Impact of Acrolein Production by Gut Bacteria on the Detoxification of Carcinogenic Heterocyclic Amines and on Intestinal Homeostasis

Abstract

Gut bacteria harboring cobalamin-dependent glycerol/diol dehydratases (GDH) (EC: 4.2.1.28/30), encoded by the genes pduCDE, can metabolize glycerol into 3-hydroxypropionaldehyde (3-HPA). 3-HPA exists in equilibrium mainly with its hydrate and its dimer, but it can also spontaneously dehydrate at physiological conditions to form acrolein. Acrolein is a highly reactive compound that may exhibit toxic effects on humans via different mechanisms including DNA adduction and it is also a wide spectrum antimicrobial. Recently, acrolein has been shown to decrease the mutagenic potential of heterocyclic amines (HCA), which are formed during high-temperature cooking of meat. HCA can form mutagenic DNA adducts and are considered an etiological factor for increased risk of colorectal cancer (CRC). The overall objective of this thesis was to investigate the potential of intestinal microbial communities and selected gut microbes for acrolein production via GDH-driven glycerol metabolism, and to gain insight on the effects that microbially produced acrolein might have on the intestinal ecosystem. First, we analyzed the GDH activity of nine in vitro cultivated model intestinal microbiota from different hosts. We hypothesized that the GDH activity depend on the abundance and distribution of GDH-active taxa and that supplementation of the GDH-positive Anaerobutyricum hallii increases GDH activity. We evaluated the GDH activity by assessing the transformation of 200 nM of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), one of the most abundant HCA in cooked meat, into its acrolein conjugate-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3',2':4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1), in presence of 100 mM of glycerol in batch fermentation after 24 hours. We also developed a quantitative PCR assay to profile the abundance and distribution of the GDH community in each microbiota. Our results showed that GDH activity was dependent on overall gdh abundance, and identified A. hallii as a key taxon in GDH metabolism. In addition, A. hallii supplementation resulted in enhanced PhIP transformation but reduced fermentation activity, likely due to acrolein accumulation. We then evaluated how the abundance of A. hallii in 20 healthy human fecal microbiota impacted PhIP transformation. We hypothesized that microbiota with high A. hallii abundance has a higher potential to transform PhIP, and that A. hallii supplementation of fecal microbiota increases PhIP transformation. We quantified the initial abundance of A. hallii in each microbiota using qPCR and assessed the transformation capacity of 200 nM of PhIP in presence of 100 mM of glycerol in fecal batch fermentation after 24 hours. Our results showed that A. hallii abundance was associated with the potential to transform PhIP as human microbiota with low A. hallii abundance (<106 per 1010 cells) did not show transformation of PhIP, while microbiota with high A. hallii abundance (>106 per 1010 cells) transformed up to 20 % of the provided PhIP. Furthermore, A. hallii supplementation significantly increased PhIP transformation in microbiota with low A. hallii abundance by up to 5 % compared to non-supplemented conditions, confirming data obtained with artificial gut microbiota from different hosts. In addition, we also showed that microbiota with high A. hallii abundance exhibited a significant decrease in metabolism likely due to acrolein accumulation. This suggests that the potential of fecal microbiota to produce acrolein is associated with the abundance of A. hallii. Acrolein is a wide-spectrum antimicrobial, therefore its production might provide a competitive advantage. Salmonella enterica Serovar Typhymurium is a gut pathogen that harbors GDH and therefore has the capacity to produce acrolein. Nevertheless, this capacity has not been reported yet. We confirmed the production of acrolein during growth of four wild-type S. Typhymurium strains, as revealed by the transformation of 200 nM of PhIP into PhIP-M1 in presence of 50 mM of glycerol in aerobic and anaerobic conditions. These results suggests that S. Typhymurium can release acrolein during growth, with strain-dependent activity Finally, we developed a model system to assess the formation of DNA adducts induced by acrolein produced by two strains of the gut commensal Limosilactobacillus reuteri. Resting cells of were with increasing glycerol concentrations (0 to 600 mM) in presence of calf-thymus DNA. Acrolein formation and adduct levels increased with glycerol concentration provided. Up to 3691 DNA adducts per 108 nucleosides were detected with 600 mM of glycerol. In addition, up to 1.5 mM of free acrolein was detected in the same conditions, suggesting that potentially more adducts could be formed. These results showed that gut bacteria can produce enough acrolein to induce DNA adducts. However, the concentrations of glycerol required to observe DNA adduct formation are likely not available in the human colon Taken together, the results of this PhD thesis suggest that the potential of intestinal communities to produce acrolein and detoxify PhIP is associated with the abundance of A. hallii. Furthermore, A. hallii supplementation increases PhIP transformation potentially reducing the risk of CRC initiation. Nevertheless, this effect was only tested in batch fermentation, a system which does not represent accurately the conditions of the human intestine. Therefore, a next step would be to test the impact of A. hallii abundance in fecal microbiota and its supplementation on PhIP transformation using a continuous fermentation system. In addition, despite the potential of A. hallii supplementation to reduce the risk of CRC initiation, the risk assessment to establish the safety of A. hallii has not been performed. Therefore, the potential safety concerns of A. hallii, must be clarified in order to contribute to the development of this bacteria as live biotherapeutic. We also showed that S. Typhymurium can produce acrolein, which might provide a competitive advantage. Nevertheless, the extent of this potential competition advantage must be clarified in vitro and in vivo. Finally, we showed that L. reuteri can produce enough acrolein to induce DNA adducts. Nevertheless, the concentrations of glycerol needed to observe the formation of DNA adducts are likely not available in the human gut. Thus, the physiological concentrations of glycerol available for gut bacteria must be clarified.