STAINING AND IN-VITRO TOXICITY OF DITHIZONE WITH CANINE, PORCINE, ANDBOVINE ISLETS

Dithizone (DTZ) is a recognized diabetogenic agent in vivo, and a supr avital stain commonly used for identification of islets to be used for transplantation. In the present studies, we compared DTZ staining of freshly isolated and cultured canine, bovine, and porcine islets, and the effect of DTZ on the function and viability of islets. Incubation with DTZ resulted in staining of canine and porcine islets, but no dis cernible staining with bovine islets. Insulin content of porcine, cani ne, and bovine islet was 2.0 +/- 0.2, 2.2 +/- 0.3, and 1.9 +/- 0.2 mU/ EIN, indicating a lack of correspondence of DTZ staining and insulin c ontent. Seven days of culture with canine islets resulted in greater t han or equal to 50% reduction of DTZ stained cells. Exposure to DTZ at 50 mu g/mL resulted in a maximal number of stained cells in preparati ons of purified islets (80-85%; counted after dispersion), a lower per centage of cells stained faintly at 20 mu g/mL (50-55%), with no disce rnible staining at 10 mu g/mL. Prolonged exposure of islets (4-48 h) t o 20 mu g/mL DTZ led to reduced insulin secretion and islet cell death . Incubation of canine or porcine islets with 100 mu g/mL of DTZ for 0 .5 h resulted in a dramatic loss of viability and diminished insulin s ecretory function, which was not reversed with continued culture. The concentration dependence of toxic effects paralleled the concentration dependence of cellular staining. The minimally effective staining con centration (20 mu g/mL) also resulted in a loss of viability. An addit ional assessment of DTZ toxicity was made using the RIN-38 beta-cell l ine, which shows no discernible staining with DTZ. Alh exposure to dit hizone resulted in a dose-dependent loss of viable RIN-38 cells. We co nclude first, that DTZ is cytotoxic to islet cells in vitro, at concen trations used for islet staining. Although the toxicity of DTZ appears to be related to its staining properties, high concentrations have to xic effects that are unrelated to staining properties. We propose that cellular accumulation of DTZ (staining), produces toxicity by concent rating DTZ to toxic levels. Secondly, we conclude that DTZ does not st ain islets of all species, despite the equivalent insulin content.