Improper use of mitomycin-C in ocular medication may result in damage to corneal cells. In this study, the toxic effects of mitomycin-C on cultured porcine keratocytes and endothelial cells were estimated by MTT, 3H-thymidine uptake and cellular counting assay methods. It was found that mitomycin-C caused a dose-dependent toxic effect to keratocytes and endothelial cells. Both cells were treated with mitomycin-C at the concentration ranging from 100, 10, 1, 0.1 to 0.01 microg/ml for 3 min, 5 min or 100 min. The 50% inhibitory dose (ID50) of mitomycin-C to keratocytes and endothelial cells as measured by MTT assay was 0.40, 0.18, 0.16 mg/ml and 0.27, 0.15, 0.14 mg/ml, respectively, after 3, 5 and 100 minutes drug treatment. The ID50 for keratocytes and endothelial cells as measured by 3H-thymidine uptake immediately, 1 day and 7 days after 100 minutes mitomycin-C treatment was 0.3, 0.0002, 143.2 microg/ml and 45.1, 101.1, 450.2 microg/ml, respectively. The ID50 for keratocytes and endothelial cells as measured by cellular counting 1 day and 7 days after mitomycin-C treatment was 232.5, 109.7 microg/ml and 239.9, 367.5 microg/ml, respectively. It is concluded that mitomycin-C is more toxic to cellular proliferation in cultured corneal keratocytes than in endothelial cells.

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The effect of substances extracted from Toona sinensis leaves with 50% alcohol solution on lipolysis was investigated in cultured 3T3-L1 differentiated adipocytes. The amount of glycerol released from cells into culture medium was used to measure lipolysis activity. Glycerol release was increased by Toona sinensis leaf extract in a dose-dependent and time-dependent manner. Following treatment of 3T3-L1 adipocyte cells with various concentrations of Toona sinensis leaf extract (0.001, 0.01, and 0.1 mg/mL) for 6 hours, the amounts of glycerol released from 3T3-L1 cells increased from a control value of 99 nmol/mg protein to 127, 144, and 154 nmol/mg protein, respectively. The lipolytic effect of Toona sinensis leaf extract was not inhibited by pretreatment of cells with cycloheximide, econazole, baicalein, or indomethacin. However, the lipolytic activity induced by Toona sinensis leaf extract was diminished by dibutyryl cyclic adenosine-5'-monophosphate (dibutyryl cAMP) and the protein kinase C inhibitor calphostin C. These results indicate that the lipolytic effect induced by Toona sinensis leaf substances may be involved in the protein kinase C pathway and may be down-regulated by cAMP.

The effects of substances extracted from Toona sinensis leaves, using 50% alcohol/water, on cellular [3H]-2-deoxyglucose uptake in differentiated cultured 3T3-L1 adipocytes were investigated. Following treatment of cells with 0.001, 0.01, or 0.1 mg/mL extracts for 60 minutes, [3H]-2-deoxyglucose uptake increased from a basal value of 0.23 nmol/min/mg protein to 0.30, 0.33, and 0.38 nmol/min/mg protein, respectively. In insulin-stimulated cells, cellular [3H]-2-deoxyglucose uptake was enhanced by Toona sinensis leaf extract from a basal value of 0.35 nmol/min/mg protein to 0.41, 0.46, and 0.52 nmol/min/mg protein, respectively. Cellular glucose uptake was also enhanced by Toona sinensis leaf extract after incubation of cells with 20 mM glucose for 48 hours. Cellular glucose uptake with a combination of Toona sinensis leaf extract and insulin was significantly inhibited by pretreatment of cells with the protein synthesis inhibitor cycloheximide and the protein kinase C inhibitor calphostin C in normal-, medium- and high-glucose media. However, the glucose uptake-enhancing effect of Toona sinensis leaf extract was not diminished by cycloheximide and calphostin C in the absence of insulin. These results indicate that enhancement of cellular glucose uptake by Toona sinensis leaf extract in basal and insulin-stimulated 3T3-L1 adipocytes may be mediated by distinct mechanisms.