Influence of human intestinal microbial metabolism on the induction of Nrf2 signaling by green tea catechins as characterized by new approach methodologies (NAMs)

Green tea, which is mainly manufactured from buds and leaves of Camellia sinensis, is one of the most widely consumed beverages around the world. Catechins are the most abundant bioactive constituents in green teas, amounting up to 30% of the total dry weight. (-)-Epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG) and (-)-epicatechin (EC) are the four major catechins among others. These catechins are believed to be responsible for various beneficial health effects that have been ascribed to green tea consumption. Though the modes of action underlying these health-promoting effects can be complicated and have not been fully understood, the Kelch-like ECH-associated protein 1/Nuclear factor E2-related factor 2 (Keap1/Nrf2) regulatory network has been reported to play a role. In the present thesis, in vitro anaerobic fecal incubation was used to characterize the microbial conversion of green tea catechins EC and EGCG. Results obtained show phenylpropane-2-ols, phenyl-valerolactones, gallic acid, pyrogallol, phenyl-valeric acids as the major metabolites, with substantial interindividual differences. Moreover, intraindividual difference in the formation of the major EC colonic metabolite 5-(3’,4’-dihydroxyphenyl)-γ-valerolactone (3,4-diHPV) was also observed, though the interindividual difference was larger than intraindividual difference. Furthermore, quantitative microbiota characterization in the fecal samples, achieved by 16S rRNA analysis, revealed substantial differences in microbiota compositions among different individuals. Correlations between specific microbial abundance and formation of certain metabolites were established. The Nrf2 signaling activation by EGCG, EC and their major microbial metabolites were tested using reporter gene assays, RT-qPCR, and proteomics technic. Results show EGCG and the microbial metabolites 3,4-diHPV and pyrogallol can induce concentration-dependent induction of Nrf2 pathway. Moreover, metabolites 3,4-diHPV and pyrogallol exhibited better Nrf2-activation potency than their respective parent compounds, EGCG and EC. Finally, the thesis also developed a human PBK model for EGCG, with sub-models for its major microbial metabolites gallic acid and pyrogallol. The model enabled prediction of in vivo kinetics of EGCG, gallic acid and pyrogallol which allowed the translation of the obtained in vitro concentration-response curves from the U2OS-Nrf2 CALUX reporter gene assay to in vivo dose-response curves for Nrf2 activation, and comparison of these data to estimated daily intake levels. Results obtained reveal, by comparison to literature data on EGCG kinetics, that the developed PBK model could adequately predict in vivo time-dependent blood concentrations of EGCG after either a single or repeated oral administration(s) of EGCG under both fasting and non-fasting conditions. The predicted in vivo dose response curve revealed that at daily intake levels of green tea or EGCG supplements, the resulting blood Cmax of EGCG was in the sub-micromolar range, concentrations at which Nrf2 activation was shown to be limited. Moreover, blood Cmax values of gallic acid and pyrogallol upon intake of EGCG were predicted to be less than 1.5% of the Cmax of EGCG, indicating that despite their higher potential for Nrf2 activation, their contribution to the overall systemic Nrf2 pathway induction upon EGCG exposure is expected to be limited. In contrast, concentrations of these metabolites in the intestinal tract may reach levels that are, expressed in EGCG equivalents for Nrf2 induction, higher than that of EGCG, and high enough to activate Nrf2 gene transcription. Taken together, combining in vitro data with a human PBK model allowed the prediction of a dose-response curve for EGCG induced Nrf2-mediated gene expression in humans, and provided insight into the contribution of gut microbial metabolites to this effect. It also provided a proof-of-principle for a NAM to study the in vivo effects of bioactive phytochemicals without the need for human intervention studies