Title
Release of arachidonic and linoleic acid metabolites in skin organ cultures as characteristics of in vitro skin irritancy
Author
van de Sandt, J.J.M.
Maas, W.J.M.
Doornink, P.C.
Rutten, A.A.J.J.L.
Instituut CIVO-Toxicologie en Voeding TNO
Publication year
1995
Abstract
In vitro techniques make a major contribution to the development of alternatives to the in vivo 'Draize' skin irritation test, and the development of sensitive and generally applicable in vitro endpoints of cutaneous 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 potent irritant benzalkonium chloride, a direct relation was established between clinical signs of irritation and in vitro release of the proinflammatory mediator 12-hydroxyeicosatetraenoic acid (12-HETE) by the exposed skin. Histological examination revealed varying degrees of epidermal damage. 12-HETE was also the predominant hydroxy fatty acid released in a dose-dependent way by rabbit skin cultures after in vitro exposure to sodium dodecyl sulfate (SDS), benzalkonium chloride (BC), and formaldehyde (FA). Human skin cultures released, in addition to 12-HETE, predominantly 15-HETE and 13-hydroxyoctadecadienoic acid (13-HODE), ω-6 oxygenase products of arachidonic acid and linoleic acid, respectively. The irritant-induced release of hydroxy fatty acids was strongly inhibited by the lipoxygenase inhibitor eicosatetraynoic acid, indicating enzyme-mediated generation of these bioactive lipids. Comparison of hydroxy fatty acid release to more established markers of cytotoxicity (leakage of the cellular enzymes, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH)) revealed that increased levels of 13-HODE, 9-HODE, 12-HETE, and ALT were specific markers of cutaneous irritancy in rabbit skin cultures. AST and LDH were more sensitive endpoints compared to ALT or hydroxy fatty acids, but failed to identify the difference in irritation capacity between SDS and BC reported in vivo. In human skin cultures, release of 12-HETE or 15-HETE occurred at irritant concentrations that did not result in enzyme leakage into the culture medium. In vitro effect levels of irritants were found comparable to the in vivo situation. We conclude that release of hydroxy fatty acids, in particular of 12-HETE, offers a mechanism-based characteristic of skin irritation which may be applicable to in vitro toxicity testing.
Subject
Biology
Animal
Animal Testing Alternatives
Arachidonic Acid
Benzalkonium Compounds
Eicosanoids
Epidermis
Female
Formaldehyde
Human
Hydroxyeicosatetraenoic Acids
In Vitro
Irritants
Linoleic Acid
Linoleic Acids
Male
Organ Culture
Rabbits
Skin
Sodium Dodecyl Sulfate
Support, Non-U.S. Gov't
Toxicity Tests
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DOI
https://doi.org/10.1093/toxsci/25.1.20
TNO identifier
67185
ISSN
1096-6080
Source
Fundamental and Applied Toxicology, 25 (1), 20-28
Document type
article