Investigating Cytoskeletal Alterations as a Potential Marker of Retinal and Lens Drug-Related Toxicity

Actin filaments play a critical role in the normal physiology of lenticular and retinal cells in the eye. Disruption of the actin cytoskeleton has been associated with retinal pathology and lens cataract formation. Ocular toxicity is an infrequent observation in drug safety studies, yet its impact to the drug development process is significant. Recognizing compounds through screening with a potential ocular safety liability is one way to prioritize development candidates while reducing development attrition. Lens epithelial cells from human, dog, and rat origins and retinal pigmented epithelium cells from human, monkey, and rat origins were cultured and investigated with immunocytochemical techniques. Cells were treated using noncytotoxic doses of the compound, fixed, stained for actin with rhodamine phalloidin, and counterstained for nuclei with TOTO-3, followed by confocal imaging. Tamoxifen and several experimental compounds known to be in vivo lens and retinal toxicants caused a reduction in F-actin fluorescence at noncytotoxic concentrations in all cells tested as observed by confocal microscopy. Developing an assay that predicts ocular toxicity helps the development process by prioritizing compounds for further investigation. Drug-induced cytoskeletal alterations may be useful as a potential safety-screening marker of retinal and lens toxicity. The knowledge of actin molecular biology and the application of other mechanistic screens to toxicology are discussed. Reducing this work to a high-throughput platform will enable chemists to select compounds with a reduced risk of ocular toxicity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63132/1/adt.2006.4.695.pd

Pentamidine reduces hERG expression to prolong the QT interval

1. Pentamidine, an antiprotozoal agent, has been traditionally known to cause QT prolongation and arrhythmias; however, its ionic mechanism has not been illustrated. 2. In a stable HEK-293 cell line, we observed a concentration-dependent inhibition of the hERG current with an IC(50) of 252 μM. 3. In freshly isolated guinea-pig ventricular myocytes, pentamidine showed no effect on the L-type calcium current at concentrations up to 300 μM, with a slight prolongation of the action potential duration at this concentration. 4. Since the effective concentrations of pentamidine on the hERG channel and APD were much higher than clinically relevant exposures (∼1 μM free or lower), we speculated that this drug might not prolong the QT interval through direct inhibition of I(Kr) channel. We therefore incubated hERG-HEK cells in 1 and 10 μM pentamidine-containing media (supplemented with 10% serum) for 48 h, and examined the hERG current densities in the vehicle control and pentamidine-treated cells. 5. In all, 36 and 85% reductions of the current densities were caused by 1- and 10-μM pentamidine treatment (P<0.001 vs control), respectively. A similar level of reduction of the hERG polypeptides and a reduced intensity of the hERG protein on the surface membrane in treated cells were observed by Western blot analysis and laser-scanning confocal microscopy, respectively. 6. Taken together, our data imply that chronic administration of pentamidine at clinically relevant exposure reduces the membrane expression of the hERG channel, which may most likely be the major mechanism of QT prolongation and torsade de pointes reported in man