Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Technická 5, 166 28, Prague 6, Czech Republic.
Department of Biology of Reproduction, Institute of Animal Science, Prague 10-Uhrineves, Czech Republic.
Sci Rep. 2023 Oct 28;13(1):18536. doi: 10.1038/s41598-023-45598-y.
Lilial (also called lysmeral) is a fragrance ingredient presented in many everyday cosmetics and household products. The concentrations of lilial in the final products is rather low. Its maximum concentration in cosmetics was limited and recently, its use in cosmetics products was prohibited in the EU due to the classification as reproductive toxicant. Additionally, according to the European Chemicals Agency, it was under assessment as one of the potential endocrine disruptors, i.e. a substance that may alter the function of the endocrine system and, as a result, cause health problems. Its ability to act as an androgen receptor agonist and the estrogenic and androgenic activity of its metabolites, to the best of our knowledge, have not yet been tested. The aim of this work was to determine the intestinal absorption, cytotoxicity, nephrotoxicity, mutagenicity, activation of cellular stress-related signal pathways and, most importantly, to test the ability to disrupt the endocrine system of lilial and its Phase I metabolites. This was tested using set of in vitro assays including resazurin assay, the CHO/HPRT mutation assay, γH2AX biomarker-based genotoxicity assay, qPCR and in vitro reporter assays based on luminescence of luciferase for estrogen, androgen, NF-κB and NRF2 signalling pathway. It was determined that neither lilial nor its metabolites have a negative effect on cell viability in the concentration range from 1 nM to 100 µM. Using human cell lines HeLa9903 and MDA-kb2, it was verified that this substance did not have agonistic activity towards estrogen or androgen receptor, respectively. Lilial metabolites, generated by incubation with the rat liver S9 fraction, did not show the ability to bind to estrogen or androgen receptors. Neither lilial nor its metabolites showed a nephrotoxic effect on human renal tubular cells (RPTEC/TERT1 line) and at the same time they were unable to activate the NF-κB and NRF2 signalling pathway at a concentration of 50 µM (HEK 293/pGL4.32 or pGL4.37). Neither lilial nor its metabolites showed mutagenic activity in the HPRT gene mutation test in CHO-K1 cells, nor were they able to cause double-strand breaks in DNA (γH2AX biomarker) in CHO-K1 and HeLa cells. In our study, no negative effects of lilial or its in vitro metabolites were observed up to 100 µM using different in vitro tests.
利拉雅黑（也称为赖玛醇）是一种存在于许多日常化妆品和家用产品中的香料成分。最终产品中利拉雅黑的浓度相当低。其在化妆品中的最大浓度受到限制，最近，由于被归类为生殖毒性物质，欧盟已禁止其在化妆品产品中的使用。此外，根据欧洲化学品管理局的说法，它被评估为一种潜在的内分泌干扰物，即可能改变内分泌系统功能并因此导致健康问题的物质。据我们所知，其作为雄激素受体激动剂的能力及其代谢物的雌激素和雄激素活性尚未经过测试。本工作的目的是确定利拉雅黑及其 I 相代谢物的肠道吸收、细胞毒性、肾毒性、致突变性、细胞应激相关信号通路的激活，最重要的是，测试其干扰内分泌系统的能力。这是通过一系列体外测定来测试的，包括resazurin 测定法、CHO/HPRT 突变测定法、基于 γH2AX 生物标志物的遗传毒性测定法、qPCR 和基于荧光素酶的雌激素、雄激素、NF-κB 和 NRF2 信号通路的体外报告测定法。结果表明，在 1 nM 至 100 µM 的浓度范围内，利拉雅黑及其代谢物对细胞活力没有负面影响。使用人宫颈癌细胞系 HeLa9903 和 MDA-kb2，验证了该物质对雌激素或雄激素受体分别没有激动活性。用大鼠肝 S9 级分孵育生成的利拉雅黑代谢物，没有显示出与雌激素或雄激素受体结合的能力。利拉雅黑及其代谢物对人肾小管细胞（RPTEC/TERT1 系）没有肾毒性作用，同时在 50 µM 浓度下（HEK 293/pGL4.32 或 pGL4.37）不能激活 NF-κB 和 NRF2 信号通路。利拉雅黑及其代谢物在 CHO-K1 细胞的 HPRT 基因突变试验中没有表现出致突变活性，也不能在 CHO-K1 和 HeLa 细胞中引起 DNA 双链断裂（γH2AX 生物标志物）。在我们的研究中，使用不同的体外试验，在高达 100 µM 的浓度下，没有观察到利拉雅黑或其体外代谢物的负面作用。
