- Purpose:
To determine the role of MDR transporters in general, and MDR1 Pgp and MRP2 in particular, in the defense against food-derived carcinogens. Previous research in our laboratory has demonstrated that Mrp2 in the rat functions in the extrusion of the most abundant food-derived heterocyclic amine, PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-B]pyridine), from the intestinal mucosa. The aim of the project is:
1.To define the mechanism by which MDR transporters extrude these compounds;
2.To extend this observation to other environmental carcinogens and to analyze the relative contribution of different MDR transporters in this process;
3.To define the effect of MDR transporter deficiency on in vivo tumor formation induced by these carcinogens;
4.To assess the relative expression of the MRPs along the human and rodent gastrointestinal tract, in order to extrapolate the results from rodents to the human situation.
- Background:
Food-derived (pro)carcinogens, such as nitrosamines, heterocyclic amines and mycotoxins, play a potentially important role in the development of cancer. The metabolic activation and detoxification of these compounds by phase I and phase II enzymes, respectively, have been studied extensively. It is surprising, however, that the entry mechanism of carcinogens into the intestinal mucosa was so far largely neglected. These compounds are amphipathic molecules and, in theory, the net entry of these molecules into the mucosa depends on the rate of diffusion or uptake into the epithelium combined with the rate of active extrusion back into the lumen or into the blood. The latter process is determined by the activity of a set of MDR transporters in the plasma membrane, including MDR1 P-glycoprotein and the family of multidrug resistance associated proteins (MRPs). If these transporters extrude unmetabolized procarcinogens, they can be regarded as "phase 0" enzymes, the function of which comes into play already before that of the metabolizing phase I and II enzymes. We have tested this hypothesis and found it to be correct: the most abundant heterocyclic amine, PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-B]pyridine) is transported by Mrp2 in the rat. The absence of this transporter in the TR- rat leads to an increased oral bioavailability and, as a consequence, to significantly higher organ contents of this carcinogen. PhIP is not an organic anion and this raises questions concerning the mechanism by which PhIP is transported. It has been shown that certain uncharged compounds can be transported via MRP1 and this transport is stimulated by GSH. We also found that modulation of intracellular GSH levels influenced the excretion of PhIP. The exact mechanism by which this happens is, however, unclear.
- Plan of investigation:
1. We will further explore the mechanism of transport of PhIP via Mrp2. Specific attention will be paid to the role of GSH. Transport will be measured in intact animals (normal vs. TR- rats), in Ussing chambers with small intestinal tissue from normal and TR- rats, in cell lines overexpressing MRP2 and in Sf9 membrane vesicles with high levels of MRP2.
2. From the different assay systems mentioned above we will choose the one that is most apt for screening of other carcinogens. This screening will not be limited to Mrp2 but we will also look at the role of Mrp1, Mrp3 and mdr1, for which knockout mice are available, or will be very soon.
3. We will assess the role of Mrp2 in carcinogen-induced tumor formation by chronic feeding of PhIP in Wistar and TR- rats. This will also be done for other carcinogens that emanate from line 2.
4. The relative expression of the MRPs along the human gastrointestinal tract will be measured by immunohistochemistry and Western blotting, in order to extrapolate results from studies with mice and rats to the human situation.
- Possible results:
It may be expected that a spectrum of environmental carcinogens is directly extruded from the intestine by MRP2 or MDR1 Pgp. This raises the possibility that these transporters constitute a first line of defense against these compounds and that individuals with polymorphisms causing reduced activity or expression will be predisposed to development of colorectal or other types of cancer. |
