7-Ethoxyresorufin O-deethylase induction in rainbow trout gill epithelium cultured on permeable supports: asymmetrical distribution of substrate metabolites.
The induction of 7-ethoxyresorufin O-deethylase (EROD) has been measured in cultured epithelia from rainbow trout gills. Epithelia incubated with water on the apical side and culture media at the basolateral side were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (betaNF), benzo[k]fluoranthene (B(k)F), and 3,3',4,4',5-pentachlorobiphenyl (PCB#126) from the water. EROD activity was measured as the formation of resorufin from 7-ethoxyresorufin over time in intact epithelia. The EC(50) values obtained after 24 h of exposure (mean+/-S.D.) were for TCDD (n=9) 4.1+/-3.2x10(-11) M, for betaNF (n=6) 1.6+/-3.8x10(-9) M, for B(k)F (n=4) 5.4+/-3.0x10(-9) M and for PCB#126 (n=4) 6.15+/-10.1x10(-9) M. When assaying for EROD activity, it was found that the resorufin concentrations differed between the apical and the basolateral compartments, indicating an asymmetrical distribution of the enzymatically formed resorufin molecules. Generally, the resorufin concentration was highest in the basolateral compartment, but there were differences between epithelia obtained from different fish individuals. Of a total of 13 preparations 10 had the highest resorufin concentration in the basolateral compartment, while in three preparations, the resorufin was uniformly distributed or slightly higher in the apical compartment. The reasons for this asymmetrical distribution of substrate metabolites are not known, and the addition of multidrug resistance inhibitors (verapamil and cyclosporin A) did not alter the asymmetrical pattern. The transepithelial electrical resistance (TER) was also measured to diagnose the tightness of the epithelia. The change from culture media to experimental water (containing TCDD, betaNF, or DMSO as control) in the apical compartment resulted in a large increase in TER, followed by a decline, measured after 24 h. The cytochrome P450 1A (CYP1A) inducers had no effect on the TER and were judged, therefore, not to affect the tightness of the epithelia.
Carlsson
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Department of Environmental Toxicology, Uppsala University, Norbyvägen 18 A, S-752 36 Uppsala, Sweden. carina.carlsson@ebc.uu.se
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D000818:Animals; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis*; D018432:Drug Resistance, Multiple; D017097:Electric Impedance; D004790:Enzyme Induction; D004847:Epithelial Cells; D005880:Gills / Q000201:enzymology*; D017686:Oncorhynchus mykiss / Q000378:metabolism*
D019363:Cytochrome P-450 CYP1A1