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

Abstract

Background: 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.

Method: 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.

Results: 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.

Conclusion: 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.

Keywords: Adaptive response; CHL/IU; Ethyl methanesulfonate; HeLa S3; Hormesis; Mitomycin C; TK6.

Fig. 1

Schematic illustration of hormetic and LNT responses. Magnitudes of responses and doses are arbitrary. In hormesis, e.g., in cytotoxicity and cell killing tests, low doses are beneficial to cells and high doses are hazardous to them so that the dose-response curve shows a reverse U-shaped curve when plotted on a linear scale (a, c, gray line). Thus, a threshold could be determined at the cross-point of the curve and the x-axis. On the other hand, LNT assumes that toxicity increases dose-proportionally and therefore the dose-response relationship is depicted as a linear line (a, red line). When the x-axis is logarithmic, the linear line becomes a curved one (c, red line). It is quite important to note that all responses in LNT come under the zero dose level (a, c, blue line). In hormesis, e.g., in mutagenicity and carcinogenicity tests, responses are opposite to cytotoxicity and cell killing tests (a, c) and show a J-shaped curve (b, d, grey line). LNT responses follow a linear line upward from left to right when plotting on a linear scale (b, red line) or a curved line when plotted on a logarithmic scale (d, red line). Here again, LNT responses never come under the zero dose level (b, d, blue line)

Fig. 2

Preliminary experimental results. CHL/IU (a, b, and c), TK6 (e and d) or HeLa S3 (f) cells were treated with mitomycin C two days after inoculation for 24 h and OD-450 was measured 2 h after addition of CCK-8. Data show OD-450 ratios (Mean ± SD). Lines connecting between the zero dose and the highest dose depict dose-response relationship expected when applying the LNT hypothesis. Although the scale of the x-axis is neither linear nor logarithmic, it is closer to logarithmic; expectations from LNT are closer to curved lines as shown in Fig. 1c and d. When the scale of the x-axis is linear, presentation of all data in a single graph is difficult. Therefore, responses within a small range (from 0 to 1 ng/mL) are depicted on a linear scale in Fig. 2a (insert), in which the blue line shows an LNT response. The number of wells were three for a and f, four for b, d, and e, and six for e. In e, the outer 36 wells were used as medium blanks, and the mean reading was subtracted from experimental readings. The control in E consisted of 12 wells

Fig. 3

Number of cells per well and hormetic expression time. Firstly, different numbers of HeLa S3 cells (2000, 4000, and 6000 cells/well) were treated with EMS for 6 (a), 12 (b), and 24 h (c) two days after inoculation. Secondly, Hela S3 cells (500, 1000, and 2000 cells/well) were treated with EMS for 9 (d), 12 (e), and 15 h (f). CCK-8 (10 μL/well) was added 2 h before OD-450 measurement. Data show OD-450 ratios (Mean ± SD)

Fig. 4

Re-examination of cell number and expression time. HeLa S3 cells (2000 cells per well) were plated and treated with MMC 48 h after plating. OD-450 was measured 9, 11, and 13 h after MMC treatment. Data show OD-450 ratios (Mean ± SD)

Fig. 5

Characteristics of CCK-8 coloration. HeLa S3 cells (2000 cells per well) were plated and treated with nine concentrations (5 to 2000 ng/mL as shown at the right of the graph) two days after inoculation. CCK-8 (5 μL/well) was added 10 h after addition of MMC and OD-450 was measured 11, 12, 13, 14, 15 and 21 h after the addition of MMC (1, 2, 3, 4, 5, and 11 h after the addition of CCK-8, respectively)