RELEASE OF ARACHIDONIC AND LINOLEIC-ACID METABOLITES IN SKIN ORGAN-CULTURES AS CHARACTERISTICS OF IN-VITRO SKIN IRRITANCY

In vitro techniques make a major contribution to the development of al ternatives to the in vivo ''Draize'' skin irritation test, and the dev elopment of sensitive and generally applicable in vitro endpoints of c utaneous toxicity is an area of intensive research. To investigate in vitro characteristics of cutaneous irritation, skin explants of rabbit and human origin were topically exposed to chemical irritants, after which the culture medium was analyzed for the presence of metabolites of both arachidonic and linoleic acid. In rabbits exposed to the poten t irritant benzalkonium chloride, a direct relation was established be tween clinical signs of irritation and in vitro release of the proinfl ammatory mediator 12-hydroxyeicosatetraenoic acid (12-HETE) by the exp osed skin. Histological examination revealed varying degrees of epider mal damage. 12-HETE was also the predominant hydroxy fatty acid releas ed in a dose-dependent way by rabbit skin cultures after in vitro expo sure to sodium dodecyl sulfate (SDS), benzalkonium chloride (BC), and formaldehyde (FA). Human skin cultures released, in addition to 12-HET E, predominantly 15-HETE and 13-hydroxyoctadecadienoic acid (13-HODE), omega-6 oxygenase products of arachidonic acid and linoleic acid, res pectively. The irritant-induced release of hydroxy fatty acids was str ongly inhibited by the lipoxygenase inhibitor eicosatetraynoic acid, i ndicating enzyme-mediated generation of these bioactive lipids. Compar ison of hydroxy fatty acid release to more established markers of cyto toxicity (leakage of the cellular enzymes, such as aspartate aminotran sferase (AST), alanine aminotransferase (ALT), and lactate dehydrogena se (LDH)) revealed that increased levels of 13-HODE, 9-HODE, 12-HETE, and ALT were specific markers of cutaneous irritancy in rabbit skin cu ltures. AST and LDH were more sensitive endpoints compared to ALT or h ydroxy fatty acids, but failed to identify the difference in irritatio n capacity between SDS and BC reported in vivo. In human skin cultures , release of 12-HETE or 15-HETE occurred at irritant concentrations th at did not result in enzyme leakage into the culture medium. In vitro effect levels of irritants were found comparable to the in vivo situat ion. We conclude that release of hydroxy fatty acids, in particular of 12-HETE, offers a mechanism-based characteristic of skin irritation w hich may be applicable to in vitro toxicity testing. (C) 1995 Society of Toxicology.