INHIBITION OF GLUTATHIONE-S-TRANSFERASE ACTIVITY IN HUMAN-MELANOMA CELLS BY ALPHA,BETA-UNSATURATED CARBONYL DERIVATIVES - EFFECTS OF ACROLEIN, CINNAMALDEHYDE, CITRAL, CROTONALDEHYDE, CURCUMIN, ETHACRYNIC-ACID,AND TRANS-2-HEXENAL

The glutathione S-transferase (GST) activity towards 1-chloro-2,4-dini trobenzene in intact human IGR-39 melanoma cells was determined by the quantification by HPLC-analysis of the excreted glutathione (GSH) con jugate (S-(2,4-dinitrophenyl) glutathione; DNPSG). The major GST subun it expressed in these melanoma cells is the K-class GST subunit P1. Us ing this system; the effect of exposure for 1 h to a series of alpha,b eta-unsaturated carbonyl compounds at non-toxic concentrations was stu died. Curcumin was the most potent inhibitor (96% inhibition at 25 mu M), while 67 and 61% inhibition at 25 mu M was observed for ethacrynic acid and trans-2-hexenal, respectively. Moderate inhibition was obser ved for cinnamaldehyde and crotonaldehyde, while no inhibition was fou nd for citral. The reactive acrolein did not inhibit the DNPSG-excreti on at 2.5 mu M, the highest non-toxic concentration. Up to about 50% G SH-depletion was found after treatment with crotonaldehyde, curcumin a nd ethacrynic acid, however the consequences for GST conjugation are p resumably small. Reversible inhibition of GST was the major mechanism of inhibition of DNPSG-excretion in melanoma cells, except in the case s of curcumin and ethacrynic acid, which compounds also inactivated GS TP1-1 by covalent modification. This was dear from the fact that depen ding on the dose between 30 and 80% inhibition was still observed afte r lysis of the cells, under which conditions reversible inhibition is absent. Intracellular levels of DNPSG remained relatively high in the case of ethacrynic acid. It is possible that ethacrynic acid also inhi bits the transport of DNPSG by inhibition of the multidrug resistance- associated protein gene encoding glutathione conjugate export pump (MR P/GS-X pump) in some way.