Collaborative study of thresholds for mutagens: Hormetic responses in the micronucleus test and gene induction by mutagenic treatments

doi:10.21203/rs.3.rs-3550460/v1

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Collaborative study of thresholds for mutagens: Hormetic responses in the micronucleus test and gene induction by mutagenic treatments

https://doi.org/10.21203/rs.3.rs-3550460/v1

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The linear no-threshold model (LNT) asserts that the genetic effects and carcinogenicity of radiation are proportional to the dose. LNT is also applied to carcinogens and mutagens. However, most experimental data show that the dose-response curve is not linear but rather a J-shaped curve, known as a hormetic response. LNT and hormesis are mutually exclusive. Which is correct? In this study, we investigated dose-response curves of mutagens in the micronucleus test using rodent cells. Since the frequency of background micronuclei was low, detecting a further decrease was difficult. When we conducted a challenge test, where cells were pre-treated with a low dose and post-treated with a high dose, clear hormetic responses were observed. Additionally, during a cross-reaction test, where cells were pre-treated with a low dose of one mutagen and post-treated with a high dose of another mutagen, unequivocal hormetic responses were detected. To investigate gene expression patterns, human lymphoma TK6 cells were treated with mitomycin C, ethyl methanesulfonate, and hydrogen peroxide, and the expression of six genes was examined by RT-PCR. Both GADD45A and p21 genes were induced in a time- and dose-dependent manner. In conclusion, the mutagens used here exhibit hormesis, indicating that the LNT model is invalid.

Biological sciences/Genetics

Earth and environmental sciences/Environmental sciences

In 1882, a young German professor named H. Schulz made a remarkable discovery. He observed that a variety of toxic agents, including heavy metal and phenol compounds, had the intriguing effect of enhancing the metabolism and proliferation of yeasts at low doses, while proving harmful and toxic at higher doses. Schulz consistently confirmed these unexpected biphasic dose-responses in his experiments. However, his innovative concept faced resistance and skepticism within academic circles, leading to his marginalization in the academic sphere [1]. It is worth noting that the exploration of chemical hormesis began over 140 years ago.

Later, Southam and Ehrlich [2] made a significant contribution to the understanding of hormesis. They demonstrated that the water-soluble extractives of western red-cedar heartwood contained a substance that exhibited toxicity to wood-decay fungi at higher doses but had a stimulating effect at lower doses. It was in this pioneering work that they introduced the term "hormesis" for the first time, defining it as follows: "The term hormesis (adj. hormetic) is proposed to designate such a stimulatory effect of subinhibitory concentrations of any toxic substance on any organism." The word "hormesis" is derived from the Greek word "hormaein," meaning "to excite." Interestingly, Southam and Ehrlich did not delve further into hormetic research, as they both pursued distinct paths after leaving the University of Idaho, where Southam had been a graduate student and Ehrlich his adviser [3].

In 1960, Townsend and Luckey [4] undertook a comprehensive effort by selecting over 100 hormetic reactions documented in pharmaceutical journals, along with a substantial list of chemicals. They introduced the term "hormoligosis," encompassing hormesis as one of its components. Later, in 2019, Luckey [5] employed the term "hormesis" in his book, where he compiled numerous hormetic responses observed across a wide range of species, including bacteria, protozoa, plants, and animals, drawing from a pool of 1,269 references.

In a separate study, Calabrese and Blain [6] conducted a survey encompassing hormetic responses induced by radiation, radionuclides, inorganic compounds, and organic compounds. Their investigation involved approximately 900 different agents. Consequently, hormesis has been consistently demonstrated and reported across numerous species, endpoints, and treatment scenarios, attesting to its widespread occurrence and significance.

As illustrated above, the exploration of hormesis emerged from investigations into the dose-response relationships of chemical substances, and its prevalence has been established across a wide spectrum of living organisms. Our collaborative research, which focuses on identifying thresholds for mutagens, can be seen as a validation test of the widespread occurrence of hormesis.

Building on our prior findings of hormesis in cellular activities [7] and cell proliferation [8], our current study extends this pattern by demonstrating that mutagens also exhibit hormetic responses in the micronucleus test. This collective body of research reinforces the idea that hormesis is a pervasive and fundamental phenomenon in biological systems.

Direct Detection of Hormetic Responses Using MMC

Hormesis is characterized by stimulation at lower doses and inhibition at higher doses, typically resulting in reverse U- or J-shaped dose-response curves [7]. When CHL/IU cells were treated with MMC, we observed a reduction in micronuclei at a dose of 0.2 ng/mL (Fig. 1A, B) and within the range of 0.2–0.78 ng/mL (Fig. 1C), but statistical significance was not detected. However, when results from nine experiments were combined, a significant difference was seen at 0.2 ng/mL 24 h after treatment (Fig. 1A, marked by a red arrowhead ─ mean ± SD of micronuclei: control, 0.83 ± 0.22; MMC 0.2 ng/mL, 0.56 ± 0.27; n = 9, P = 0.023). These results indicate that hormetic responses in the micronucleus test, in which the background incidence is as low as around 1% or less, are difficult to identify directly. Comparison of responses at 24 and 48 h suggests that 24 h is better for micronucleus detection (Fig. 1A vs. B; C vs. D).

Notably, cellular growth parameters (RCC and RPD) were stimulated at doses of 0.2 ng/mL (Fig. 1A) and 0.78 ng/mL (Fig. 1C), aligning with hormetic responses observed in a previous study [8]. RICC deviated from RCC and RPD (Fig. 1A), but this was exceptional, and the three parameters usually showed similar patterns as shown in Fig. 1B and supplementary information (S-Fig. 1, 2). Interestingly, MN (%) was significantly reduced at 0.2 ng/mL of MMC (S-Fig. 1).

[Fig. 1.]

Hormetic Responses by Challenge Tests Using EMS and AF-2

Detecting hormetic responses directly in the micronucleus test, where background incidence is low (Fig. 1), posed challenges. Consequently, we conducted challenge tests: cells initially exposed to low-dose levels insufficient to induce micronuclei were later exposed to high-dose levels (Fig. 2). Both EMS and AF-2 exhibited hormetic responses. We also observed hormetic responses when cells were treated with MMC and colchicine (supplementary information, S-Fig. 3, 4).

Since the same test compound induces the opposite reactions depending on doses in hormesis, selection of dose levels for pre-treatments and post-treatments is critically important in challenge tests. In this sense, pre-treatments with 1.0 and 2.0 µg/mL of AF-2 were not very appropriate, because these doses induced significantly micronuclei (P < 0.05, Fig. 2B). Although cells were pretreated with micronucleus-inducing doses, hermetic responses were observed (Fig. 2B).

[Fig. 2.]

Hormetic Responses in Cross-Reaction Tests Using AF-2 and MMC

Following the observation of hormetic responses in challenge tests (Fig. 2), we conducted cross-reaction tests (Fig. 3). In these tests, cells were first exposed to low-dose levels of one mutagen, insufficient to induce micronuclei, and then challenged with a high-dose level of another mutagen, sufficient to induce micronuclei. In Fig. 3A, cells were pretreated with AF-2 and then challenged with MMC, resulting in hormetic responses. Conversely, in Fig. 3B, cells were pretreated with MMC and then challenged with AF-2, also demonstrating hormesis. Notably, even a low dose of 0.25 ng/mL of MMC induced a hormetic response in Fig. 3B. This observation is reminiscent of the MMC test, where a hormetic response was seen at 0.2 ng/mL (supplementary information S-Fig. 1) and when nine tests were combined (Fig. 1A). Additionally, hormetic responses were detected when cells were pretreated with MMC and then challenged with EMS, and vice versa (supplementary information S-Fig. 5, 6).

[Fig. 3.]

Gene Expression in TK6 Cells Treated with MMC

After confirming hormesis induction by several mutagens (Fig. 1–3), RT-PCR was used to examine whether gene expression changes occurred. The roles of the seven selected genes are briefly described in Materials and Methods.

First, we estimated the effect of MMC on the gene expression of six genes other than the reference gene (Fig. 4). Cytotoxicity and p21 gene expression began to increase slightly at 500 ng/mL for 4 h exposure (Fig. 4A, B). At 10 h of exposure, cytotoxicity and GADD45A gene expression began to increase dose-dependently at 200 ng/mL, and p21 gene expression began to increase at 50 ng/mL (Fig. 4A, C). At 24 h exposure time, the starting dose level of cytotoxicity, GADD45A gene expression level, and p21 gene expression level were approximately 20, 50, and 10 ng/mL, respectively (Fig. 4A, D).

[Fig. 4.]

As shown in Fig. 1, MMC significantly induces micronuclei at a concentration of 6.3 ng/mL after 24 h of exposure. Notably, cytotoxicity and gene expression changes occur at higher dose levels than those required for micronucleus induction. The p21 gene encodes a protein known as an inhibitor of cyclin-dependent kinases (CDKs), which leads to the cessation of cell proliferation. On the other hand, GADD45A encodes a protein that functions as a stress sensor for various cellular processes such as cell cycle arrest, DNA repair, and apoptosis in response to genotoxic stress. Therefore, it appears that the cytotoxic doses of MMC induce these genes associated with cell cycle arrest and DNA repair. In cases where repair is unsuccessful, apoptosis may occur to eliminate damaged cells. Notably, p21 responds more sensitively to MMC compared to GADD45A.

Gene Expression in TK6 Cells Treated with EMS

The effect of EMS on the gene expression of 6 genes was estimated by measuring the mRNA levels relative to the housekeeping gene GAPDH (Fig. 5). At a 4-h exposure time, almost no cytotoxicity and no gene induction were observed with EMS treatment (Fig. 5A, C). At 10 h of exposure, cytotoxicity and gene expression of p21 began to increase at 50 µg/mL and 20 µg/mL, respectively. Gene expression of GADD45A began to increase at 200 µg/mL (Fig. 5A, D). For the exposure time of 24 h, cytotoxicity and gene expression of p21 and GADD45A began to increase at approximately 20 µg/mL (Fig. 5A, E). Genes induced by EMS were p21 and GADD45A as well as MMC. It is noteworthy that the dose-response of p21 expression showed a J-shaped curve, even at 4 h exposure time (Fig. 5B). If this is true, it is indicated that hormesis could occur without gene expression.

[Fig. 5.]

Gene Expression in TK6 Cells Treated with H₂O₂

The effect of H₂O₂ on the gene expression of six genes was estimated by measuring the mRNA levels relative to the housekeeping gene GAPDH (Fig. 6). Cytotoxicity and gene expression of p21 and GADD45A began to increase slightly at 10 µg/mL after 4 h of exposure (Fig. 6A, B). At 10 h exposure time, cytotoxicity and gene expression of p21 and GADD45A began to increase clearly and dose-dependently at approximately 10 µg/mL (Fig. 6A, C). Little increase in p21 and GADD45A gene expression was observed at 24 h exposure time, probably because the treatment was too toxic (Fig. 6A, D). Thus, cytotoxic dose levels of H₂O₂ induced some genes associated with cell cycle arrest and DNA repair as well as MMC and EMS.

[Fig. 6.]

The LNT posited that the dose-response for ionizing radiation-induced mutations was linear, extending down to single ionizations. It suggested that all genetic damage was cumulative and irreversible. LNT was recommended by the US National Academy of Sciences (NAS) in 1956 [10], which led to the abolition of the previous threshold model, set at around 500 mGy/year in 1934. LNT primarily relied on Drosophila data to predict genetic effects in humans. Intriguingly, human data from Hiroshima and Nagasaki were deliberately excluded from its consideration [11]. In 2006, NAS reaffirmed the use of LNT for assessing cancer risk from radiation, citing data from Hiroshima and Nagasaki [12]. However, Sutou [13] raised concerns, highlighting eight drawbacks in NAS's presentation. Indeed, survivors of atomic bombings in these cities have shown longer lifespans and lower cancer risk [14]. Accumulated data suggest that low-dose radiation is essential, mid-dose radiation may have a hormetic effect, and high-dose radiation is hazardous [15]. Hormesis and LNT are fundamentally incompatible. If hormesis holds true, LNT is invalidated, and vice versa. Our studies lend support to the validity of hormesis as a model.

The US Environmental Protection Agency (EPA) came into existence in 1970 and sought guidance from NAS on how to regulate carcinogens. NAS recommended the application of the LNT model to carcinogens [16]. Given the difficulties in directly establishing thresholds, our research has focused on investigating hormetic dose-responses. In previous studies [7, 8], we demonstrated the feasibility of establishing thresholds in both cell activity and cell proliferation tests. In the present study, we have confirmed that the mutagens we tested induce hormesis in the micronucleus test, adding a piece of evidence to the growing body of proof against LNT.

Detecting hormetic responses is difficult when the baseline micronuclei frequency is low (Fig. 1). To overcome this difficulty, we employed challenge tests (Fig. 2) and cross-reaction tests (Fig. 3), because Preconditioning is hormesis [17]. Both challenge and cross-reaction tests confirmed the induction of hormesis by mutagens (Fig. 2, 3). Of particular significance is the observation that hormesis occurs not only among different chemicals but also between chemicals and radiation sources [18, 19]. This suggests that hormesis is not divided into distinct categories of chemical and radiation hormesis. Instead, it indicates that living organisms employ common defense mechanisms to respond to a range of external stressors and challenges.

The Keap1-Nrf2 pathway functions as a cellular defense system against oxidative and xenobiotic stresses caused by ROS and electrophiles, respectively. Under normal, unstressed conditions, Nrf2, a transcription factor, is continually captured by Keap1 and subsequently degraded via the ubiquitin-proteasome pathway. However, when cells are exposed to ROS or electrophiles, this degradation of Nrf2 is halted. As a result, Nrf2 moves to the nucleus, where it promotes the transcription of over 100 genes associated with antioxidation and detoxification. Ionizing radiation itself is imperceptible, but its primary biological impact lies in the generation of reactive oxygen species (ROS). ROS function as signal mediators and are recognized by the Keap1-Nrf2 system, one of the two major systems responsible for regulating ROS [20].

Another major system responsible for handling ROS is the NF-κB/IκB system [21]. NF-κB proteins constitute a family of transcription factors that regulate the expression of numerous genes, pivotal not only in ROS management but also in inflammation, immunity, cell growth, differentiation, development, and apoptosis. Oxygen, despite its essential role in sustaining life, can paradoxically be one of the most toxic substances in our environment. Mitochondria, crucial cellular components, are a significant source of ROS production, estimated at a staggering 10⁹ ROS/cell/day [22]. To put this into perspective, the rate of double-strand DNA breaks per cell per day caused by background radiation (1 mGy) is calculated to be 10^− 4, whereas those induced by endogenous ROS are calculated to be 10^− 1. This highlights that endogenous ROS pose a substantially greater hazard than natural radiation.

Given this context, it's reasonable to assume that many of the enzymes induced by the NF-κB/IκB system play vital roles in hormetic responses. The Keap1-Nrf2 and NF-κB/IκB systems govern numerous genes, and they do not function independently. For instance, enzymes like glutathione-S-transferase, which regulates ROS levels along with other enzymes like superoxide dismutase, catalase, and glutathione peroxidase, are induced by both systems. This interplay underscores the complexity of cellular responses to oxidative stress.

We conducted an assessment of gene expression changes after mutagenic treatments using RT-PCR. The genes examined included GAPDH (control), p21, GADD45A, TOP2A, MCM, TP53, and GSTP1, which play roles in cell cycle regulation, apoptosis, DNA repair, and other crucial cellular processes. Among these six genes, p21 and GADD45A exhibited dose- and time-dependent induction in response to MMC, EMS, and H₂O₂ treatments. Notably, this induction was observed at relatively higher dose levels where cytotoxic effects became apparent, whereas micronuclei induction occurred at lower dose levels. This observation suggests that hormesis occurs under physiological conditions and that cellular responses, accompanied by gene induction, manifest at higher dose levels coinciding with cytotoxicity. While it may be tempting to simplify this induction as a response to halt the cell cycle for DNA repair, it's essential to acknowledge the limitations of our study. We examined a relatively small number of genes, and our analysis relied on a single detection system. Therefore, the full complexity of cellular responses to these mutagenic treatments may not be fully captured in this simplified explanation.

Calabrese and Baldwin [23] have defined two distinct types of hormesis: Direct Stimulation Hormesis (DSH) and Overcompensation Stimulation Hormesis (OCSH). DSH represents a steady-state adaptive response, reflective of normal physiological dynamics. In contrast, OCSH begins with the disruption of homeostasis, followed by modest overcompensation, reestablishment of homeostasis, and the adaptive nature of the process. DSH typically results in a dose-response curve that exhibits a reverse U-shaped or J-shaped pattern, as observed in our prior studies [7, 8]. On the other hand, when OCSH occurs, it leads to various curve shapes, including an S-shaped curve consisting of concave and convex segments, a J-shaped curve, or a typical reverse U-shaped curve. Given the involvement of numerous mechanisms in hormesis induction, the expression patterns are intricate, and responses are both time- and dose-dependent.

Cell

Chinese hamster cells (CHL/IU) with a doubling time of 18 hours were obtained from the National Institute of Health Science, where CHL/IU was established. CHL/IU cells were cultured in 6 cm diameter Petri dishes. Two types of media were used: Eagle's minimal essential medium (MEM) and Dulbecco's modified MEM, both supplemented with penicillin and streptomycin (25 µg/mL) and enriched with either 10% calf serum or fetal calf serum (FCS). Mouse lymphoma cells (L5178Y) were purchased from the American Type Culture Collection (ATCC). L5178Y cells were grown in Petri dishes for subculturing and in well-plates for testing. These cells were maintained in RPMI 1640 medium, supplemented with FCS and antibiotics. Human lymphoblast thymidine kinase heterozygote cells (TK6) were also purchased from ATCC. TK6 cells were cultured with RPMI 1640 medium supplemented with 10% FCS in 25 cm² flasks.

Chemicals

Acridine orange (AO, CAS: 494-38-2), Mitomycin C (MMC, CAS: 1950-07-7), (Z)-2-(2-furyl)-3-(5-nitro-2-furyl) prop-2-enamide (AF-2, CAS: 3688-53-7), and hydrogen peroxide (H₂O₂) were procured from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Ethyl methanesulfonate (EMS, CAS: 62-50-0) was obtained from NAKALAI TESQUE, INC., Kyoto, Japan. Phosphate buffered saline (PBS) and colchicine (CAS: 64-86-8) were supplied by Sigma-Aldrich Co. LLC, St. Louis, MO. Cell counting kit-8 (CCK-8) was obtained from DOJINDO LABORATORIES, Kumamoto, Japan. To administer these chemicals to the cells, a solution was prepared by either directly dissolving them in the culture medium or creating a 10-fold concentrated drug solution. The latter was then used to generate serial dilutions with a dilution factor of 2. Subsequently, the prepared solution was added to the cell culture medium at a ratio of 1:10.

Observation of Micronuclei in CHL/IU Cells by Giemsa Staining

CHL/IU cells in Petri dishes were harvested by trypsinization (0.25%, 2 mL). These cells were subjected to treatment with a hypotonic solution (0.135 M KCl) for 5 min at room temperature. After the hypotonic treatment, cells were fixed using a cold fixative (0.5 mL, methanol: acetic acid = 3:1) and then collected via centrifugation at 500 rpm for 5 min. Cells were subsequently subjected to additional rounds of fixation with 5 mL of the fixative, repeating this step three times. After the rounds, they were stored in a refrigerator for over 1 hour. Finally, the fixed cells were suspended in 1 mL of a cold fixative (methanol: acetic acid = 99:1). A few drops of this cell suspension were placed on a glass slide, which was positioned on a hot plate set at 37°C. The air-dried cells on the slide were stained using Giemsa's solution (4% Giemsa's fluid in PBS) for a duration of 15 to 30 min. Two plates were prepared for each treatment, and a total of 1,000 cells were examined per plate under a microscope to detect micronuclei. To assess the toxicity of a test chemical, the number of cells was quantified using a cell counter, specifically the ScepterTM2.0 (Merck Millipore Ltd., Burlington, MA).

Observation of Micronuclei in CHL/IU Cells by AO Staining

An aliquot (5 mL) from a cell suspension (0.8 x 10⁴ cells/mL) was added to plastic dishes with a diameter of 6 cm and treated with MMC (two plates per treatment) after 48 h. Specimens were prepared 24 and 48 h after treatment. Cells were removed by trypsinization (0.05% trypsin-0.02% EDTA) and collected by centrifugation at 1,000 rpm for 5 min at 20°C. Hypotonic treatment was done with 2 mL of 0.075 M KCl for 2–5 min. About 5 mL of a fixative (acetic acid: methanol = 1:3) was added to the cells. After 2 fixations, cells were collected by centrifugation and resuspended in 1 mL of a fixative (acetic acid: methanol = 1:99). A few drops of the cell suspension were placed on a glass slide on a 37°C hot plate. AO solution (40 µg/mL) was added to the slide just before microscopic examination.

Preparation of AO-Coated Slides for Supravital Staining Method

An AO stock solution was prepared by dissolving 10 mg of AO in 1 mL of PBS. The stock solution was diluted to a concentration of 1 mg/mL with PBS just before use. An aliquot (20 µL) of the solution was placed between two glass slides, and they were separated by pulling them apart horizontally in opposite directions. The AO-coated slides were quickly dried using a hair dryer. These AO-coated slides can be stored at room temperature in a dark environment for an extended period until needed. An aliquot (20 µL) of a cell suspension was placed onto the AO-coated slide, which was then covered with a cover slip (24 x 40 mm). Excess fluid was removed by gently pressing the slide between a piece of paper tissue. In cases where cell numbers are expected to be low due to toxic treatments, transfer a larger volume of the cell suspension (e.g., 100 µL) to an Eppendorf tube. Cells were concentrated by centrifugation at 3,000 rpm for 30 sec and then resuspended in a smaller volume of medium (e.g., 20 µL). Code numbers were sealed with a "Post-it" note. Micronuclei were examined under a fluorescence microscope using a B excitation unit (450–490 nm) in conjunction with a broadband filter that cuts off light at wavelengths less than 520 nm.

Examination of Gene Expression by RT-PCR

TK6 cells were initially plated at a density of 15,000 cells per 100 µL per well in three separate 96-well plates. After 24 h, 10 µL of cells from each plate were taken and added to four wells. These cells were then treated with test chemical solutions (MMC, EMS, or H₂O₂). After 4, 10, and 24 h of treatment, CCK-8 (10 µL) was added to three out of the four wells from each set. OD₄₅₀ was measured 2 h after the addition of CCK-8 using a plate reader (SH-1100R, Corona Electric Co., Ltd).

Extraction and Analysis of Gene Expression by RT-PCR

Cells were harvested from one of the four wells in each treatment group. RNA was extracted from the collected cells using the QIAzol reagent and the RNeasy Mini Kit, both provided by QIAGEN K.K., Tokyo. cDNA was synthesized from 100 ng of the extracted RNA using the High-Capacity RNA-to-cDNA Kit, manufactured by Thermo Fisher Scientific Inc. RT-PCR was performed using a thermal cycler (ViiA7 Real-Time PCR system from Thermo Fisher Scientific Inc.). The reaction mixture consisted of 5 µL of cDNA, 5 µL of primer mix, and 10 µL of reaction solution per well, with two reactions conducted per sample. The PCR conditions included 40 cycles: initial denaturation at 95°C for 10 min, denaturation at 95°C for 15 sec, followed by annealing and extension at 60°C for 1 min. A standard curve was generated by serially diluting control cDNA with a dilution factor of 5, ranging from 0.1 to 0.00016. Each data point was obtained through the ΔCq method, and the relative gene expression level compared to the control was determined.

Seven Genes Used for Gene Expression Analyses

GAPDH (glyceraldehyde-3-phosphate dehydrogenase) codes for an enzyme that catalyzes the sixth step of glycolysis. It is commonly used as a reference gene in gene expression studies due to its stable expression across various conditions. p21 (cyclin-dependent kinase Inhibitor 1), also known as CDK-interacting protein 1, plays a crucial role in inhibiting cyclin-dependent kinases (CDKs), leading to cell cycle arrest. It acts as a regulator of cell cycle progression. GADD45A (growth arrest and DNA damage inducible alpha) encodes a protein belonging to the GADD45 family. GADD45A is involved in various cellular processes, including apoptosis induction, inhibition of microtubule stability, and translocation of Bim (Bcl-2 interacting mediator of cell death) to mitochondria. It functions as a stress sensor, responding to genotoxic stress by regulating cell cycle arrest, DNA repair, and apoptosis. TOP2A (DNA topoisomerase 2-alpha) encodes an enzyme responsible for the transient breaking and rejoining of two strands of duplex DNA. This activity relieves topological DNA stress during transcription, chromosome condensation, and chromatid separation. MCM (minichromosome maintenance protein) is a gene family consisting of six proteins (MCM 2–7) that are essential replication initiation factors in eukaryotic cells. They play a crucial role in assembling the prereplication complex at replication origins during the early G₁ phase, contributing significantly to cell cycle progression. TP53 (tumor protein p53) codes for the tumor suppressor protein p53. P53 is involved in numerous cellular activities, including cell cycle arrest, DNA repair, apoptosis induction, and inhibition of angiogenesis. It functions as a key regulator of various processes associated with cancer prevention. GSTP1 (glutathione S-transferase Pi 1) encodes a member of the large glutathione S-transferase family. These enzymes play a critical role in detoxification by catalyzing the conjugation of hydrophobic and electrophilic compounds with reduced glutathione. The examination of the expression levels of these genes provides valuable insights into the cellular responses to different treatments and their potential effects on various cellular processes, including cell cycle regulation, DNA repair, and detoxification.

Statistics

To evaluate statistical differences, a table developed by Kastenbaum and Bowman [9] was used. Additionally, when necessary, Student's t-test was employed to compare the means of two groups.

Ethical Approval or Consent

All experiments here were performed in accordance with relevant guidelines and regulations.

Acknowledgements

All authors are members of the Collaborative Study Group of Thresholds for Mutagens in JEMS.MMS. Regrettably, we mourn the passing of KT on June 28, 2023, due to colon cancer at the age of 57. On a positive note, SS received the International Distinguished Scientist Award on July 1, 2023, from Asia's Science, Technology, and Research Awards 2023 in recognition of his contributions to radiation and chemical hormesis research. Additionally, the Asia International Research Awards Congress 2023 provided SS with the Outstanding Researcher Award on October 1, 2023 for his contributions to radiation and chemical hormesis research.

Authors' contribution

In addition to communicating via email, participants engaged in discussions about the study during the biannual meeting of JEMS.MMS. The micronucleus test using CHL/IU cells with Giemsa staining was conducted by HS. The micronucleus test using CHL/IU cells with AO staining and gene expression experiments were carried out by AK and KK. The micronucleus test using L5178Y cells was performed by SS and TK. The original manuscript was prepared by SS, and all authors reviewed and approved the final version.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Research funding

Each organization to which participants belong supported research fund individually.

Ethics approval

The regulations of the Safety and Ethics Committee stipulate that research involving human or human-derived materials (such as human tissues, cells, blood, saliva, urine, etc.) must undergo review by the Committee and may only be conducted after obtaining approval. However, there are exceptions to this rule, and it is specified that human cells widely used in society and obtainable from public institutions (for example, HeLa S3 cells and TK6 cells) are not subject to this restriction. In our case, as we used TK6 cells, the experiments were conducted without undergoing review by the Safety and Ethics Committee accordingly.

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No competing interests reported.

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Collaborative study of thresholds for mutagens: Hormetic responses in the micronucleus test and gene induction by mutagenic treatments

Status:

Version 1

Abstract

Figures

Introduction

Results

Direct Detection of Hormetic Responses Using MMC

Hormetic Responses by Challenge Tests Using EMS and AF-2

Hormetic Responses in Cross-Reaction Tests Using AF-2 and MMC

Gene Expression in TK6 Cells Treated with MMC

Gene Expression in TK6 Cells Treated with EMS

Gene Expression in TK6 Cells Treated with H₂O₂

Discussion

Methods

Cell

Chemicals

Observation of Micronuclei in CHL/IU Cells by Giemsa Staining

Observation of Micronuclei in CHL/IU Cells by AO Staining

Preparation of AO-Coated Slides for Supravital Staining Method

Examination of Gene Expression by RT-PCR

Extraction and Analysis of Gene Expression by RT-PCR

Seven Genes Used for Gene Expression Analyses

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Collaborative study of thresholds for mutagens: Hormetic responses in the micronucleus test and gene induction by mutagenic treatments

Status:

Version 1

Abstract

Figures

Introduction

Results

Direct Detection of Hormetic Responses Using MMC

Hormetic Responses by Challenge Tests Using EMS and AF-2

Hormetic Responses in Cross-Reaction Tests Using AF-2 and MMC

Gene Expression in TK6 Cells Treated with MMC

Gene Expression in TK6 Cells Treated with EMS

Gene Expression in TK6 Cells Treated with H2O2

Discussion

Methods

Cell

Chemicals

Observation of Micronuclei in CHL/IU Cells by Giemsa Staining

Observation of Micronuclei in CHL/IU Cells by AO Staining

Preparation of AO-Coated Slides for Supravital Staining Method

Examination of Gene Expression by RT-PCR

Extraction and Analysis of Gene Expression by RT-PCR

Seven Genes Used for Gene Expression Analyses

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Gene Expression in TK6 Cells Treated with H₂O₂