Involvement of an organic anion transporter (canalicular multispecific organic anion transporter/multidrug resistance-associated protein 2) in gastrointestinal secretion of glutathione conjugates in rats.

We investigated the role of cMOAT/MRP2 (canalicular multispecific organic anion transporter/multidrug resistance-associated protein 2) in the intestinal secretion of organic anions by comparing the behavior in Sprague-Dawley (SD) rats and Eisai hyperbilirubinemic rat (EHBR) whose cMOAT/MRP2 is hereditarily defective. After i.v. administration of 1-chloro-2,4-dinitrobenzene (30 micromol/kg), the biliary and intestinal excretion of its glutathione conjugate 2, 4-dinitrophenyl-S-glutathione (DNP-SG), a substrate for cMOAT/MRP2, was significantly reduced in EHBR compared with SD rats. This result also was confirmed by Ussing chamber studies; DNP-SG showed 1.5-fold greater serosal-to-mucosal flux compared with the mucosal-to-serosal flux in SD rats, whereas a similar flux was observed in both directions in EHBR. In addition, metabolic inhibitors reduced the preferential serosal-to-mucosal flux of DNP-SG in SD rats. In everted sac studies, intestinal secretion clearance, defined as the efflux rate of DNP-SG into the mucosal side divided by the area under the curve on the serosal side, was significantly lower in the jejunum of EHBR than that in SD rats. Northern blot analyses demonstrated the highest mRNA level of cMOAT/MRP2 in the jejunum, which is in good agreement with the results of the everted sac studies. These results suggest that cMOAT/MRP2 is involved in the secretion of organic anions in the small intestine.

Polymorphism in the glutathione conjugation activity of human erythrocytes towards ethylene dibromide and 1,2-epoxy-3-(p-nitrophenoxy)-propane.

In this study a polymorphism in the conjugating activity of human erythrocyte cytosol towards the dihaloethane, ethylene dibromide (EDB; 1,2-dibromoethane) was found. Two out of 12 human erythrocyte cytosols did not catalyze the formation of glutathione (GSH) conjugates of [1,2-14C]EDB. Ten cytosols formed the S,S'-ethylenebis(GSH) conjugate at a rate ranging from 0.5 to 3.2 (mean 1.76 +/- 0.95) pmol min-1 (mg protein)-1. The activity of the cytosols towards EDB was compared with the activity towards 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP) and 1-chloro-2,4-dinitrobenzene (CDNB). The GSH conjugates formed from EDB, EPNP and CDNB were all quantified by HPLC. Every cytosol was active with the classical GST substrate CDNB (2.04 +/- 0.74 nmol min-1 (mg protein)-1). The two samples not showing any detectable activity towards EDB were also inactive towards EPNP: The activity towards EDB correlated significantly with EPNP (rs = 0.90, P < 0.005; Spearman's rank correlation), but not with CDNB (rs = 0.36, P > 0.10). In the incubations with EPNP, the alpha-, mu-, and pi- class glutathione S-transferase (GST) inhibitor S-hexyl(GSH) was included, indicating that the class-theta GST is the principal GST class conjugating EDB in erythrocyte cytosol. The apparent polymorphism of GST-theta which has recently been recognized to be crucial for several mono- and dihalomethanes, will thus also have considerable implications for the risk assessment of EDB.

Formation and transport of xenobiotic glutathione-S-conjugates in red cells.

In vitro studies with freshly drawn human erythrocytes showed 4-dimethylaminophenol, a cyanide antidote, to be rapidly metabolized with the formation of a transient S,S-(2-dimethylamino-5-hydroxy-1,3-phenylene)bis-glutathione conjugate and a stable S,S,S-(2-dimethylamino-5-hydroxy-1,3,4-phenylene)tris-glutathione conjugate. The stable tri-glutathionyl derivative was actively transported across the red cell membrane with an apparent Vmax = 1 nmol/min/ml red cell suspension (15 g hemoglobin/100 ml) and Km = 0.5 mM. The transport system was strictly unidirectional, inhibited completely by sodium fluoride and reduced to one-fifth by lowering the temperature from 37 to 22 degrees. Similarly S-(2,4-dinitrophenyl)-glutathione, the glutathione-S-transferase mediated glutathione-S-conjugate with 1-chloro-2,4-dinitrobenzene, was unidirectionally transported, a process which was inhibited by sodium fluoride. Kinetic analysis revealed two different transport processes: Vmax = 0.9 nmol/min/ml, Km = 1.4 microM and Vmax = 4.5 nmol/min/ml, Km = 700 microM. Mutual inhibition of the low affinity transport system was found for both glutathione-S-conjugates. The apparent energies of activation for all these transport processes and for GSSG were identical (70 kJ/mol) suggesting at least one common carrier for the excretion of the three glutathione-S-conjugates.

Detoxification of xenobiotics by glutathione S-transferases in erythrocytes: the transport of the conjugate of glutathione and 1-chloro-2,4-dinitrobenzene.

The incubation of human erythrocytes with 1-chloro-2,4-dinitrobenzene (CDNB) results in almost quantitative conjugation of glutathione (GSH) to form S-(2,4-dinitrophenyl) glutathione. The reaction is catalysed by erythrocyte glutathione S-transferase. During the present studies we have identified the conjugate in the incubation medium of CDNB-treated erythrocytes, indicating that the conjugate of GSH and CDNB is transported out by the erythrocytes. Quantitation of the conjugate in the incubation medium by amino acid analysis and thin layer chromatography indicates that the erythrocytes transport the conjugate at an approximate rate of 140 nmol/h/ml erythrocytes. The transport of the conjugate is inhibited by sodium fluoride. Exhaustion of ATP from the erythrocytes results in a significant decrease in the rate of transport which is restored with the regeneration of ATP by incubating the erythrocytes with adenine and inosine. This indicates that the transport of conjugate is an energy dependent process.

Evidence for glutathione-S-transferase activity in human blood platelets.

Data will be presented pointing to the presence of glutathione-S-transferase activity in human blood platelets and the possible involvement of this enzyme in the process of platelet activation. Using 1-chloro-2,4-dinitrobenzene as a synthetic substrate of the glutathione-S-transferase a rapid dose-dependent depletion of platelet glutathione was measured. The formed GSH-CDNB conjugate was separated by thin-layer chromatography. Hints to the formation of leukotriene-like substances by glutathione-S-transferase catalysed reaction were obtained using the specific leukotriene C antagonist FPL 55 712 in aggregation studies.