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

P-glycoproteins (P-gps) encoded by multidrug resistance 1 (mdr 1) genes are ATP-dependent transporters located in the cytoplasmic membrane which media te the efflux of a broad spectrum of hydrophobic compounds from the cell. T he tissue distribution of P-gps suggests their role in the organismal defen se against xenobiotics by effecting xenobiotic excretion and reducing xenob iotic 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 polluta nts was studied - the imidazole fungicide prochloraz and the alkylphenolic surfactant nonylphenol diethoxylate (NP2EO). Using a monoclonal antibody (m AB C219) directed against a conserved P-gp epitope, an immunoreactive prote in of 160 kDa was detected in immunoblots of liver extracts from control tr out. In sections of control trout livers, immunohistochemistry with the mAB C219 resulted in specific staining of bile canaliculi. In juvenile trout e xposed for 7 days to sublethal concentrations of prochloraz (0.027 muM; 0.2 7 muM) or NP2EO (0.32 muM; 1.30 muM). no changes in levels of hepatic P-gp( s) were found in immunoblot and immunochemical investigations. The efflux o f the fluorescent mdr1 substrate rhodamine 123 (Rh123) from cultured isolat ed trout hepatocytes was partly inhibited by verapamil and vinblastine, com pounds known to interfere with mdr1-dependent transport. This demonstrates the presence of a mdr1-like mechanism in trout liver which is probably invo lved in the biliary excretion of hydrophobic xenobiotics. Non-cytotoxic con centrations of prochloraz and NP2EO were tested for effects on the efflux o f Rh123 from trout hepatocytes. Prochloraz was a potent inhibitor of the md r1-like mechanism, being effective at 0.3 muM and above. NP2EO inhibited Rh 123 efflux only at the highest concentration tested (31.6 muM). The accumul ation and elimination of C-14-prochloraz by cultured trout hepatocytes was not affected by mdr1-type substrates (Rh123, vinblastine) and a mdr1 inhibi tor (verapamil). This shows that prochloraz is, despite its inhibitory pote ncy, not a substrate of the mdr1-like mechanism in trout liver. The inhibit ion by prochloraz and NP2EO of the mdr1-like mechanism in trout hepatocytes suggests that water pollutants can interfere with P-gp-function in fish an d thus may impair the organismal defense against xenobiotics. (C) 2001 Else vier Science B.V. All rights reserved.