van de Sandt J J, Maas W J, Doornink P C, Rutten A A
TNO Nutrition and Food Research Institute, Division of Toxicology, Zeist, The Netherlands.
Fundam Appl Toxicol. 1995 Apr;25(1):20-8. doi: 10.1006/faat.1995.1036.
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), omega-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.
体外技术对体内“德雷兹”皮肤刺激试验替代方法的发展做出了重大贡献，开发敏感且普遍适用的皮肤毒性体外终点是一个深入研究的领域。为了研究皮肤刺激的体外特征，将兔源和人源的皮肤外植体局部暴露于化学刺激物，之后分析培养基中花生四烯酸和亚油酸代谢物的存在情况。在暴露于强效刺激物苯扎氯铵的兔子中，建立了刺激的临床体征与暴露皮肤体外释放促炎介质12-羟基二十碳四烯酸（12-HETE）之间的直接关系。组织学检查显示出不同程度的表皮损伤。在体外暴露于十二烷基硫酸钠（SDS）、苯扎氯铵（BC）和甲醛（FA）后，12-HETE也是兔皮肤培养物中以剂量依赖性方式释放的主要羟基脂肪酸。除12-HETE外，人皮肤培养物主要释放15-HETE和13-羟基十八碳二烯酸（13-HODE），分别是花生四烯酸和亚油酸的ω-6加氧酶产物。脂氧合酶抑制剂二十碳四炔酸强烈抑制刺激诱导的羟基脂肪酸释放，表明这些生物活性脂质是由酶介导生成的。将羟基脂肪酸释放与更成熟的细胞毒性标志物（细胞酶如天冬氨酸转氨酶（AST）、丙氨酸转氨酶（ALT）和乳酸脱氢酶（LDH）的泄漏）进行比较，结果显示13-HODE、9-HODE、12-HETE和ALT水平升高是兔皮肤培养物中皮肤刺激性的特异性标志物。
