Abstract
The glutathione S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene in intact human IGR-39 melanoma cells was determined by the quantification by HPLC-analysis of the excreted glutathione (GSH) conjugate (S-(2,4-dinitrophenyl)glutathione; DNPSG). The major GST subunit expressed in these melanoma cells is the pi-class GST subunit P1. Using this system, the effect of exposure for 1 h to a series of alpha, beta-unsaturated carbonyl compounds at non-toxic concentrations was studied. Curcumin was the most potent inhibitor (96% inhibition at 25 microM), while 67 and 61% inhibition at 25 microM was observed for ethacrynic acid and trans-2-hexenal, respectively. Moderate inhibition was observed for cinnamaldehyde and crotonaldehyde, while no inhibition was found for citral. The reactive acrolein did not inhibit the DNPSG-excretion at 2.5 microM, the highest non-toxic concentration. Up to about 50% GSH-depletion was found after treatment with crotonaldehyde, curcumin and ethacrynic acid, however the consequences for GST conjugation are presumably small. Reversible inhibition of GST was the major mechanism of inhibition of DNPSG-excretion in melanoma cells, except in the cases of curcumin and ethacrynic acid, which compounds also inactivated GSTP1-1 by covalent modification. This was clear from the fact that depending on the dose between 30 and 80% inhibition was still observed after lysis of the cells, under which conditions reversible inhibition was is absent. Intracellular levels of DNPSG remained relatively high in the case of ethacrynic acid. It is possible that ethacrynic acid also inhibits the transport of DNPSG by inhibition of the multidrug resistance-associated protein gene encoding glutathione conjugate export pump (MRP/GS-X pump) in some way.
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