Inhibitors of the multixenobiotic resistance mechanism in natural waters: In vivo demonstration of their effects

Xenobiotics in water that can inhibit the multixenobiotic resistance (MXR) defense mechanism in water organisms, the chemosensitizers, may cause severe ecotoxicological effects. We determined their concentrations in polluted and unpolluted river waters and seawaters using different methods. Here we present the method that enables direct determination of MXR-inhibitory potential in natural waters. This method measures the modulating potential of river water or seawater on the rate of rhodamine B (RB) accumulation in gills of a freshwater clam (Dreissena polymorpha) or a marine mussel (Mytilus galloprovincialis), respectively. The sensitivity of the method with RB even enabled the determination of MXR inhibitors by measuring their potential to modulate the rate of efflux of RB from gills of mussels. The concentrations of MXR inhibitors found by these methods (expressed in mu M of verapamil-equivalents) were higher in natural waters from polluted rivers or in natural seawaters from polluted marine sites than in natural waters from unpolluted rivers or in natural seawaters from unpolluted sites. Polluted waters enhanced accumulation or decreased efflux rate of RB, a good substrate of P-glycoprotein, demonstrating that the complex mixture of chemicals present in polluted waters contains MXR-inhibiting potential. In addition, the efflux version of the method with RB has the clear advantage of not requiring that organisms be killed and so allows repeated use of the same individuals. In addition, it is the simplest and the most reliable method for measuring the activity of MXR in these organisms.