Sutou Shizuyo, Koeda Akiko, Komatsu Kana, Shiragiku Toshiyuki, Seki Hiroshi, Yamakage Kohji, Niitsuma Takeru, Kudo Toshiyuki, Wakata Akihiro
1School of Pharmacy, Shujitsu University, 1-6-1 Nishigawara, Naka-ku, Okayama-shi, 703-8516 Japan.
Ina Research Inc., 2148-188 Nishiminowa, Ina-shi, Nagano-ken, 399-4501 Japan.
Genes Environ. 2018 Oct 8;40:20. doi: 10.1186/s41021-018-0108-1. eCollection 2018.
According to the linear no-threshold model (LNT), even the smallest amount of radiation is hazardous. Although the LNT is not based on solid data, this hypothesis has been applied to mutagens and carcinogens. As a result, it has been postulated that there are no thresholds for these chemicals. To demonstrate negativity by experiments is practically impossible, because negative data may leave behind the possibility that additional data might make the resolution power high enough to change negativity to positivity. Furthermore, additional data collection may be endless and we may be trapped in agnosticism. When hormesis is established, in which biological responses are higher at low-doses and lower at high-doses than the control, thresholds could be established between the low- and high-doses. Before examination of thresholds in chemical mutagenesis, hormetic responses in cytotoxicity were tested using cultured mammalian cells.
Human cells (HeLa S3 and TK6) or Chinese hamster cells (CHL/IU) were cultured in 96-well plates and treated with mitomycin C (MMC) or ethyl methanesulfonate (EMS) at various dose levels and optical density was measured after addition of a reagent to detect cellular activity. In hormetic responses, data might fluctuate to and fro; therefore, experimental conditions were examined from various aspects to eliminate confounding factors including cell numbers, detection time, the edge effect of 96-well plates, and measurement time after addition of the reagent for detection.
The dose response relationship was never linear. Cellular activities after treatment with MMC or EMS were generally higher at lower doses levels and lower at higher doses than the control, showing hormesis and allowing the establishment of thresholds. Dose response curves sometimes showed two or three peaks, probably reflecting different cellular responses.
Hormetic responses in cytotoxicity tests were observed and thresholds could be established. Based on the results of this investigation, we put forward a tentative protocol to detect chemical hormesis in cytotoxicity tests, i.e., inoculate 2000 cells per well, add various doses of a test chemical 48 h after inoculation, add a detection dye 10 h after treatment, and measure optical density 2 h after addition of the reagent for detection.
根据线性无阈模型（LNT），即使是最小剂量的辐射也是有害的。尽管LNT并非基于确凿的数据，但这一假设已被应用于诱变剂和致癌物。因此，有人推测这些化学物质不存在阈值。通过实验证明阴性实际上是不可能的，因为阴性数据可能会留下这样的可能性，即额外的数据可能会使分辨能力足够高，从而将阴性转变为阳性。此外，额外的数据收集可能是无止境的，我们可能会陷入不可知论。当建立了兴奋效应（即低剂量时生物反应高于对照组，高剂量时低于对照组）时，就可以在低剂量和高剂量之间建立阈值。在研究化学诱变中的阈值之前，先使用培养的哺乳动物细胞测试了细胞毒性中的兴奋效应反应。
将人细胞（HeLa S3和TK6）或中国仓鼠细胞（CHL/IU）培养在96孔板中，并用不同剂量水平的丝裂霉素C（MMC）或甲基磺酸乙酯（EMS）处理，在加入检测细胞活性的试剂后测量光密度。在兴奋效应反应中，数据可能会来回波动；因此，从各个方面检查实验条件以消除混杂因素，包括细胞数量、检测时间、96孔板的边缘效应以及加入检测试剂后的测量时间。
剂量反应关系从未呈线性。用MMC或EMS处理后的细胞活性通常在较低剂量水平时高于对照组，在较高剂量时低于对照组，显示出兴奋效应并允许建立阈值。剂量反应曲线有时会出现两个或三个峰值，可能反映了不同的细胞反应。
在细胞毒性测试中观察到了兴奋效应反应，并可以建立阈值。基于本研究结果，我们提出了一种在细胞毒性测试中检测化学兴奋效应的初步方案，即每孔接种2000个细胞，接种后48小时加入不同剂量的测试化学品，处理后10小时加入检测染料，加入检测试剂后2小时测量光密度。
