diseases. Humans get in contact with hundreds of chemicals every day, thus the gut microbiota is daily exposed to many factors that could alter its status. The field of gut microbiota toxicology is still relatively new, more data are needed to evaluate the impact of chemicals on human health. Given this, an already validated method was used to study the effects that different substances have on the gut microbiota.

We employed an in vitro method for the culture of gut microbiota that maintains the functional and compositional profiles of in vivo gut microbiota. Then, stool samples from healthy donors were used to test the impact of chemicals on the gut microbiota using metaproteomic and metabolomic analyses. The in vitro gut microbiota models were treated separately with the several chemicals, including PFOA, six replacement PFAS, Glyphosate and Cypermethrin, at different concentrations. Control samples grown without chemicals were also prepared and analyzed. The analysis was performed on a UHPLC Vanquish System coupled with an Orbitrap Q Exactive Plus. The results of the analysis were elaborated using Metalab MAG software.

The metaproteomic analyses of the in vitro experiments were used to perform both phylogenetic and functional analysis. For example, at the level of phyla only the stimulation with Glyphosate led to statistically significant changes. For the other taxonomic levels investigated -orders, genera and species- all the chemicals led to significant alterations. Almost all modulated taxa were previously studied either to be important for the health and stability of the gut (decreased levels after treatments) or involved in some inflammatory processes (increased levels after treatments). The functional analysis of the treated samples treated showed a clear distinct pattern, at the molecular functions and biological processes levels, in respect with the control group. Finally, the stimulation of the gut microbiota with the six replacement PFAS induced significant microbiota changes.

The in-vitro approach coupled with metaproteomics is a valuable tool to investigate the impact of chemicals on the gut microbiota.