In vitro and in vivo reversible and irreversible inhibition of rat glutathione S-transferase isoenzymes by caffeic acid and its 2-S-glutathionyl conjugate

Abstract

The reversible and irreversible inhibition of glutathione S-transferases (GST) by caffeic acid [3-(3,4-dihydroxyphenyl)-2-propenoic acid] was studied in vitro using purified rat isoenzymes, and in vivo in male Wistar (WU) rats. The concentrations of caffeic acid that inhibited reversibly 50% of the activity of different GST isoenzymes towards 1-chloro-2,4-dinitrobenzene (CDNB) (I50 values) were 58 (GST 4-4), 360 (GST 3-3) and 470 microM (GST 7-7), and higher than 640 microM for GST isoenzymes of the alpha class (GST 1-1 and 2-2). The major glutathione conjugate of caffeic acid, 2-S-glutathionylcaffeic acid (2-GSCA), was a much more 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 microM (GST 2-2). On the other hand, caffeic acid was a much more efficient irreversible inhibitor of GST than 2-GSCA. In this respect, GST 7-7 was by far the most sensitive enzyme. The remaining activity towards CDNB (expressed as percentage of control) after incubating 1.25 microM-GST with 100 microM-caffeic acid for 6 hr at 37 degrees C was 34 (GST 2-2), 24 (GST 1-1), 23 (GST 4-4), 10 (GST 3-3) and 5% (GST 7-7). Almost no irreversible inhibition of GST 1-1 and 3-3 occurred during incubation with 2-GSCA. Incubation of caffeic acid with liver microsomes from dexamethasone-induced rats catalysed the oxidation of caffeic acid about 18 times more effectively as compared with the spontaneous oxidation, as determined 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 weight) on the cytosolic GST activity towards CDNB was studied 18 hr after dosing in the liver, kidney and intestinal mucosa. A marginal but significant linear relationship was found between the amount of caffeic acid dosed and the irreversible inhibition of GST activity in the liver, with a maximum of about 14% inhibition in the highest dose group. This inhibition coincided with a small decrease in the mu-class GST subunits, which was only significant for GST subunit 4.