Multixenobiotic resistance, P-glycoprotein, and chemosensitizers

Abstract

Aquatic organisms are inherently equipped with a mechanism that rescues them by pumping potentially toxic xenobiotics out of the cells before they express their deleterious potential. The presence of this multixenobiotic resistance (MXR) mechanism may explain, at least partly, the simultaneous resistance of many aquatic organisms to the presence of different chemicals in polluted waters (Kurelec, 1992). This ATP-dependent membrane P-glycoprotein (Pgp) pump, identical to Pgp in a mechanism of multidrug resistance (MDR) in tumour cells (Endicott and Ling, 1989 ; Gottesman and Pastan, 1993), was confirmed in aquatic organisms by biochemical ("binding" ; Kurelec and Pivcevic, 1989), molecular (immunohistochemical, Western, Northern ; Toomey and Epel, 1993 ; Minier et al., 1993 ; Kurelec et al., 1992 ; Cornwall et al., 1995), physiological (verapamil-sensitivity ; Kurelec and Pivcevic, 1991 ; Minier and Moore, 1996 ; Galgani et al., 1996) and toxicological (modulation of toxicity ; Waldmann et al., 1993) methods. The inducibility of MXR by xenobiotics and its wide taxonomic distribution suggests its role as a general biological first-line defence mechanism (Kurelec, 1997a), and also suggests that it could be used as a biomarker for exposure to pollution. Supporting this hypothesis are the finding that exposure to polluted water induces the expression of both MXR activity and protein titer (Kurelec et al., 1996). Also, there is a correlation between the concentration of identified pollutants and activity of the transporter (Eufemia and Epel, 1998). Such induction of MXR increases the resistance of individuals or populations to polluted waters. Therefore, measurement of the level of MXR-induction in individuals and populations could be used also as a biomarker of (non)susceptibility (Kurelec, 1997b). Such biomarkers may considerably improve the characterisation of exposure, a crucial segment in the process of environmental risk assessment (Muller et al., 1996). However, before the MXR can be adopted as a valid biomarker, background information on seasonal, geographical, other stressors, and population variables needs to be accumulated. An additional property of the MXR mechanism is that a wide variety of chemicals which are not necessarily toxic by themselves can be substrates for the transporter. These chemicals can compete with more toxic xenobiotics and effectively inhibit the MXR-mechanism, thus circumventing the MXR defence. The concentration of such MXR-inhibitors/competitive substrates, which we refer to as "chemosensitizers" is significantly higher in polluted than in unpolluted waters (Smital and Kurelec, 1997). The concentrations present were able to enhance the accumulation, and therefore the effects, of xenobiotics in experimentally exposed aquatic organisms. For example, there was increased accumulation of carcinogenic aromatic amines in mussel, with subsequent enhancement in the production of their mutagenic metabolites Kurelec et al., 1997), the induction of single strand breaks in DNA (Waldmann et al., 1993) and the induction of DNA adducts (Kurelec, 1992). These results demonstrate the ecotoxicological significance of this new class of hazardous chemicals and confirms the importance of the control of MXR-inhibitors in ecological risk assessment studies (Kurelec et al., 1998). In this paper we describe a variety of methods that measure the activity of MXR using an accumulation assay with rhodamine B in whole organisms as well as in isolated cells and tissues. We also describe methodology that measures the level of expression of the MXR transport protein by immunochemical (Western) methods. In addition, we describe two methods which allow measurement of the concentration of chemosensitizers either in environmental samples, by measurement of the modulation of the rate of rhodamine B accumulation in a culture of NIH 3T3 cells stable transfected with a human MDR1 gene, or in native waters by measurement the modulation of the rate of efflux of rhodamine B in aquatic species under in vivo conditions.