Differentiation of normal human keratinocytes influences hexavalent chromium uptake and distribution and the ability of cells to withstand Cr(VI) cytotoxicity.

Abstract

The degree of differentiation of normal human keratinocytes determines the biology of the cells to a large extent. We have previously documented that keratinocytes from different donors differ significantly in their ability to withstand hexavalent chromium [Cr(VI)]-induced cytotoxicity. Several factors may contribute to this differing donor sensitivity to Cr(VI). The aims of this study were to investigate to what extent keratinocyte differentiation might influence Cr(VI) uptake and the ability of cells to withstand Cr(VI)-induced cytotoxicity. Keratinocytes from different donors were cultured under identical conditions and exposed to Cr(VI) (as potassium dichromate) at different points during their maturation process. The degree of differentiation of the cells was assessed using a quantitative assay for involucrin and related to the Cr(VI) cytotoxicity experienced by the cells. Chromium content was measured in whole cell, cytosolic and particulate fractions. While proliferative keratinocytes exposed to Cr(VI) showed a high degree of cytotoxicity to dichromate exposure, the more differentiated cells showed significantly less cytotoxicity but a higher uptake of the metal ion into the cells. The relative percentage of cytosolic chromium was high in the proliferative cells and decreased as the cells matured, suggesting that differentiated cultures were binding most of the chromium to the particulate fraction. Total chromium also increased during differentiation. The use of the channel-blocking agent 4, 4'-diisothiocyanate-2-2'-stilbenedisulphonic acid confirmed the spatial differences of chromium accumulation in the phenotypically different cultures, in that it prevented Cr(VI) entry into the proliferative cells and attenuated dichromate cytotoxicity in these cultures, but had no effect on the Cr(VI) uptake in differentiated cells, nor did it reduce its cytotoxicity. These data support the hypothesis that the upper differentiated layers of the epidermis are able to offer considerable physical protection to the lower proliferative layers from chemical pro-oxidants.