Validation of a morphometric analysis procedure using indomethacin-induced alterations in cultured hepatocytes

Morphometric analysis was used to quantitate indomethacin-induced morphological changes in primary cultures of neonatal rat hepatocytes by comparison with standard assessments of functional integrity (i.e. enzyme release, urea levels, and dye exclusion). For this procedural validation, indomethacin concentrations were selected to correspond to the therapeutic plasma levels of rheumatoid arthritic or systemic lupus erythematosus patients having liver cell injury secondary to prolonged, high-dose administration of this nonsteroidal antiinflammatory agent. Primary cultures of neonatal rat hepatocytes were exposed for 12 h to 0, 100, 500 or 1000 microM indomethacin and subjected to a double-blind morphometric analysis procedure modified for use on cultured cells. This procedure systematically converted two-dimensional morphologic information into three-dimensional numerical data for statistical analysis. These optical measurements provided an accurate analysis following 4 h of measurements. When the concentration of indomethacin was increased from 0 to 1000 microM, the relative volume percent of Type I cells decreased. Therefore these cells, which were indistinguishable from healthy, untreated control cells, become less numerous as toxicant level increased. In contrast, the relative volume percent values of other progressively more damaged cell types (i.e. Types II, III, and IV cells) increased with elevation of indomethacin levels. Morphometric assessments paralleled functional assessments of indomethacin-induced cytotoxicity (r2: 0.88-0.99). Therefore, the present study validated this morphometric analysis procedure using a rigorous cellular exposure which caused substantial cell injury and subsequent lifting of 23% cells from the substrate. Combined with previous validation studies involving cadmium, erythromycin and benoxaprofen, the present study showed that morphometric analysis is a rapid, accurate method for the measurement of cell injury in cultured parenchymal hepatocytes.