Measurement of eicosanoid release in keratinocyte cultures to investigate skin irritation and tumour promoting activity

Abstract

Skin irritation, inflammation and hyperplasia appear to be intimately associated with the phenomenon of tumour promotion, but the mechanism of action remains elusive. Prostaglandins and leukotrienes play an important role in skin inflammation and prostaglandin E 2 (PGE 2) modulates several events associated with phorbol ester-induced tumour promotion. This study investigated the release of eicosanoids (PGE 2 and leukotriene B 4) and markers of cytotoxicity [neutral red (NR) uptake and intracellular acid phosphatase (AP) activity], after exposure of rat tongue epithelial (RTE) keratinocyte cultures to chemicals of different irritating and tumour promoting activity. The potent phorbol ester tumour promoter phorbol-12-myristate-13-acetate (PMA), and the less potent, structurally related diterpene ester, mezerein (MEZ) were compared with the known skin irritant sodium dodecyl sulfate (SDS). Cytotoxicity data reflected the in vivo skin irritation potential of the test chemicals and intracellular AP activity was increased after exposure to SDS (160 μg/ml), but did not appear to be increased by the more cytotoxic chemicals PMA and MEZ. Extracellular levels of PGE 2 were increased (200 to > 1000% of control levels) after an 18-hr exposure to PMA or MEZ over a concentration range of 0.01 to 20 μg/ml (NR 50 values 8.0 ± 6.6 and 15.5 ± 4.8 μg/ml, respectively). These data indicated that PGE 2 release occurred in the absence of cytotoxicity. In contrast, SDS only elicited PGE 2 release after exposure to 80 μg/ml (a cytotoxic dose level, NR 50 82.5 ± 9.9 μg/ml). The potency of a chemical to elicit PGE 2 release in keratinocytes in the absence of a cytotoxic response may reflect intracellular pathways intimately associated with the initiation of an inflammatory response and possibly with tumour promoting activity.