Impact of Gut Microbiota on the Metabolism of Carcinogenic Dietary Heterocyclic Amines

Abstract

Human are constantly exposed to potentially toxic chemicals from the environment, diet, and therapeutic interventions. However, the gut harbors a diverse community of microorganisms, which may alter the exposure and toxicity of these chemicals. Heterocyclic amines (HCAs) are mutagens presented in meat cooked at high temperature, and they are strongly associated with the increased risk of colorectal cancer. Bacterial transformation of HCAs may alter their structures and thus their toxicity, but little is known regarding the influence of gut microbiota on the risk associated with these chemicals due to the existing knowledge gap regarding the microbiota-chemical interactions. The work described in this thesis concerns the chemical and biochemical mechanisms of commensal gut bacterial transformation of HCAs and their liver metabolites, as well as toxicological and physiological relevance of the microbial transformations. The knowledge obtained provides mechanistic insights on how gut microbiota alter chemical toxicity and supports gut microbiota as a factor in the risk assessment of toxic chemicals. Chapter 1 introduces the background information concerning the microbial metabolism of glycerol in the human gut and its relevance to HCA transformation. In addition, an overview of the potential targets of gut bacteria-derived acrolein is given. In Chapter 2, we aimed to address the generality of bacterial conjugation of HCAs with acrolein. MeIQx is an imidazoquinoxaline mutagen ten times more mutagenic than PhIP toward bacterial DNA in vitro assays. E. hallii, Lactobacillus reuteri, and Lactobacillus rossiae were found to convert MeIQx to a new microbial metabolite characterized on the basis of HRMS and NMR as 9-hydroxyl-2,7-dimethyl-7,9,10,11-tetrahydropyrimido-[2′,1′:2,3]imidazo[4,5-f]quinoxaline (MeIQx-M1). Acrolein derived from the decomposition of 3-HPA, which is a product of glycerol reduction mediated by GDH activity, was identified as the active compound responsible for the formation of MeIQx-M1. MeIQx-M1 appears to have slightly reduced cytotoxic potency toward human colon epithelial cells, and diminished mutagenic potential toward bacteria after metabolic activation. In Chapter 3, the physiological and toxicological relevance of the microbial transformation of MeIQx to MeIQx-M1 was characterized. To address whether the microbial transformation influences the intestinal transport of MeIQx, the intestinal uptake of MeIQx and its metabolite MeIQx-M1 was quantified with ex vivo rat intestinal segments, however, only negligible amounts of both MeIQx and MeIQx-M1 were transported. In addition, neither MeIQx nor MeIQx-M1 were cytotoxic towards liver HepaRG cells at dietary levels or higher concentrations. Physiologically based pharmacokinetic modeling suggests that increased microbial transformation of MeIQx can reduce plasma levels of MeIQx, potentially contributing to reduced systemic exposure of MeIQx in human. In Chapter 4, the impact of commensal gut microbes on the transformation of HCA liver metabolites, especially glucuronide conjugates was investigated. The aim was to gain knowledge regarding how gut microbes coordinate to transform HCA-glucuronides to release HCAs and further convert them to HCA-M1s. The beta-glucuronidase (GUSB) produced by species belong to Eubacterium and Faecalibacterium catalyzed the re-activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine N2-β-D-glucuronide (PhIP-G), a liver metabolite of PhIP, to form PhIP. GDH of Flavonifractor plautii, Blautia obeum, E. hallii, and L. reuteri contributed the detoxification of PhIP to form PhIP-M1. Co-culture of GUSB+ strain and GDH+ strain led to the conversion of PhIP-G to PhIP and PhIP-M1. These results for the first time demonstrate the gut microbial transformation of PhIP-G to PhIP-M1, providing a mechanistic model for reducing PhIP exposure from enterohepatic circulation by linking the modification of a carcinogen with the metabolic activity of predominant gut microbes. In Chapter 5, the chemical scope of the reaction of acrolein with HCAs was expanded. The aim of this study was to gain knowledge on the reactivity of heterocyclic guanines (HCGs) with α,β-unsaturated aldehydes (UAs). Results suggest that HCGs react with acrolein to form products bearing a hydroxyltetrahydropyrimidine ring. Substitution on both α and β positions of acrolein dramatically reduced reactivity and acrolein had the highest reactivity among the tested UAs. Moreover, two drugs bearing a guanidine moiety can readily react with acrolein with 491-721 times faster than that of DNA base with acrolein. These findings suggest the guanidine conjugation with acrolein is a general reaction of HCGs mediated by bacterial acrolein and HCGs are scavengers of acrolein. Chapter 6 summarizes the findings and gives an outlook. A critical discussion of achievements and limitations is provided and ongoing future directions are presented.