The effect of dichlorotriazinyl aminofluorescein on human keratocytes in vitro

Dichlorotriazinyl aminofluorescein (DTAF) has been used to stain corneal stromal collagen as part of in vivo animal experiments for many years. Toxicity of this drug, if present, might alter the observed wound healing. To determine if this drug has any deleterious effect on keratocytes, we evaluated it in vitro. Human keratocytes prepared in 24-well plates were exposed to varying concentrations of DTAF (10(-4), 10(-3), 10(-2), 1, 10, 10(2) microgram/ml). Exposure times of 1 hour and 24 hours at each concentration of DTAF were evaluated. The cell number was measured 1 and 3 days after initiation of exposure to DTAF using a Coulter counter. Keratocyte proliferation was not affected by 1-hour exposure to DTAF, but keratocyte proliferation measured 3 days after initiation of exposure to DTAF for 24 hours was inhibited in a dose-dependent manner (p = 0.02) and was significantly inhibited at concentrations of 10 and 100 microgram/ml (p < 0.05). Fluorescent microscopy showed binding of DTAF to keratocytes. We have demonstrated that prolonged exposure to DTAF inhibits proliferation of cultured keratocytes. These results suggest that DTAF-induced cytotoxicity may alter net production of collagen in the corneal stroma in animal models.

Some photoresponses of isolated tissue preparations to ultraviolet light in the presence of photosensitizers.

Responses of isolated tissue preparations to ultraviolet (UV) light were studied with and without the presence of photosensitizers like eosin, fluorescein and sodium nitrite. Exposure to UV light in the presence of sodium nitrite induced consistent relaxation of rat duodenum. The photorelaxation was found to be related to the concentration of sodium nitrite. Adrenergic or cholinergic mechanisms do not seem to be involved. The isolated rat duodenum preparation exhibited quantitatively consistent photoresponse for 3 to 4 hr at its normal tone obviating the need for additional spasmogens as needed with other preparations. The preparation is a suitable test model for the study of photobiologic response evoked by UV light.