The differential cytotoxicity of methotrexate in rat hepatocyte monolayer and spheroid cultures

It is important to assess the usefulness of long-term irt vitro liver model s for studying chronic toxicity, since acute assays may not reflect the in vivo situation. A potential long-term hepatocyte culture (i.e, liver sphero ids) was investigated and compared to primary rat hepatocyte monolayer cult ures following exposure to methotrexate (MTX), a well-documented chronic he patotoxin. Following up to 7 days' treatment with MTX, cultures were morpho logically assessed and assayed for enzyme leakage, intracellular reduced gl utathione (GSH) and adenosine triphosphate (ATP). Spheroids maintained high er concentrations of GSH over the 14-day culture and ATP was maintained, bu t at a concentration not significantly different from monolayer cultures. T reatment of monolayer cultures resulted in concentration-related decreases in GSH and ATP, accompanied by enzyme leakage, In contrast, only. ATP was a ffected following treatment of spheroids for 7 days, Spheroids appeared to be less sensitive to exposure to MTX, when compared with monolayer cultures , This may result from the maintenance of cellular functions, or from the l ack of compound penetration into the three-dimensional spheroid structure. Therefore, the usefulness of spheroids to chronic in vitro toxicity testing may be limited. (C) 2000 Elsevier Science Ltd. All rights reserved.