Tanya Bagrij

Cells exposed to calcein acetoxymethyl ester (calcein AM) in the growth medium become fluorescent following cleavage of calcein AM by cellular esterases to produce the fluorescent derivative calcein. It has previously been shown by others that multidrug resistant cells which overexpress P-glycoprotein accumulate much less fluorescent calcein than the corresponding parental cells. We have now examined the transport of calcein in multidrug resistant cells which overexpress an alternative transporter, the multidrug resistance-associated protein (MRP). Accumulation of calcein fluorescence was greatly reduced in the MRP-overexpressing human lung cancer cell lines COR-L23/R and MOR/R compared with their parental lines. Energy depletion resulted in a considerably increased accumulation in the resistant lines. Treatment of resistant cells with buthionine sulfoximine (BSO), which depletes cellular glutathione (GSH), did not affect calcein accumulation, in marked contrast to our previous results for daunorubicin or the fluorescent probe rhodamine 123. Genistein, verapamil, cyclosporin A and ouabain were also each able to modify, to some extent, accumulation of daunorubicin, whilst having essentially no effect on calcein accumulation. However, the organic anion transport inhibitor probenecid was able to increase accumulation of both calcein and daunorubicin in the resistant cells. Genistein and verapamil treatment preferentially reduced the GSH content of resistant cells, whilst probenecid did not. However, probenecid caused a clear decrease in release of GSH from resistant cells into the medium.

C57B1/6 male normal mice were treated with 5-FU (200 mg/kg i.p.) alone or in combination with a bolus injection of uridine (2 x 3500 mg/kg i.p.) in order to study the potential rescue effect of uridine on 5-FU-induced gastrointestinal toxicity. 5-FU alone inhibited the activity of different enzymes (thymidine-kinase, alkaline-phosphatase, sucrase and maltase) which were selected as the early biochemical markers for the injured small intestinal mucosa. The nadir of the enzyme activities was between 24-96 hrs after 5-FU administration, and the complete regeneration took a week. In the combination of 5-FU plus uridine bolus injection the seriousness of gastrointestinal damage caused by 5-FU was significantly (p < 0.05) milder and the recovery time was shorter by 2 days. Comparing the rescue effect of two dose schedules of uridine, both high dose (2 x 3500 mg/kg) or repeated lower doses of uridine (7 x 800 mg/kg) resulted in a similar protection from the gastrointestinal side effect of 5-FU.