Changes in cytosolic calcium, bleb formation, and cell death in neural crest cells treated with isotretinoin and 4-oxo-isotretinoin

Information regarding the cytopathologic mechanism of action of the retinoids isotretinoin (IR) and 4-oxo-isotretinoin (4-OIR)] on neural crest cells (NCCs) in culture was sought. Those pathophysiologic alterations in cell metabolism studied were: cell blebbing (xieosis), free radical formation, cell viability, and cellular calcium homeostasis. Cells were treated with IR or 4-OIR in the presence of high (1.4 mM) and low (5.0 microM) levels of extracellular calcium ions. Recently developed techniques utilizing fluorescent molecular probes for calcium analyses, i.e., Fura 2AM, were used to study the effects of these drugs on the cytosolic calcium concentration of NCCs. The effects of IR and 4-OIR on NCC viability, Ca++]int, were contrasted with the effects of certain sulfhydryl drugs (HgCl2, NEM, PCMBS) and calcium ionophores (ionomycin, A23187), agents known to perturb cell membranes, increase cytosolic calcium loads, and induce cell injury and subsequent cell death. Both retinoids were shown to induce an increase in the generation of superoxide radicals (SO) and increase the influx of calcium ions by the NCCs, thus increasing Ca++]int by several hundred percent within 5 to 10 min. The liberation of SO was calcium dependent. These early effects were accompanied by an increase in cell blebbing activity. Also, a significant decrease in NCC viability was seen as early as 10 min after the addition of IR or 4-OIR to the incubation medium. 4-OIR proved to be the more potent of the two retinoids tested. The severity of these effects on NCC metabolism was dependent on medium calcium concentration with all changes being increased in the presence of the higher extracellular calcium levels. From the data presented it appears as though the retinoids cause a rapid elevation in cytosolic Ca++]int possibly by purturbing the integrity of the cell membrane, denaturing membrane Ca-ATPase activity, or both. Retinoid-induced changes in membrane activity are evidenced by increased surface blebbing and superoxide formation. The prolonged elevation of intracellular Ca++] may be directly related to depressed NCC viability and thus explain the known teratogenic effects of these drugs and their relationship to ectomesenchymal cell hypoplasia and craniofacial dysmorphogenesis.