Prochloraz and nonylphenol diethoxylate inhibit an mdr1-like activity in vitro, but do not alter hepatic levels of P-glycoprotein in trout exposed in vivo

Abstract

P-glycoproteins (P-gps) encoded by multidrug resistance 1 (mdr1) genes are ATP-dependent transporters located in the cytoplasmic membrane which mediate the efflux of a broad spectrum of hydrophobic compounds from the cell. The tissue distribution of P-gps suggests their role in the organismal defense against xenobiotics by effecting xenobiotic excretion and reducing xenobiotic uptake. In the present work, the interaction of P-gp(s) in the liver and in primary cultured hepatocytes of rainbow trout with two model pollutants was studied - the imidazole fungicide prochloraz and the alkylphenolic surfactant nonylphenol diethoxylate (NP2EO). Using a monoclonal antibody (mAB C219) directed against a conserved P-gp epitope, an immunoreactive protein of 160 kDa was detected in immunoblots of liver extracts from control trout. In sections of control trout livers, immunohistochemistry with the mAB C219 resulted in specific staining of bile canaliculi. In juvenile trout exposed for 7 days to sublethal concentrations of prochloraz (0.027 microM; 0.27 microM) or NP2EO (0.32 microM; 1.30 microM), no changes in levels of hepatic P-gp(s) were found in immunoblot and immunochemical investigations. The efflux of the fluorescent mdr 1 substrate rhodamine 123 (Rh123) from cultured isolated trout hepatocytes was partly inhibited by verapamil and vinblastine, compounds known to interfere with mdr 1-dependent transport. This demonstrates the presence of a mdr1-like mechanism in trout liver which is probably involved in the biliary excretion of hydrophobic xenobiotics. Non-cytotoxic concentrations of prochloraz and NP2EO were tested for effects on the efflux of Rh123 from trout hepatocytes. Prochloraz was a potent inhibitor of the mdr1-like mechanism, being effective at 0.3 microM and above. NP2EO inhibited Rh123 efflux only at the highest concentration tested (31.6 microM). The accumulation and elimination of 14C-prochloraz by cultured trout hepatocytes was not affected by mdr 1-type substrates (Rh123, vinblastine) and a mdr 1 inhibitor (verapamil). This shows that prochloraz is, despite its inhibitory potency, not a substrate of the mdr1-like mechanism in trout liver. The inhibition by prochloraz and NP2EO of the md r1-like mechanism in trout hepatocytes suggests that water pollutants can interfere with P-gp-function in fish and thus may impair the organismal defense against xenobiotics.