This proposal is designed to test the hypothesis that 60 Hz electromagnetic fields (EMF) alter differentiated cellular function of human skin-derived keratinocytes. Field strengths commonly found in the environment will be applied to both human fibroblasts and keratinocytes in tissue culture so that they can determine if measurable changes in differentiation-specific protein expression occur. These have already been documented in skin-derived fibroblasts exposed to 20 Hz EMFs, and their first objective will be to replicate this result and determine if similar changes in protein expression are generated by 60 Hz EMFs as well. Using identical exposure conditions, they will next test the hypothesis that differentiation-specific function is, likewise, enhanced in the EMF-exposed human keratinocyte. The expression of both early and late differentiation markers (keratin 1 and involucrin) in exposed cells will be quantitated in keratinocytes. The second phase of their work will examine the mechanisms by which the observed changes in gene expression, differentiation, growth, and migration occur. Specifically, the PKC and [Ca(2+)](i) arms of the intracellular signalling system will be probed, since these signals have already been linked to control of the cellular functions under investigation. Changes in PKC isoform expression and subcellular localization in EMF- exposed cells will be examined and the hypothesis that EMF-induced changes in PKC activity alter differentiated function will be tested by the inclusion of PKC inhibitors in EMF-exposed cultures. Changes in [Ca(2+)](i), if observed in EMF-exposed cells, imply either an alteration in the intracellular [Ca(2+)](i) control mechanism, or changes in ion flux across the cell membrane, or possibly both. These possibilities will be investigated by patch clamp analysis of ion fluxes and fura-2 fluorescence ratio imaging of [Ca(2+)](i) in EMF-exposed cells, and quantitation of Ins(1,4,5)P(3), the messenger which releases Ca(2+) from intracellular stores. Understanding the mechanisms controlling any observed phenomenon, such as alteration in [Ca(2+)](i) is essential in defining the role of EMF on cellular function. The possibility that changes in cell phenotype in EMF-exposed cells is mediated through the stress response will also be investigated. They will focus on one stress protein initially, HSP27, because their preliminary work indicates that it is translocated to the nucleus after 60 HZ field exposure. The final phase of these studies will evaluate the significance of the cellular changes induced in cells exposed to ac fields in vitro to the intact organism. Composite skin grafts will be constructed of human keratinocytes and fibroblasts exposed to EMF of field strengths which alter cell behavior in vitro. These skin composites will be grafted onto recipient nude, athymic mice, and their growth, differentiation, and ability to repair cutaneous would will be monitored over time. This approach offers a considerable advantage in assessing the ultimate risk of very low EMF exposures in human skin, without the need to employ human subjects directly.
