Staining and in Vitro Toxicity of Dithizone with Canine, Porcine, and Bovine Islets

Abstract

Dithizone (DTZ) is a recognized diabetogenic agent in vivo, and a supravital 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 discernible staining with bovine islets. Insulin content of porcine, canine, 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 content. Seven days of culture with canine islets resulted in ≥50% reduction of DTZ stained cells. Exposure to DTZ at 50 μg/mL resulted in a maximal number of stained cells in preparations of purified islets (80-85%; counted after dispersion), a lower percentage of cells stained faintly at 20 μg/mL (50-55%), with no discernible staining at 10 μg/mL. Prolonged exposure of islets (4-48 h) to 20 μg/mL DTZ led to reduced insulin secretion and islet cell death. Incubation of canine or porcine islets with 100 μg/mL of DTZ for 0.5 h resulted in a dramatic loss of viability and diminished insulin secretory 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 concentration (20 μg/mL) also resulted in a loss of viability. An additional assessment of DTZ toxicity was made using the RIN-38 β-cell line, which shows no discernible staining with DTZ. A 1 h exposure to dithizone resulted in a dose-dependent loss of viable RIN-38 cells. We conclude first, that DTZ is cytotoxic to islet cells in vitro, at concentrations used for islet staining. Although the toxicity of DTZ appears to be related to its staining properties, high concentrations have toxic effects that are unrelated to staining properties. We propose that cellular accumulation of DTZ (staining), produces toxicity by concentrating DTZ to toxic levels. Secondly, we conclude that DTZ does not stain islets of all species, despite the equivalent insulin content.