Effect of (mixtures of) flavonoids on the in vitro and in vivo bioavailability of 2-mino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) : a biologically based modelling approach

The transport of food ingredients across the intestinal epithelium is an important factor determining the absorption upon oral intake. Uptake of compounds in the intestine may be influenced by transport proteins such as the ATP binding cassette transporters (ABC transporters). ABC transporters have been shown to be involved in the efflux of several food related compounds and in the efflux of xenobiotics. The pro-carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was found to be transported by efflux transporters from the intestinal cells back into the intestinal lumen and in this way its uptake into the body could be prevented. Also it has been shown that flavonoids, present in fruits, vegetables, nuts, wine and tea are capable of inhibiting the efflux of substrates by ABC transporters. The aim of this thesis was to investigate the possible effect of flavonoids and mixtures thereof on the transport of PhIP across the intestinal barrier and to predict the absorption of PhIP through kinetically modelling. The results presented in this thesis show that different flavonoids are capable of increasing the apical to basolateral PhIP transport through Caco-2 monolayers, an in vitro model for the intestinal epithelium. It is also shown that the flavonoids can do so by acting as inhibitors of the apical ABC transporters in the intestinal cells. Using typical inhibitors for the ABC transporters experiments in Caco-2 monolayers revealed that especially MRP2 and BCRP are involved in the apical excretion of PhIP and these transporters can be inhibited by the flavonoids. Furthermore, an in silico model describing this process taking into account passive diffusion and active transport of PhIP was developed. Using the in silico model it could be demonstrated that for several flavonoids, including flavone, kaempferol chrysoeriol, myricetin, luteolin, naringenin, quercetin and apigenin, their apparent Ki values for inhibition of the active transport to the apical side are in the 5 to 50 μM range and thus within the physiological concentration range that may be achieved within the intestine upon supplement intake. Additional experiments revealed that several binary flavonoid mixtures and one mixture containing five model flavonoids increased the apical to basolateral PhIP transport through the Caco-2 monolayer. Assuming competitive inhibition of the apparent active transporter by the flavonoids and concentration-additivity for their inhibiting effect, the kinetic model could be extended and thus adequately described the experimental values obtained for the flavonoid mixtures. This illustrates that the effect of different flavonoids present in the diet is additive and from this it can be concluded that the effects of flavonoids on PhIP bioavailability can even be expected at levels achieved upon normal dietary intake. Finally, it was demonstrated that the observation that quercetin increases the transport of PhIP through Caco-2 monolayers in vitro could be confirmed in an in vivo rat model. Co-administration of PhIP and quercetin significantly increased the blood AUC(0-8hr) of PhIP in rats to 131±14% of the AUC(0-8hr) for rats dosed with PhIP alone. Therefore, all together the studies presented in this thesis point to a flavonoid-mediated increase of the bioavailability of PhIP and, thus, a possible adverse effect of these supposed beneficial food ingredients when present in combination with the pro-carcinogen PhIP.