Kim, Jae Kyeom2013-08-262013-08-262013-06https://hdl.handle.net/11299/155896University of Minnesota Ph.D. dissertation. June 2013. Major: Nutrition. Advisor: Sabrina P. Trudo. 1 computer file (PDF); xi, 234 pages, appendices A14-B.Apiaceous vegetables and their furanocoumarins inhibit activating enzymes for the food-born carcinogen PhIP, while cruciferous vegetables and their indole-3-carbinol (I3C) and phenethyl isothiocyanate (PEITC) induce PhIP-activating and -detoxifying enzymes. The experiments described in Chapter 2 and Chapter 3 were conducted to probe if these vegetables and their phytochemicals exert synergistic protection against PhIP genotoxicity owing to their complementary impacts on PhIP metabolism. Further, it was also aimed to make a direct comparison regarding the chemopreventive effects between intact vegetable feeding and purified compound feeding by matching the actual levels of active compounds, exposed to rats in each study. In the feeding study in Chapter 2, male Wistar rats were fed diets supplemented with cruciferous (21%, wt/wt), apiaceous (21%, wt/wt), and combination of both cruciferous and apiaceous vegetables (10.5% wt/wt of cruciferous and 10.5% wt/wt of apiaceous) for seven days. And it was found that the apiaceous vegetable group had the most protective effect against PhIP genotoxicity (approximately 20% reduction in PhIP-DNA adducts) followed by cruciferous vegetable and combination of vegetable groups indicating that expected synergistic potential through the combination of the vegetables was not shown. However, given the lack of inhibitory effects of apiaceous vegetables on the activity of PhIP-activating enzymes and N2-hydroxylated PhIP metabolites, the hypothesis on synergism of apiaceous and cruciferous vegetables is not completely rejected. Several experimental approaches can be pursued in future studies to achieve the suppression of PhIP-activating enzymes and test the hypothesis originally intended: 1) higher dose of apiaceous vegetable supplementation, 2) utilization of specific mechanistic inhibitors of PhIP activating enzymes, and 3) genetically engineered animal strains (e.g., CYP1A1 null mice). However, it should also be considered that such modulation in PhIP metabolism with either high dose of vegetable supplementation or exposure to specific inhibitors is achievable and realistic via habitual diets. In the experiments described in Chapter 2 and Chapter 3, we utilized the same animal strain (i.e., male Wistar rats) with identical experimental conditions including the level of PhIP exposure in order to compare protective potency between fresh vegetables and purified active compounds present in corresponding vegetable groups; however it was unfeasible to make one-to-one comparison because of statistical difference in PhIP-DNA adduct levels of control groups between studies. Thus, protective potential was estimated by looking at the effects of diet supplementation against the respective PhIP positive control group in each study. Cruciferous vegetable feeding and PEITC+I3C feeding showed approximately 17% (P=0.07) and 44% (P<0.0001) reduction of PhIP-DNA adduct formation, respectively, which is reasonable considering higher bioavailability of purified compounds as opposed to innate forms present in vegetables. However, furanocoumarin supplementation failed to elicit similar protection, demonstrated by apiaceous vegetables in Chapter 2. On the other hand, using a metabolomics approach, we detected 17 and 16 urinary PhIP metabolites in the vegetable feeding and phytochemical feeding studies, respectively. Of note, methylation was one of the major metabolic pathways for PhIP based on the relative abundance of the methylated metabolites detected in both studies and one of the methylated PhIP metabolites was not matched with any previously reported reference, suggesting a novel metabolite. As aforementioned in previous chapters, methylation is generally considered as a toxification step since it makes metabolites more lipophilic than the parent compound. However, interestingly, it was shown that apiaceous vegetable feeding increased methylated PhIP metabolites and these were negatively correlated with PhIP-DNA adducts in the colon; this association between methylation of PhIP metabolites and PhIP-DNA adducts was not expected but is somewhat biologically plausible. Of three methylated PhIP metabolites, two of them were methylated on the N2 position of PhIP; it is possible that methylation would influence the substrate-enzyme specificity, thereby providing less chance for PhIP to be activated to carcinogenic metabolites. In contrast, in the feeding study of Chapter 3, with purified compound supplementation, furanocoumarins (i.e., bioactive compounds of apiaceous vegetables) did not change the levels of methylated PhIP metabolites. Considering the abundance of methylated PhIP metabolites, further investigations on N-methyl transferases regarding their potential role in PhIP metabolism are warranted. In both chapters, enzyme activity results were mostly in agreement with the metabolite profile analysis while we found overall lack of correlation between enzyme activity and protein expression results. In particular, CYP1A1 and CYP1A2 activity in Chapter 3 were significantly increased in PEITC and I3C feeding (approximately 9.3-and 3.6-fold, respectively) without changes in their protein expression which is somewhat unexpected. However this lack of correlation between protein expression and enzyme activity was observed in other studies as well. For example, as aforementioned, utilizing a similar study design (e.g., rat strain, supplemented phytochemical components, and duration of exposure), it was found that PEITC supplementation significantly increased both CYP1A1 and CYP1A2 activity while protein expression levels were not changed during 4, 10, and 30 days of feeding periods. Such inconsistency was even more pronounced in CYP2B; although 30 days of treatment with PEITC increased enzyme activity approximately 3-fold but did not change the protein expression level, this suggests the possibility that incorporation of PEITC may influence these enzymes via different regulatory mechanisms such as post-translational modification. In fact, there is some indication that CYPs may undergo post-translational modification. To shed further light on this inconsistency, further studies are warranted, particularly regarding additional regulatory mechanisms of the respective gene expression and possible roles of phytochemicals. Collectively, we originally hypothesized that chemopreventive effects of vegetables and their active compounds would be achieved via modulation of phase I and phase II enzymes which are responsible for PhIP activation and detoxification. And as a matter of fact, supplementation of cruciferous vegetables and their active compounds resulted in modulation of both phase I and phase II enzymes; these modulations seem to favor PhIP detoxification. Even though apiaceous vegetable supplementation was effective in lowering PhIP-DNA adducts, such protection was not likely owing to furanocoumarins present in them or suppression of PhIP activation, taking into account the lack of effects of apiaceous vegetables on CYP1A activity and expression. It is not likely that impacts of vegetables are solely limited to their modulation in biotransformation enzymes. For example, there is evidence indicating another possible underlying mechanism of chemoprevention achievable through vegetables against PhIP, modulation of phase III detoxification. In particular, multidrug resistant-associated protein 2 (MRP2) is known to play a crucial role in PhIP metabolism as they extrude parent and PhIP metabolites from the gut mucosa. Previously, it was demonstrated that active compounds present in vegetables (e.g., flavonoids) can modulate PhIP metabolism via 1) induction of protein expression level of MRP2, 2) inhibition of the activity of MRP2, and 3) acting as substrates thereby modulating biliary or renal excretion of MRP2 substrates. Therefore, investigations in regard to the impact of apiaceous vegetables on phase III detoxification would be meritorious to explain the protection of apiaceous vegetables against PhIP genotoxicity.en-USThesis or Dissertation