CS Mott Center for Human Growth and Development, Reproductive Endocrinology and Infertility, Department of Ob/Gyn, Wayne State University School of Medicine, Detroit, Michigan, USA.
Reproductive Stress 3M, Inc., Grosse Pointe Farms, Michigan, USA.
Stem Cells Dev. 2022 Jun;31(11-12):296-310. doi: 10.1089/scd.2021.0330.
Lowest observable adverse effects level (LOAEL) is a standard point-of-departure dose in toxicology. However, first observable adverse effects level (FOAEL) was recently reported and is used, in this study, as one criterion to detect a mutagenic stimulus in a live imager. Fluorescence ubiquitinated cell cycle indicator (FUCCI) embryonic stem cells (ESC) are green in the S-G2-M phase of the cell cycle and not green in G1-phase. Standard media change here is a mild stress that delays G1-phase and media change increases green 2.5- to 5-fold. Since stress is mild, media change rapidly increases green cell number, but higher stresses of environmental toxicants and positive control hyperosmotic stress suppress increased green after media change. Perfluoro-octanoic acid (PFOA) and diethyl phthalate (DEP) previously suppressed progression of nongreen to green cell cycle progression. Here, bisphenol A (BPA), cortisol, and positive control hyperosmotic sorbitol also suppress green fluorescence, but benzo(a)pyrene (BaP) at high doses (10 μM) increases green fluorescence throughout the 74-h exposure. Since any stress can affect many cell cycle phases, messenger RNA (mRNA) markers are best interpreted in ratios as dose-dependent mutagens increase in G2/G1 and nonmutagens increase G1/G2. After 74-h exposure, RNAseq detects G1 and G2 markers and increasing BaP doses increase G2/G1 ratios but increasing hyperosmotic sorbitol and PFOA doses increase G1/G2 marker ratios. BaP causes rapid green increase in FOAEL at 2 h of stimulus, whereas retinoic acid caused significant green fluorescence increases only late in culture. Using a live imager to establish FOAEL and G2 delay with FUCCI ESC is a new method to allow commercial and basic developmental biologists to detect drugs and environmental stimuli that are mutagenic. Furthermore, it can be used to test compounds that prevent mutations. In longitudinal studies, uniquely provided by this viable reporter and live imager protocol, follow-up can be done to test whether the preventative compound itself causes harm.
最低可见不良反应水平 (LOAEL) 是毒理学中的标准起始剂量。然而，最近报道了最早可见不良反应水平 (FOAEL)，并在本研究中用作在活体成像仪中检测致突变刺激的一个标准。荧光泛素细胞周期指示剂 (FUCCI) 胚胎干细胞 (ESC) 在细胞周期的 S-G2-M 期呈绿色，而在 G1 期则不呈绿色。这里的标准培养基换液是一种轻度应激，会延迟 G1 期，而培养基换液会使绿色增加 2.5 到 5 倍。由于应激是轻度的，培养基换液会迅速增加绿色细胞数量，但环境毒物和阳性对照高渗应激的更高应激会抑制培养基换液后绿色的增加。全氟辛酸 (PFOA) 和邻苯二甲酸二乙酯 (DEP) 先前抑制了非绿色向绿色细胞周期进展。在此，双酚 A (BPA)、皮质醇和阳性对照高渗山梨醇也抑制绿色荧光，但高剂量 (10 μM) 的苯并 (a) 芘 (BaP) 在整个 74 小时暴露过程中增加绿色荧光。由于任何应激都可能影响许多细胞周期阶段，因此最好将信使 RNA (mRNA) 标志物解释为比值，因为剂量依赖性诱变剂会增加 G2/G1，而非诱变剂会增加 G1/G2。在 74 小时暴露后，RNAseq 检测到 G1 和 G2 标志物，随着 BaP 剂量的增加，G2/G1 比值增加，而随着高渗山梨醇和 PFOA 剂量的增加，G1/G2 标志物比值增加。BaP 在刺激的 2 小时内引起 FOAEL 的快速绿色增加，而视黄酸仅在培养后期才引起显著的绿色荧光增加。使用活体成像仪建立 FUCCI ESC 的 FOAEL 和 G2 延迟是一种新方法，可使商业和基础发育生物学家能够检测具有致突变性的药物和环境刺激物。此外，它可用于测试预防突变的化合物。在纵向研究中，通过这种可行的报告基因和活体成像仪方案提供了独特的后续，可以测试预防化合物本身是否会造成伤害。
