Conjugation of chlorambucil with GSH by GST purified from human colon adenocarcinoma cells and its inhibition by plant polyphenols.

Chlorambucil (CMB) combines with glutathione (GSH) spontaneously in vitro to form monochloromonoglutathionyl CMB (MG-CMB). This was identified and quantified by an HPLC-UV method. Glutathione S-transferase (GST) purified from human colon adenocarcinoma cells increased the formation of the conjugate significantly. The GST-mediated conjugation, represented by the difference between total and spontaneous conjugation showed Michaelis-Menten kinetics with apparent Km and Vmax values of 0.2 mM and 75.8 nmol/min/mg for CMB and 5.2 mM and 127.0 nmol/min/mg for GSH respectively. Unexpectedly, we found in our study that both the spontaneous and the enzymatic conjugation of chlorambucil with GSH were affected markedly by a change in pH from 6.0 to 8.0. The optimum for the enzymatic conjugation was about 7.0, above which the spontaneous conjugation increased rapidly, while the enzymatic conjugation became lower. The plant polyphenols namely tannic acid, butein, quercetin, morin, 2-hydroxychalcone and 2'-hydroxychalcone at 40 microM inhibited the GST-mediated conjugation of CMB with GSH by 38 to 62%. Their action in this respect may contribute to sensitisation of tumour cells to anticancer drugs.