Collaborative study of thresholds for mutagens: proposal of a typical protocol for detection of hormetic responses in cytotoxicity tests

Abstract

BackgroundAccording 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.MethodHuman 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.ResultsThe 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.ConclusionHormetic 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.

Collaborative Study of Thresholds for Mutagens: Hormetic Responses in Cell Proliferation Tests Using Human and Murine Lymphoid Cells

“…In the previous report, adherent cells were cultured in a 96-well plate and the edge effect—different culture conditions between outer and inner wells because water evaporates more rapidly from outer wells than inner ones—had to be taken into consideration. 3 Although the effect would only have been minimal, the outer 24 wells were not used. In the present study, 48- or 24-well plates were used, since 2 to 4 or more cell samples can be taken consecutively from a single well.…”

“…Detailed rationale is discussed in our previous paper (see Figure 1A ). 3 Although precise and sophisticated analyses of hormetic responses have been published to date, they are not suitable for application to the present tests, in which dose-response patterns fluctuate largely and change transiently as shown by fallen S-shaped and J-shaped curves in addition to typical reverse U-shaped curves. 5 , 6 Here, paying attention only to reverse U-shaped curves, we propose tentative, simplified method focusing on the maximum hormetic response.…”

“…Higher sensitivity of murine cells was hinted in the previous study in which murine CHL cells started to show suppression of cell activity at around 2 ng/mL (see Figure 2A and C ), while human TK6 cells started at around 20 ng/mL (see Figure 2D and E ). 3 …”

“…As members of the Japanese Environmental Mutagen and Genome Society (JEMS), the Collaborative Study Group of Thresholds for Mutagens was organized to confirm hormetic responses using cell activity tests. 3 In this study, we used several mutagens and chemicals to determine whether or not they show hormesis in in vitro cell proliferation tests. For this, we used human and murine cultured lymphoid cells, because suspended cells were convenient for cell counting.…”

The prospective mathematical idea satisfying both radiation hormesis under low radiation doses and linear non-threshold theory under high radiation doses

“…Certainly, a proportional relationship has been described at ≥100 mSv, but it has not been confirmed whether there is a proportional relationship for cases of < 100 mSv [1]. In addition, the radiation hormesis theory is thought to actually have beneficial effects on health [2][3][4][5][6][7][8][9][10]. Therefore, I wondered if there was an idea that LNT and hormesis could hold at the same time.…”