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We have recently reported that methotrexate (MTX) causes degenerative as well as reactive-like astroglial changes and alters the cell cycle kinetics of astrocytes in vitro. To further characterize the nature of the reactive-like changes that were noted by light and electron microscopy following MTX exposure, the glial fibrillary acidic protein (GFAP) content of astrocytes in culture was investigated by enzyme-linked immunosorbent assay, flow cytometry and double-immunofluorescent staining. An increase in GFAP content which did not correlate with drug dosage or DNA synthesis was noted in the MTX-treated cultures. It is postulated that this increase in GFAP content of astrocytes reflects an adaptive response to MTX-induced injury and partly explains the gliosis that is seen in methotrexate encephalopathy.

Relatively pure population of astrocytes in primary culture were examined by flow cytometry at various time intervals after exposure to 1, 10 and 100 microM of methotrexate (MTX). The percentage population of cells in different phases of the cell cycle was determined using propidium iodide (PI) to measure cellular DNA content. DNA synthesis was assessed by measuring the fluorescence intensity of FITC-conjugated antibody to bromodeoxyuridine (BrdUrd). The two parameters were correlated to determine the location of BrdUrd-incorporating cells within the cell cycle. Exposure of astrocytes to MTX caused a consistent increase in number of cells in S-phase which correlated with the increase in BrdUrd incorporation. These studies provide supportive evidence that the astrogliosis seen in MTX encephalopathy may be due to a primary involvement of astrocytes.

Recent studies on the animal model suggest that astrocytes may be a primary target for methotrexate (MTX) toxicity. To establish whether the astroglial alterations are due to a direct toxic effect of the drug, we studied the morphologic alterations, mitotic index, viability and growth rate of astrocytes in primary culture after exposure to varying concentrations of MTX in the absence or presence of dibutyryl cyclic AMP (dBcAMP). Dense bodies and cellular debris were noted by light and electron microscopy, and became more prominent with increasing doses and greater frequency of treatment. Degenerating cells and areas of necrosis were seen at higher concentrations. These changes became less conspicuous when MTX was given concurrently with dBcAMP. Large reactive-like astrocytes were also seen after MTX administration both in the absence or presence of dBcAMP. Mitotic rate inhibition was noted at all concentrations but was not dose-related. Cell viability was reduced and remained low up to 48 h after withdrawal of MTX and correlated well with drug concentration, although growth rate did not vary significantly from the control. Our findings show that pure populations of astrocytes can be adversely affected by MTX especially in the absence of bBcAMP, while also causing reactive-like changes in some cells. This report provides further evidence that astrocytes may be a primary target for MTX toxicity and suggests that the gliosis seen in MTX encephalopathy may in part be related to MTX-induced astrocytic injury.