Morphometric evaluation of dark and clear epidermal basal cells during early 2-stage chemical skin carcinogenesis in the hairless mouse using two different fixation methods.

Ultrastructural morphometric characteristics of basal keratinocytes in hairless mouse epidermis were analyzed statistically. The following variables were assessed: (i) low versus physiological osmolality during fixation, (ii) alterations induced by a 2-stage carcinogenesis regimen using DMBA and TPA, (iii) criteria for a cell being dark versus being clear, (iv) inter-observer variation. The results show that with low fixation osmolality most basal cells swell and become electron lucent. The few cells which apparently do not swell stand out as shrunken electron dense dark cells. Morphometrically they are more differentiated than clear cells, but do share many features with the basal cell type which appears after fixation in a buffer of physiological osmolality. Iso-osmolality during fixation seems to induce a homogeneous basal cell population of relatively electron dense cells without typical dark and clear elements. Treatment with DMBA/TPA induces not only intercellular edema and reduced desmosomal contacts, but causes injury to the plasma membrane leading to hydropic changes in the cells. This general intra- and intercellular DMBA/TPA induced hydration might induce secondary compression of some of the cells, leading to an increased number of compressed dark cells. It is, however, only after fixation in low buffer osmolality that these effects of DMBA/TPA are statistically significant and clearly observable. The inter-person variation was, apart from a few instances, either not statistically significant or did not interfere with the other effects. We did not find clear arguments in favor of the view that dark cells are primitive epidermal stem cells. They seem only to reflect non-specific toxic effects of tumor promoters, which appear only under certain fixation conditions, that have been used by most authors. The results suggest that dark and clear cells are mainly a consequence of the degree of cellular hydration.