Glutathione conjugation of chlorambucil: Measurement and modulation by plant polyphenols

Chlorambucil (CMB), an anticancer drug, was cytotoxic at concentrations of 5-20 mu M to human colon adenocarcinoma cells. It inhibited [C-14]thymidine uptake in a dose-dependent manner. Both effects were potentiated by simultaneous exposure of the cells to 10 mu M plant polyphenols. In an attempt to explain the possible mechanism of action of the polyphenols in relation to these observations, an HPLC-radiometric method was developed to measure the conjugation of CMB with glutathione in these cells and to monitor the export of monochloromonoglutathionyl CMB (MG-CMB), its main glutathione conjugate. At micromolar concentrations, five polyphenols, namely quercetin, butein, tannic acid, 2'-hydroxychalcone and morin, inhibited the efflux of CMB significantly; an inhibition of 40% was observed with 10 mu M quercetin. The glutathione S-transferase (GST) activity of the cancer cells, measured with 1-chloro-2,4-dinitrobenzene, was also inhibited by the polyphenols. Their combined action on GST and on the efflux of MG-CMB conjugate could provide an enhanced positive modulation of sensitivity of the tumour cells to CMB.