Development of a method for assessing micronucleus induction in a 3D human skin model (EpiDerm™)
Development of a method for assessing micronucleus induction in a 3D human skin model (EpiDerm™)
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DOI：
10.1016/j.mrgentox.2006.04.016
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发表时间：
2006-09-05
影响因子：
1.9
通讯作者：
Aardema, Marilyn J.
中科院分区：
文献类型：
--
作者：
Curren, Rodger D.;Mun, Greg C.;Aardema, Marilyn J.
为了满足欧盟化妆品指令第7修正案的要求，化妆品制造商需要使用非动物方法确定成分的安全性。从2009年开始，将不允许对化妆品成分进行体内遗传毒性试验。由于皮肤暴露量相对较高，因此皮肤是许多化妆品关注的目标区域。因此，进行非动物、基于皮肤的遗传毒性试验是有益的，尤其是在体外使用人类皮肤的试验。在本文中，我们描述了使用EpiDerm（TM）工程化人皮肤构建体（MatTek Corp.，阿什兰，马萨诸塞州）。我们描述了从3D皮肤模型中分离单细胞和处理细胞用于微核（MN）显微镜分析的方法。此外，由于对EpiDerm TM模型中角质形成细胞分裂的动力学知之甚少，我们评估了细胞松弛素B（Cyt-B）是否可用于区分分裂细胞群，从而在双核细胞中开展微核试验。我们发现双核细胞的频率随着时间和Cyt-B浓度的增加而增加。暴露48小时后，可重复获得30-50%的双核细胞。最后，我们使用模型遗传毒物丝裂霉素C（MMC）和硫酸长春碱（VB）评价微核诱导。在该模型中，MN的背景频率非常低且具有重现性，MMC和VB均诱导微核细胞频率出现统计学显著性增加。这些是开发用于人类组织中染色体损伤的常规“体内样”测定的初始步骤。希望其他研究人员利用这些方法来进一步了解这种潜在的有价值的新的非动物方法。(c)2006 Elsevier B. V.保留所有权利。
To meet the requirements of the EU 7th Amendment to the Cosmetics Directive, manufacturers of cosmetics products will need to ascertain the safety of ingredients using non-animal methods. Starting in 2009, in vivo genotoxicity tests for cosmetics ingredients will not be allowed. Skin is a target area of interest for many cosmetic products because of its relatively high exposure. Therefore, it would be beneficial to have a non-animal, skin-based genotoxicity assay, especially one that utilized human skin in vitro. In this paper, we describe the development of a reproducible micronucleus assay that uses EpiDerm (TM) engineered human skin constructs (MatTek Corp., Ashland, MA). We describe methods for isolating single cells from the 3D skin model and for processing the cells for microscopic analysis of micronuclei (MN). In addition, since little was known about the kinetics of the dividing keratinocytes in the EpiDerm TM model, we evaluated whether cytochalasin B (Cyt-B) could be used to distinguish the population of dividing cells allowing the development of a micronucleus assay in binucleated cells. We found that the frequency of binucleated cells increased both with time and with increasing concentration of Cyt-B. After a 48-h exposure, 30-50% binucleated cells were reproducibly obtained. Finally, we evaluated micronucleus induction using the model genotoxicants mitomycin C (MMC) and vinblastine sulfate (VB). The background frequency of MN is very low and reproducible in this model, and statistically significant increases in the frequency of micronucleated cells were induced by both MMC and VB. These are initial steps in developing a routine "in vivo-like" assay for chromosomal damage in human tissue. It is hoped that other investigators utilize these methods to further the understanding of this potentially valuable new non-animal method. (c) 2006 Elsevier B.V. All rights reserved.