IN-VITRO AND IN-VIVO REVERSIBLE AND IRREVERSIBLE INHIBITION OF RAT GLUTATHIONE-S-TRANSFERASE ISOENZYMES BY CAFFEIC ACID AND ITS 2-S-GLUTATHIONYL CONJUGATE

The reversible and irreversible inhibition of glutathione S-transferas es (GST) by caffeic acid [3-(3,4-dihydroxyphenyl)-2-propenoic acid] wa s studied in vitro using purified rat isoenzymes, and in vivo in male Wistar (WU) rats. The concentrations of caffeic acid that inhibited re versibly 50% of the activity of different GST isoenzymes towards 1-chl oro-2,4-dinitrobenzene (CDNB) (I50 values) were 58 (GST 4-4), 360 (GST 3-3) and 470 mum (GST 7-7), and higher than 640 mum for GST isoenzyme s of the alpha class (GST 1-1 and 2-2). The major glutathione conjugat e of caffeic acid, 2-S-glutathionylcaffeic acid (2-GSCA), was a much m ore potent reversible inhibitor of GST, with I50 values of 7.1 (GST 3- 3), 13 (GST 1-1), 26 (GST 4-4), 36 (GST 7-7) and more than 125 mum (GS T 2-2). On the other hand, caffeic acid was a much more efficient irre versible inhibitor of GST than 2-GSCA. In this respect, GST 7-7 was by far the most sensitive enzyme, The remaining activity towards CDNB (e xpressed as percentage of control) after incubating 1.25 muM-GST with 100 muM-caffeic acid for 6 hr at 37-degrees-C was 34 (GST 2-2), 24 (GS T 1- 1), 23 (GST 4-4), 10 (GST 3-3) and 5% (GST 7-7). Almost no irreve rsible inhibition of GST 1-1 and 3-3 occurred during incubation with 2 -GSCA. Incubation of caffeic acid with liver microsomes from dexametha sone-induced rats catalysed the oxidation of caffeic acid about 18 tim es more effectively as compared with the spontaneous oxidation, as det ermined by the formation of GSH conjugates from caffeic acid. In vivo, the effect of single oral doses of caffeic acid (50-500 mg/kg body we ight) on the cytosolic GST activity towards CDNB was studied 18 hr aft er dosing in the liver, kidney and intestinal mucosa. A marginal but s ignificant linear relationship was found between the amount of caffeic acid dosed and the irreversible inhibition of GST activity in the liv er, with a maximum of about 14% inhibition in the highest dose group. This inhibition coincided with a small decrease in the mu-class GST su bunits, which was only significant for GST subunit 4.