In vitro assessment of human nuclear hormone receptor activity and cytotoxicity of the flame retardant mixture FM 550 and its triarylphosphate and brominated components

Firemaster^® 550 (FM 550) is a mixture of brominated and triarylphosphate flame retardants used in polyurethane foam-based products. The primary components are also used in numerous other applications and are thus common household and industrial contaminants. Our previous animal studies suggested that FM 550 exposure may alter metabolism and cause weight gain. Employing human nuclear receptor (NR) luciferase reporter assays, the goal of this study was to evaluate the agonist actions of FM 550 and its constituent compounds at NRs with known roles in establishing or regulating energy balance. FM 550 was found to have significant agonist activity only at the master regulator of adipocyte differentiation PPARγ. As a result, the concentration response relationships and relative activities of FM 550 at PPARγ were investigated in more detail with the contribution of each chemical component defined and compared to the activities of the prototypical PPARγ environmental ligands triphenyltin and tributyltin. The resulting data indicated that the primary metabolic disruptive effects of FM 550 were likely mediated by the activity of the triarylphosphates at PPARγ, and have identified TPP as a candidate metabolic disruptor that also acts as a cytotoxicant.     

Variants in TRIM44 Cause Aniridia by Impairing PAX6 Expression

Congenital aniridia is a genetic disorder that manifests as iris hypoplasia and other associated ocular complications. Mutations in the paired box 6 (PAX6) gene are considered the major cause of aniridia. In this study, we identified four mutations exclusively presented in aniridia patients from a four‐generation Chinese pedigree, including two single nucleotide substitutions in the 3′UTR of PAX6 (NM_000280.4:c.[*76G>A; *2977C>A]) and two missense mutations in tripartite motif containing 44 (TRIM44, NM_017583.4:c.[191C>A; 463G>A]), which lead to amino acid changes p.S64Y and p.G155R, respectively. Bioinformatic analyses revealed that the two 3′UTR mutations of PAX6 disrupted microRNA binding motifs in the wildtype 3′UTR sequence. Luciferase reporter assay and Western blotting with predicted microRNAs showed that the two 3′UTR mutations could only increase or have no effect on the expression of PAX6. Therefore, they would not be the cause of aniridia that resulted from PAX6 deficiency. Instead, we found that overexpression of TRIM44 significantly reduced the expression of PAX6 in human lens epithelial cells, and the p.G155R mutant exhibited much stronger effect than the wildtype form. We conclude that inhibition of PAX6 expression by mutant TRIM44 is a novel pathogenic mechanism for aniridia.     

TNF-α 3'-UTR双荧光素酶报告基因系统的构建及丹参酮类药物筛选

目的构建并鉴定p GL3-TNF-α3'端非翻译区(UTR)双荧光素酶报告基因(DLR)表达系统,以此基于调控TNF-α转录后水平对丹参酮类成分进行筛选。方法反转录人脐静脉内皮细胞HUVEC的mRNA成c DNA,以之为模板,PCR扩增含TNF-α3'-UTR的DNA片段全长,经酶切后连接至荧光素酶报告载体p GL3-control上,构建出p GL3-TNF-α3'UTR全长的荧光素酶报告基因载体并进行鉴定。将所构建的p GL3-TNF-α3'UTR与p SVRenilla质粒组成双荧光素酶报告系统共转染至单核巨噬细胞RAW264.7,经脂多糖诱导后利用该双荧光素酶报告基因表达系统判定丹参酮类成分对TNF-α转录后调控是否有影响。结果成功构建p GL3-TNF-α3'UTR荧光素酶报告基因,克隆获得的DNA片段大小及序列与Genbank报道的一致。脂多糖(LPS)可以明显诱导转入p GL3-TNF-α3'UTR载体细胞的荧光强度。丹参酮类成分中隐丹参酮可以明显降低LPS诱导的p GL3-TNF-α3'UTR载体细胞的荧光素酶活性。结论成功构建含TNF-α3'UTR区的双荧光素酶报告基因表达系统,并以此从丹参酮类化合物中筛选出隐丹参酮,可以对TNF-α的转录后水平进行抑制调控。     

Sprouty1载体的构建及对NF-κB转录活性的调控影响

目的构建pCMV-MYC-SPRY1表达载体,应用荧光素酶报告基因实验研究pCMV-MYC-SPRY1对NF-κB信号转导通路的影响,以及对HEK293细胞增殖调控的研究。方法采用PCR技术在肝文库里扩增SPRY1编码的目的基因片段,将其构建到真核表达pCMV-MYC载体上,用荧光素酶报告基因实验验证其对下游报告基因NF-κB转录活性的影响以及用GENMEDMTS细胞增殖检测试剂盒检测pCMV-MYC-SPRY1对HEK293细胞增殖的影响。结果成功构建pCMV-MYC-SPRY1表达载体,表明SPRY1负调控NF-κB的活性,对HEK293细胞增殖具有抑制作用。结论 pCMV-MYC-SPRY1表达载体的成功构建及对NF-κB转导通路的研究,为进一步研究SPRY1基因编码的蛋白质的功能打下了基础。     

人支气管上皮细胞中苯并芘诱导TNF-α表达的分子机制

目的：构建肿瘤坏死因子-α（TNF-α）启动子双荧光素酶报告基因载体,研究苯并芘（B[a]P）暴露对TNF-αm RNA表达的调控机制。方法：采用Real time-PCR技术检测B[a]P暴露下,人支气管上皮细胞（Beas-2B）中TNF-αm RNA随时间的变化趋势。通过分子克隆技术构建TNF-α启动子双荧光素酶报告基因载体并检测其活性。进一步检测Beas-2B细胞和人肾上皮细胞293T细胞暴露于B[a]P环境下,TNF-α启动子活性的变化趋势。结果：Real time-PCR检测B[a]P暴露下,24 h内,Beas-2B细胞中TNF-αm RNA表达量随时间延长升高。且B[a]P刺激Beas-2B细胞24 h内,TNF-α启动子活性也呈升高趋势。B[a]P刺激293T细胞,TNF-α启动子活性也会升高。结论：成功构建了TNF-α启动子双荧光素酶报告基因载体。而且,B[a]P能够促进TNF-αm RNA的转录从而促进TNF-αm RNA的表达,且promoter1要比promoter2活性强,没有细胞特异性。     

CYP3A4＊1G基因多态性及功能的初步探讨

采用双荧光素酶报告基因系统分析CYP3A4＊ 1G多态性对CYP3A4基因转录活性的影响.构建含CYP3A4＊ 1G突变位点的荧光素酶基因表达载体,用Lipofectamine 2000转染HepG2细胞,化学发光法检测荧光素酶活性. 应用双荧光素酶报告基因系统检测显示,pGL3-promoter-A质粒转染后荧光素酶活性显著高于pGL3-promoter-G（P=0.022＜ 0.05）. CYP3A4＊ 1G能够增强荧光素酶基因的表达,可能增强CYP3 A4基因的转录活性.     

The intra- and inter-laboratory reproducibility and predictivity of the KeratinoSens assay to predict skin sensitizers in vitro: Results of a ring-study in five laboratories

Due to regulatory constraints and ethical considerations, research on alternatives to animal testing to predict the skin sensitization potential of novel chemicals has gained a high priority. Accordingly, different in vitro, in silico and in chemico approaches have been described in the scientific literature to achieve this goal. To replace regulatory approved animal tests, these alternatives need to be transferable to other labs, their within and between laboratory reproducibility must be assured, and their predictivity should be high. The KeratinoSens assay is a cell-based reporter gene assay to screen substances with a full dose-response assessment. It is based on a stable transgenic keratinocyte cell line. The induction of a luciferase gene under the control of the antioxidant response element (ARE) derived from the human AKR1C2 gene is determined. Here we report on the results of a ring-study with five laboratories performing the KeratinoSens assay on a set of 28 test substances. The assay was found to be easily transferable to all laboratories. Overall both the qualitative (sensitizer/non-sensitizer categorization) and the quantitative (concentration for significant gene induction) results were reproducible between laboratories. A detailed analysis of the transferability, the within- and between laboratory reproducibility and the predictivity is presented.