Fluorometric Assay for Intracellular pH in Human Epidermal Keratinocytes

摘要

Sulfur mustard (HD) is a powerful alkylating agent with cytotoxic, mutagenic, and vesicant properties. Pathology in humans results from ocular, respiratory and cutaneous exposure. Basal cells of the epidermis of the skin are damaged by exposure to HD, causing large, slow healing blisters. To protect individuals from the effects of HD, the mechanism by which it induces pathology must be determined. In this report, a fluorometric assay of intracellular pH was developed and used to examine the role of intracellular pH in cutaneous cellular toxicity. HD undergoes a hydrolysis reaction in aqueous environments, generating thiodiglycol, protons, and chloride ions. One hypothesis of HD injury postulates the formation of hydrochloric acid (HCl), which could acidify surrounding cell and tissue milieu. All cellular metabolic pathways are dependent on carefully buffered systems that maintain the intracellular pH within strict boundaries. Any disruption in pH regulation has the potential to result in cell or tissue injury. The effect of HD on intracellular pH had not been previously examined. The value of fluorescent assays is in their sensitivity and specificity. Using fluorescent probes capable of crossing the cell membrane and becoming trapped within the cytoplasm, we were able to directly determine proton concentrations within the cytoplasm and could determine intracellular pH levels before and after exposure to HD in vitro. While small changes in pH were noted, they were not determined to be of biological significance. We suggest, therefore, that vesicating agents do not produce their toxicity through alteration of intracellular pH.