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Potent Bile Salt and Organic Anion Inhibition of Methotrexate Uptake and Accumulation in the Freshly Isolated Rat Hepatocyte¹

Department of Medicine, Medical College of Virginia, Richmond, Virginia 23298

Influx of [³H]methotrexate into freshly isolated hepatocytes is mediated by high- and low-affinity processes, both of which are inhibited by the bile salts, cholate, taurocholate, and deoxycholate, and the organic anions, bromosulfophthalein and rose bengal. At 100 µM concentrations, these compounds inhibit 1 µM [³H]methotrexate influx by 70 to 90%. Dixon plots established a similar K_i (20 µM) for taurocholate inhibition of the high- and low-affinity influx routes for methotrexate, although the kinetics of this inhibition has not, as yet, been established. Bile salt inhibition of methotrexate influx requires the simultaneous presence of the bile salt and methotrexate at the cell membrane. Hence, pretreatment of hepatocytes with taurocholate, with subsequent removal of the bile salt from the extracellular compartment, does not alter methotrexate influx. Methotrexate at high concentration was found to inhibit the influx of [³H]taurocholate, suggesting the possibility of shared transport site(s) for bile salts and methotrexate. Influx of the naturally occurring folate, 5-methyltetrahydrofolate, is also inhibited by the bile salts and bromosulfophthalein, although to a lesser extent than influx of methotrexate. Cholate and taurocholate reduce not only methotrexate influx but also the net level of intracellular methotrexate accumulation, with a proportional reduction in the synthesis of methotrexate polyglutamate derivatives. Deoxycholate and bromosulfophthalein inhibit methotrexate polyglutamate synthesis to a greater extent than inhibition of total drug uptake. The presence of albumin in the buffer markedly reduces bromosulfophthalein inhibition of MTX influx, but has a much lesser effect on taurocholate inhibition of methotrexate influx, presumably related to the much higher affinity of albumin for bromosulfophthalein than for taurocholate.

These studies suggest that the level of bile salts in the hepatic sinusoids under physiological conditions may influence uptake and accumulation of methotrexate and its polyglutamate derivatives in hepatic parenchymal cells and may, therefore, influence the potential of this agent for producing hepatotoxicity in chemotherapeutic regimens. This raises the possibility that alterations in this interaction, in terms of regulation of bile salt pool size or time of administration of methotrexate relative to meals, may yield useful approaches for reducing methotrexate accumulation and metabolism in the liver and thereby minimize methotrexate hepatotoxicity especially in long-term low-dose regimens.

¹ Supported by Grants CA-16906 and AM-18976 from the NIH.

² Supported by Training Grant HL-07110 from the NIH.

³ To whom requests for reprints should be addressed.

Received 11/ 5/79. Accepted 2/19/80.

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