Characteristics of methotrexate polyglutamate formation in cultured hepatic cells

Abstract

Upon exposure of primary monolayer cultures of hepatocytes and H35 hepatoma cells, methptrexate (MTX) is taken up by carrier-mediated mechanisms and converted to γ-glutamyl derivatives with one to four residues being added. Under conditions that result in 90% or greater conversion, the primary metabolite in both cell types is MTX with three additional glutamates (4-NH 2-10-CH 3PteGlu 4). When the time-dependent synthesis of MTX polyglutamates (4-NH 2-10-CH 3PteGlu 2 and higher) at extracellular concentrations of 10 and 100 μ m methotrexate is measured, both cell types exhibit linear synthesis for 4 to 6 hr, at which time an apparent steady state intracellular concentration of approximately 40 μ m is reached. The concentration of MTX polyglutamate synthesized is not due a restriction in MTX since the hepatocytes and H35 cells accumulated 400 and 138 μ m intracellular methotrexate, respectively, after 24 h in the presence of 100 μ m extracellular MTX. Examination of MTX polyglutamate formation following a 24-h incubation showed concentration dependence with respect to intra- and extracellular MTX. Saturation was reached at a medium concentration of approximately 2 μ m with both cell types which corresponded to 10 to 12 μ m intracellular MTX. Placement of cells at steady state in medium lacking MTX results in the rapid equilibration of all free intracellular MTX with the medium. The MTX polyglutamates leave the cell by a slow loss of intact polyglutamates and also by intracellular cleavage to MTX followed by efflux. The longer-chain-length γ-glutamyl derivatives (Glu 4–5) are more avidly retained by the cells than the shorter ones (Glu 2–3).