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Conversion of Methotrexate to 7-Hydroxymethotrexate and 7-Hydroxymethotrexate Polyglutamates in Cultured Rat Hepatic Cells

Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201

The formation of 7-hydroxymethotrexate and its diglutamate has been established in rat hepatic parenchymal cells in culture. The formation of 7-hydroxymethotrexate increases with the extracellular methotrexate concentration (1–50 µM) and with time over a 24-h period. The majority of the 7-hydroxy derivative is found in the medium after 6- and 24-h incubations at all concentrations examined. At high methotrexate concentration (50 µM) 7% of the total extracellular methotrexate was the 7-hydroxy derivative. 7-Hydroxymethotrexate diglutamate is accumulated within the cell, although longer chain length derivatives are not observed. The inability to form longer chain length polyglutamate derivatives is consistent with a limited capacity of hepatocytes to convert radiolabeled 7-hydroxymethotrexate to the tri- and tetraglutamates although the diglutamate is readily formed. A hepatoma cell line has an extremely limited capacity to form 7-hydroxy derivatives from methotrexate but form measurable amounts of mono-, di-, and triglutamates of 7-hydroxymethotrexate. 7-Hydroxymethotrexate was a good substrate for glutamylation in the hepatoma cells with large amounts of tri- through pentaglutamate derivatives. These studies confirm that 7-hydroxymethotrexate and the diglutamate must be considered when evaluating methotrexate pharmacology and demonstrate that this will be necessary with regard to methotrexate hepatotoxicity.

¹ Recipient of NIH Grants CA25933 and CA34314 by the National Cancer Institute, USPHS, Department of Health and Human Service. To whom requests for reprints should be addressed.

Received 1/13/86. Revised 4/ 9/86. Accepted 4/16/86.