Cellular transport of microcystin-LR in rainbow trout (Oncorhynchusmykiss) across the intestinal wall: Possible involvement of multidrugresistance-associated proteins

FLAVIA BIECZYNSKI; JULIETA S. DE ANNA; MACARENA PIREZ; BEATRÍZ M. BRENA; SILVINA S.M. VILLANUEVA; CARLOS M. LUQUET

AQUATIC TOXICOLOGY

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2014 p. 97 - 106

0166-445X

We studied Abcc mediated-transport in middle and posterior intestine of the rainbow trout, Oncorhynchusmykiss. Luminal and serosal transport were evaluated in everted and non-everted intestinal sacs,respectively, incubated with 1-chloro-2,4-dinitrobenzene (CDNB; 200 M). CDNB enters the cells and isconjugated with glutathione via glutathione S-transferase (GST) to form 2,4-dinitrophenyl-S-glutathione(DNP-SG), a known Abcc substrate. DNP-SG concentration in the bath was recorded every 10 min, in orderto calculate the mass-specific transport rate. For evaluating the possible involvement of Abcc proteins inmicrocystin-LR (MCLR) transport, 1.135 M MCLR was added to the bath or inside the sacs, in evertedor non-everted preparations, respectively. Both luminal and serosal DNP-SG efflux were significantlyinhibited by MCLR. A concentration?response curve obtained using strips from middle intestine yieldedan IC50value of 1.33 M MCLR. The Abcc inhibitor, MK571 produced concentration-dependent inhibitionof DNP-SG similar to that produced by MCLR. Since competition of MCLR and CDNB as GST substratescould bias the DNP-SG transport results, we evaluated the effects of MCLR on calcein efflux, which does notdepend on GST activity. We applied the non-fluorescent, cell-permeant compound calcein-AM (0.25 M)to middle intestinal strips and recorded the efflux of its hydrolysis product, the fluorescent Abcc substratecalcein. 2.27 M MCLR and 3 M MK571 inhibited calcein efflux (17.39 and 20.2%, respectively). Finally,MCLR interaction with Abcc transporters was evaluated by measuring its toxic intracellular effects. Mid-dle intestinal segments were incubated in saline solution with 1.135 M MCLR (MC1), 2.27 M MCLR(MC2), 3 M MK571 (MK) or 1.135 M MCLR + 3 M MK571 (MC1/MK). After 1 h, GSH concentration,protein phosphatase 1 and 2A (PP1, PP2A) and GST activities were measured in each segment. MC1didnot produce significant effect while MC1/MK and MC2 significantly inhibited PP1and PP2A in similarproportions (34?49%). MK alone significantly increased PP2A activity (40%) with no effect in any othervariable. GST activity and GSH concentration were not affected by any treatment. Concentration?responsecurves for MCLR (1.135 to 13.62 M) alone or plus 3 or 6 M MK571 were obtained using PP1 activity asresponse variable. The IC50values were 1.0, 0.52, and 0.37 M, respectively. Our results suggest that O.mykiss enterocytes are capable of eliminating MCLR by GST-mediated conjugation and luminal excretionthrough an Abcc-like apical transporter. This mechanism would prevent toxic effects and reduce the toxinuptake into the blood, which is likely mediated by basolateral Abccs.