Hormesis redefined: Insights from application of δ plot quantification of the Yonezawa effect to dose responses in the micronucleus test

Hormesis redefined: Insights from application of δ plot quantification of the Yonezawa effect to dose responses in the micronucleus test | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hormesis redefined: Insights from application of δ plot quantification of the Yonezawa effect to dose responses in the micronucleus test Shizuyo Sutou, Akiko Koeda, Kana Komatsu, Toshiyuki Shiragiku, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5109349/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cells and organisms respond dynamically to environmental factors like radiation and chemicals. These responses vary based on detection systems, leading to terms such as adaptive response, biphasic response, and hormesis. In micronucleus tests using cultured cells, obtaining a typical J-shaped dose-response curve, a hallmark of hormesis, was challenging due to low background micronucleus frequency. We conducted challenge and cross-challenge tests. In challenge tests, cells were pre-treated with low priming doses and then post-treated with a high challenging dose. In cross-challenge tests, cells were pre-treated with one chemical at low doses and then post-treated with a high dose of another chemical. Both tests showed clear suppression of micronucleus induction by high doses following pre-treatments. Our paper reporting hormesis in the micronucleus test was initially rejected, with reviewers claiming we detected an adaptive response rather than hormesis. Believing these concepts to be equivalent, we re-analyzed our data using the δ plot, which quantifies the Yonezawa effect, a type of radiation adaptive response. The analysis showed our results fit effectively with the δ plot. Since the Yonezawa effect aligns with the definition of hormesis, our findings could be termed as such. Other adaptive responses fitting the δ plot could also be considered hormesis. Biological sciences/Genetics Earth and environmental sciences/Environmental sciences Health sciences/Health care Adaptive response Challenging dose Cross-adaptive response Hormetic Preconditioning Priming dose Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Throughout the approximately 4 billion years of evolutionary history, organisms have been continuously exposed to radiation. Radiation levels 4 billion years ago were nearly 10 times higher than they are today [1]. It has been suggested that radiation may have contributed to both chemical and biological evolution [2]. All organisms are radioactive to some extent because they continuously take in radioactive elements such as K-40 and C-14. In humans, internal radiation exposure alone amounts to approximately 9,000 Bq/sec, and when external exposure is included, it totals about 20,000 Bq/sec [3, 4]. The primary effect of low-dose radiation is its reaction with water to produce reactive oxygen species (ROS), which means that the biological effects of low-dose radiation are mediated through ROS as chemical agents. ROS act as signaling molecules, and the Keap1-Nrf2 pathway is capable of detecting them [5]. This pathway contributes to biological defense against oxidative stress derived from ROS and electrophilic foreign substances. The NF-κB/IκB system is also involved in ROS regulation but is broadly associated with inflammation, immunity, cell division, differentiation, development, and apoptosis [6]. Organisms that have evolved alongside radiation naturally exhibit genetic responses to it [7]. Luckey's comprehensive work, which cites 1,269 references, includes numerous examples of radiation hormesis across various life forms, including bacteria, protozoa, plants, and animals [8]. On the other hand, it is estimated that we are exposed to tens of thousands of chemical substances. There are numerous examples of cells and organisms exhibiting hormesis in response to these chemicals, showing stimulation at low doses and inhibition at high doses. Southam and Ehrlich discovered that an extract from wood showed growth stimulation at low doses and growth inhibition at high doses for certain types of fungi, and they named this phenomenon "hormesis" [9]. Subsequently, Townsend and Luckey searched pharmaceutical literature and compiled a list of over 100 substances that showed biphasic responses with stimulation at low doses and inhibition at high doses [10]. Calabrese and Blain expanded on this work, documenting over 900 hormetic responses for radiation, radioactive substances, inorganic compounds, and organic compounds [11]. Based on these extensive studies, hormesis is now considered to be a ubiquitous phenomenon in the biological world. Our collaborative study group within the Mammalian Mutagenicity Study Group (MMS) of the Japanese Environmental Mutagen and Genome Society (JEMS) investigated thresholds for mutagens. Using cell viability and proliferation as endpoints, we observed reverse U-shaped hormetic responses [12, 13]. However, when examining hormesis in micronucleus tests, typical J-shaped hormetic responses were difficult to obtain due to the low natural frequency of micronuclei (around 1%). To address this, we conducted challenge tests involving low-dose pre-treatment followed by high-dose post-treatment. We also performed cross-challenge tests using different substances for pre- and post-treatment. Both approaches demonstrated suppression of micronucleus induction by post-treatments. We submitted a manuscript claiming hormetic responses in micronucleus tests, which was published as a preprint [14]. However, the paper was rejected, with reviewers suggesting we were observing adaptive responses rather than hormesis. We subsequently revised the terminology from "hormetic" to "adaptive" and successfully published in a different journal [15]. Given our view that hormesis and adaptive responses are essentially equivalent, this distinction seemed arbitrary. We therefore reexamined raw micronucleus test data obtained in the previous study [15] using the δ plot, which quantifies the Yonezawa effect―a type of radiation adaptive response. We propose that other adaptive responses fitting the δ plot could also be classified as hormesis. Results I. Rationale: the scheme of the Yonezawa Effect Fig. 1 illustrates the schematic representation of the Yonezawa effect, also known as the priming dose effect [17, 18]. Fig. 1 II. Difficulty in detecting hormesis with a typical biphasic, J-shaped dose response curve Before conducting the micronucleus tests, dose-finding experiments are typically performed. Fig. 2 illustrates dose-finding tests with MMC (A) and EMS (B). Both compounds exhibited a slight reduction in micronuclei at low doses, suggesting a J-shaped dose-response curve, which is characteristic of hormesis. However, this reduction was not statistically significant, and hormesis could not be confirmed with certainty. Given that the background level of micronuclei is around 1%, detecting a further reduction due to a hormetic response proves challenging. Additionally, high-dose treatments with EMS led to the appearance of numerous pycnotic cells and cells with amorphous nuclei, especially at 2,000 μg/mL, complicating the examination of micronuclei. A similar result, suggesting a J-shaped dose-response curve, was obtained in a dose-finding test with colchicine (see Supplementary Fig. 1). Fig. 2. III. Challenge tests with AF-2 and EMS Detecting hormetic responses directly in the micronucleus test, where the background incidence is low, proved challenging (Fig. 2). Therefore, challenge tests were conducted in which cells initially exposed to low doses (priming doses) insufficient to induce micronuclei were subsequently exposed to a high dose (challenging dose) capable of inducing micronuclei (Fig. 3). Both AF-2 (Fig. 3A) and EMS (Fig. 3B) demonstrated that micronucleus induction by the challenging dose was suppressed by the priming doses. It is noteworthy that higher priming doses resulted in greater suppression of micronuclei. Similar results were observed in a challenge test with MMC (see Supplementary Fig. 2). Fig. 3. IV. Cross-challenge tests with AF-2 and MMC In addition to the challenge tests (Fig. 3), cross-challenge tests were conducted using AF-2 and MMC, where cells were first primed with AF-2 and subsequently challenged with MMC (Fig. 4A). The results of these cross-challenge tests exhibited similar response patterns to those observed in the challenge tests (Fig. 3). Likewise, when cells were initially primed with MMC and then challenged with AF-2, comparable results were obtained (Fig. 4B). In both cases, higher priming doses led to greater suppression of micronuclei under the current experimental conditions. Similar results were observed in cross-challenge tests involving EMS and MMC (see Supplementary Fig. 3). Fig. 4 Discussion Yonezawa et al. demonstrated a radiation-induced adaptive response in mice, where an initial low-dose radiation (priming dose, D 1 in Fig. 1) reduced acute lethality from a subsequent high-dose radiation (challenging dose, D 2 in Fig. 1). The survival rates of mice that received both D 1 and D 2 were higher than those that received only D 2 . Key factors in the Yonezawa effect include D 1 , D 2 , the time interval between D 1 and D 2 , and the endpoint (mouse survival). The framework of the Yonezawa Effect, as shown in Fig. 1, can essentially be applied to the micronucleus test (Figs. 3 and 4). Therefore, the present micronucleus test can be considered a chemically induced adaptive response. However, the micronucleus test described here differs from the mouse survival test in several key ways. (1) The appearance of micronuclei is time- and dose-dependent—micronuclei tend to appear earlier with low-dose treatments, later with high-dose treatments, and disappear after a prolonged period. This necessitates determining the optimal expression time. The micronucleus test using suspended cells is particularly convenient, as samples can be collected consecutively (e.g., 18, 24, 30, and 36 h after treatment) from the same culture to estimate the optimal expression time. (2) The continuous presence of chemicals in cell cultures may affect both cell division and micronucleus formation, as demonstrated in the EMS experiment (Fig. 2B), especially at higher doses. Some chemicals, such as microtubule disruptors like colchicine, complicate the micronucleus test due to their specific effects on cell division. In such cases, the chemicals can be removed by washing, as shown in the MMC experiment (Fig. 2A). (3) When the toxicity of a test chemical reduces cell numbers, the cells can be concentrated through brief centrifugation, as described in the "Preparation of AO-Coated Slides for Supravital Staining" section in Materials and Methods. (4) Supravitally stained cells can be stored at -80°C for a month or longer until examination. Dose-response relationships in biological processes are often non-linear, and dose-response curves have been described as biphasic, bell-shaped, U-shaped, inverted U-shaped, J-shaped, diphasic, bimodal, bidirectional, sinusoidal, and more [20]. Over 50 researchers studying these diverse dose-response patterns have recommended adopting more consistent terminology. They proposed the common use of the term "hormesis," including specific categories like physiological conditioning hormesis, radiation conditioning hormesis, and chemical conditioning hormesis when preconditioning is involved [21]. Later, Calabrese, the lead author of the referenced paper, argued that "Preconditioning is hormesis" [22, 23]. The "Yonezawa Effect" is an example of preconditioning, where a small preconditioning dose administered before a high (challenging) dose protects against the harmful effects of the latter, and this can be considered a hormetic response. Since the δ plot shown in Fig. 1 is essentially applicable to the adaptive response observed in the micronucleus test, this test can also be considered a form of hormesis. The δ plot can also be effectively applied to cross-adaptive responses between radiation and chemicals [24, 25, 26]. These adaptive responses can similarly be referred to as hormesis. Methods Cells Mouse lymphoma cells (L5178Y) were obtained from the JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition, Japan. Cells were maintained in RPMI 1640 medium supplemented with penicillin and streptomycin (25 μg/mL) and enriched with 10% fetal calf serum. Cells were kept in a 5% CO 2 incubator at 37°C. Cell counts were performed using a hemocytometer. Chemicals Acridine orange (AO, CAS: 494-38-2), Mitomycin C (MMC, CAS: 1950-07-7), and (Z)-2-(2-furyl)-3-(5-nitro-2-furyl) prop-2-enamide (AF-2, CAS: 3688-53-7) 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) was supplied by Sigma-Aldrich Co. LLC, St. Louis, MO. To administer chemicals to the cells, solutions were prepared by either directly dissolving them in the culture medium or creating a 10-fold concentrated chemical solution. The latter was then used to generate serial dilutions with a dilution factor of 2. Subsequently, the prepared solutions were added to the cell culture medium at a ratio of 1:10. 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, which were then 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 pieces of paper tissue. In cases where cell numbers were expected to be low due to toxic treatments, a larger volume of the cell suspension (e.g., 100 μL) was transferred 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. Statistics To evaluate statistical differences, a table developed by Kastenbaum and Bowman was used [16]. For easy comparison, a computer-generated table was created, displaying significance levels of 0.01 and 0.05, P-values ranging from 0.01 to 0.99, along with the corresponding number of micronuclei from 1 to 1000. Declarations Acknowledgements: On July 1, 2023, the Asia Research Awards, an organization headquartered in India, awarded SS the “International Distinguished Scientist Award” among scientists for his research on “biological effects of low-dose radiation.'' On October 1, 2023, the organization awarded the “Asia's Outstanding Researcher Award” to SS among scientists, researchers, doctors, and professors for his work on "research on radiation hormesis." As a year-round recipient, the organization awarded SS “the International Innovative Scientist of the Year Award” on March 16, 2024 for his research on "radiation and chemical hormesis." Author contributions: Although the micronucleus test data using L5178Y cells were collected by SS, these raw data were presented in a previous paper and are shared by all the authors of reference [15]. The original manuscript was prepared by SS, and all authors reviewed and approved the final version. Ethics approval and consent to participate : Not applicable. Consent for publication : Not applicable. Data availability : 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. Funding : This work is not supported by third parties. References Karam, P.A. & Leslie, S.A. Calculations of background beta-gamma radiation dose through geologic time. Health Phys . 77, 662-667 (1999). Sutou, S. Low-dose radiation effects. Curr. Opin. Toxicol . 2022;30:100329. Sutou, S. Hormetic effects of low-dose radiation in Low dose radiation in health and disease (eds. Pandy, B.N. & Huilgol, N.G.) 1-16 (Narosa Publishing House, 2024). Scott, B.R. et al. Radiation-stimulated epigenetic reprogramming of adaptive-response genes in the lung: an evolutionary gift for mounting adaptive protection against lung cancer. Dose-Response 7, 104-131 (2009). Suzuki, M., Otsuki, A., Keleku-Lukwete, N. & Yamamoto, M. Overview of redox regulation by Keap1–Nrf2 system in toxicology and cancer. Curr. Opin. Toxicol. 1, 29–36 (2016). Morgan, M.J. & Liu Z.G. Crosstalk of reactive oxygen species and NF-κB signaling. Cell Res. 21, 103-1015 (2011). Jargin, S.V. Hormesis and radiation safety norms: Comments for an update. Hum. Exp. Toxicol . 37, 1233-1243 (2018). Luckey, T.D. Hormesis with ionizing radiation. 1-222 (CRC Press, 2019). Southam, C.M. & Ehrlich, J. Effects of extract of western red-cedar heartwood on certain wood decaying fungi in culture. Phytopathol. 33, 517–524 (1943). Townsend, J.F. & Luckey ,T.D. Hormoligosis in pharmacology. J. Amer. Med. Assoc . 173, 1960, 44-48 (1960). Calabrese, E.J, & Blain ,R. The occurrence of hormetic dose responses in the toxicological literature, the hormesis database: an overview. Toxicol. Appl. Pharmacol. 202, 289-301 (2005). Sutou, S. et al. Collaborative study of thresholds for mutagens: Proposal of a typical protocol for detection of hormetic responses in cytotoxicity tests. Genes Environ. 2018 Oct 8;40:20. Sutou, S. et al. Collaborative study of thresholds for mutagens: Hormetic responses in cell proliferation tests using human and murine lymphoid cells. Dose-Response . 2021 Jun 29;19(2):15593258211028473. Sutou, S. et al. Collaborative study of thresholds for mutagens: Hormetic responses in the micronucleus test and gene induction by mutagenic treatments. https://www.researchsquare.com/article/rs-3550460/v1 (2013). Sutou, S. et al. Collaborative study of thresholds for mutagens: Adaptive responses in the micronucleus test and gene induction by mutagenic treatments. Dose-Response 2024 May 7;22(2):15593258241252040. Kastenbaum, M.A. & Bowman, K.O. Tables for determining the statistical significance of mutation frequencies. Mutat. Res .9, 527-549 (1970). Yonezawa, M., Takeda, A. & Misonoh, J. Acquired radioresistance after low dose X-irradiation in mice. J. Radiat. Res. 31, 256-262 (1990). Yonezawa, M., Misonoh, J. & Hosokawa, Y. Two types of X-ray-induced radioresistance in mice: presence of 4 dose ranges with distinct biological effects. Mutat. Res. 358, 237-243 (1996). Fornalski, K.W. et al. The radiation adaptive response and priming dose influence: the quantification of the Raper-Yonezawa effect and its three-parameter model for postradiation DNA lesions and mutations. Radiat, Environ. Biophys. 61, 221-239 (2022). Calabrese, E.J. & Baldwin, L.A. Defining hormesis. Hum. Exp. Toxicol. 21, 91-97 (2002). Calabrese, E.J. et al. Biological stress response terminology: Integrating the concepts of adaptive response and preconditioning stress within a hormetic dose-response framework. Toxicol. Appl. Pharmacol. 222, 122-128 (2007). Calabrese, E.J. Preconditioning is hormesis part I: Documentation, dose-response features and mechanistic foundation. Pharmacol. Res. 110, 242-264 (2016). Calabrese, E.J. Preconditioning is hormesis part II: How the conditioning dose mediates protection: Dose optimization within temporal and mechanistic frameworks. Pharmacol. Res. 110, 265-275 (2016). Wolff, S., Afzal, V., Wienke, J.K, Olivier, G. & Michaeli, A. Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA. Intern. J. Radiat. Biol. 53, 39–49 (1988). Sakai, K., Nomura, T. & Ina, Y. Enhancement of bio-protective functions by low dose/dose-rate radiation. Dose-Response 4, 327-332 (2006). Kakinuma, S., Yamauchi. K., Amasaki, Y., Nishimura, M. & Shimada, Y. Low-dose radiation attenuates chemical mutagenesis in vivo – cross adaptation– J. Radiat. Res. 5, 401-405 (2009). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5109349","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":426645478,"identity":"6feab936-c3dd-4166-a7f3-d5d1b2b699dd","order_by":0,"name":"Shizuyo Sutou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYNCCCoYEBoaDDSAmMxHKQWrOgLU0NhCvhbENpIWBsYEoJ8nPyD/4mHdeXZ7BwcPtD3+2MbCbE9JicCOZ2Zh32+FigwMHG5t52xiYLQnZZSCRzCbNu+1A4gaQFqALmQ0OEHQYSMucOrCWxp/EaGG4AdLSwAzW0sBLjBaDM4+NDeccO5w4E6hlNs85CcJ+kW9PfPjgTU1dYt+N4w8+/iizSSYYYgggAXaQRLIB8Vr4IQ6yI0HLKBgFo2AUjBAAAFh2RZ713oUgAAAAAElFTkSuQmCC","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Shizuyo","middleName":"","lastName":"Sutou","suffix":""},{"id":426645479,"identity":"3b36006e-379a-49f9-85d3-765ac5e319b1","order_by":1,"name":"Akiko Koeda","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Akiko","middleName":"","lastName":"Koeda","suffix":""},{"id":426645480,"identity":"91a88c08-faab-4085-b6f9-766cfb348af5","order_by":2,"name":"Kana Komatsu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kana","middleName":"","lastName":"Komatsu","suffix":""},{"id":426645481,"identity":"217f0de1-4e48-4418-b6e1-1dbcd2c52403","order_by":3,"name":"Toshiyuki Shiragiku","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Toshiyuki","middleName":"","lastName":"Shiragiku","suffix":""},{"id":426645482,"identity":"fe51cb6c-b57d-486b-b797-034b628bd026","order_by":4,"name":"Hiroshi Seki","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Seki","suffix":""},{"id":426645483,"identity":"fa360661-d7a6-4523-9b6d-6f13cd728f10","order_by":5,"name":"Toshiyuki Kudo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Toshiyuki","middleName":"","lastName":"Kudo","suffix":""}],"badges":[],"createdAt":"2024-09-18 10:42:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5109349/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5109349/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78627566,"identity":"95d39820-b29c-4f33-ae20-7d5e7b78d054","added_by":"auto","created_at":"2025-03-17 02:44:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":40247,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the Yonezawa effect, slightly adapted for the micronucleus version from Fornalski et al. [19]. Cells were treated with a priming dose (D\u003csub\u003e1\u003c/sub\u003e) that does not induce micronuclei and, after a certain period, were exposed to a micronucleus-inducing dose (D\u003csub\u003e2\u003c/sub\u003e). The parameter δ represents the difference between the number of micronuclei produced by a single dose of D\u003csub\u003e2\u003c/sub\u003e (without the priming dose) and the number produced by the combination of D\u003csub\u003e1\u003c/sub\u003e + D\u003csub\u003e2\u003c/sub\u003e (with the priming dose). δ is quantified by the equation δ = 1 − N\u003csub\u003e1+2\u003c/sub\u003e/N\u003csub\u003e2\u003c/sub\u003e, where N\u003csub\u003e2\u003c/sub\u003e is the number of micronuclei induced by D\u003csub\u003e2\u003c/sub\u003e alone and N\u003csub\u003e1+2 \u003c/sub\u003eis the number induced by the combination of D\u003csub\u003e1\u003c/sub\u003e + D\u003csub\u003e2\u003c/sub\u003e. In this study, the micronucleus parameter is expressed as the micronucleus ratio, which is the ratio of the number of micronuclei induced by the challenge treatments to the number of micronuclei in the control, with the control ratio set to unity.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5109349/v1/de0e672e7c3606efdcfcfee7.jpg"},{"id":78627238,"identity":"05a82bd3-b925-4911-a9bd-e37a6147578a","added_by":"auto","created_at":"2025-03-17 02:37:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":56051,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDose-finding tests with MMC (A) and EMS (B).\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e\u0026nbsp;L5178Y cells (2.5 × 10\u003csup\u003e5\u003c/sup\u003e cells/well/0.5 mL) were treated with MMC for 24 h in a 24-well plate. Following treatment, cells were transferred to an Eppendorf tube, centrifuged at 3,000 rpm for 30 sec, and cultured in 500 μL of fresh medium. Twelve hours after washing, 200 μL of the cell suspension was transferred to an Eppendorf tube. Cells were again centrifuged at 3,000 rpm for 30 seconds, suspended in 50 μL of fresh medium, and 20 μL were used to prepare specimens. Two specimens were prepared from each treatment, and 1,000 cells per specimen were examined. The incidence of micronuclei per 1,000 cells was as follows: 7 and 7, 8 and 8, 5 and 6, 5 and 7, 5 and 6, and 14 and 15 for 0, 0.125, 0.25, 0.5, 1.0, and 2.0 ng/mL, respectively. \u003cstrong\u003eB:\u003c/strong\u003e\u0026nbsp;L5178Y cells (2 × 10\u003csup\u003e5\u003c/sup\u003e cells/well/0.5 mL) were plated in a 24-well plate. EMS was added to the cultures 24 h after plating. Specimens were prepared 30 h after EMS addition, and 1,000 cells per specimen were examined. The incidence of micronuclei per 1,000 cells was as follows: 12, 8, 7, 10, 16, 56, 160, and 13 for 0, 31.3, 62.5, 125, 250, 500, 1,000, and 2,000 μg/mL, respectively. Asterisks * and ** denote statistically significant differences between treatments and the control at P \u0026lt; 0.05 and P \u0026lt; 0.01, respectively.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5109349/v1/b3fc853f2b7cd7ca36d21a6a.jpg"},{"id":78627236,"identity":"8b3a0e61-d67c-427b-a1e4-0480ea9d9e33","added_by":"auto","created_at":"2025-03-17 02:36:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChallenge tests with AF-2 (A) and EMS (B).\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e\u0026nbsp;L5178Y cells (2.5 × 10\u003csup\u003e5\u003c/sup\u003e cells/well/0.5 mL) were treated with priming doses of AF-2 at 0.25, 0.5, and 1.0 μg/mL, followed by a challenging dose of 20 μg/mL after 12 h. Cells transferred to an Eppendorf tube were then centrifuged at 3,000 rpm for 30 sec and cultured in 500 μL of fresh medium. Specimens were prepared 18 h after washing. Two specimens were prepared from each treatment, and 1,000 cells per specimen were examined. The incidence of micronuclei in the control was 7/2,000 cells (0.35%). Applying the Yonezawa effect scheme (Fig. 1) to Fig. 3A, the δ values for priming doses of 0.25, 0.5, and 1.0 μg/mL were 0.202, 0.270, and 0.405, respectively.\u003cstrong\u003e B:\u003c/strong\u003e\u0026nbsp;The method was the same as in A, except that cells were pretreated with EMS at 12.5, 25, and 50 μg/mL and challenged with 500 μg/mL. The incidence of micronuclei in the control was 19/2,000 cells (0.95%). Applying the Yonezawa effect scheme (Fig. 1) to Fig. 3B, the δ values for priming doses of 12.5, 25, and 50 μg/mL were 0.141, 0.239, and 0.301, respectively.\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eAsterisks * and ** denote statistically significant differences between treatments and the control at P \u0026lt; 0.05 and P \u0026lt; 0.01, respectively.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5109349/v1/b176cc882890fb7769b5e0fa.jpg"},{"id":78627239,"identity":"127716f9-454c-4f5b-9d37-b6ec5920c7bf","added_by":"auto","created_at":"2025-03-17 02:37:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":73180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCross-challenge tests with AF-2 and MMC. \u003c/strong\u003e\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e L5178Y cells (2.5 x 10⁵ cells/well/0.5 mL) were treated with priming doses of AF-2 at concentrations of 0.25, 0.5, and 1.0 μg/mL. After 12 h, the cells were challenged with MMC at a concentration of 20 ng/mL. Cells transferred to an Eppendorf tube were centrifuged at 3,000 rpm for 30 seconds and then cultured in 500 μL of fresh medium. Specimens were prepared 18 h after washing. Two specimens were prepared per treatment, and 1,000 cells per specimen were examined. The incidence of micronuclei in the control group was 11/2,000 cells (0.55%). When applying the Yonezawa effect (Fig. 1) to the combination of AF-2 and MMC (Fig. 4A), the δ values for priming doses of 0.25, 0.5, and 1.0 μg/mL of AF-2 were 0.253, 0.269, and 0.284, respectively. \u003cstrong\u003eB:\u003c/strong\u003e The procedure was the same as in A, except that cells were pretreated with 0.25, 0.5, and 1.0 ng/mL of MMC and challenged with AF-2 at 20 μg/mL. The incidence of micronuclei in the control group was 9/2,000 cells (0.45%). The δ values for priming doses of 0.25, 0.5, and 1.0 ng/mL of MMC were 0.470, 0.514, and 0.647, respectively. Asterisks * and ** indicate statistically significant differences between treatments and the control, with P \u0026lt; 0.05 and P \u0026lt; 0.01, respectively.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5109349/v1/0f38c911e0f7ce791aa7c83f.jpg"},{"id":78627791,"identity":"bc25a40c-0d97-45f9-b800-22a262155008","added_by":"auto","created_at":"2025-03-17 02:53:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":829795,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5109349/v1/d849a607-d2b4-4601-9f41-67d58363d806.pdf"},{"id":78627240,"identity":"64629668-9161-42b6-8a6b-ad929458dd15","added_by":"auto","created_at":"2025-03-17 02:37:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":238826,"visible":true,"origin":"","legend":"","description":"","filename":"SRSupplematerials2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5109349/v1/4ff59c8eb5b5424dfb473f71.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eHormesis redefined: Insights from application of δ plot quantification of the Yonezawa effect to dose responses in the micronucleus test\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThroughout the approximately 4\u0026nbsp;billion years of evolutionary history, organisms have been continuously exposed to radiation. Radiation levels 4\u0026nbsp;billion years ago were nearly 10 times higher than they are today [1]. It has been suggested that radiation may have contributed to both chemical and biological evolution [2]. All organisms are radioactive to some extent because they continuously take in radioactive elements such as K-40 and C-14. In humans, internal radiation exposure alone amounts to approximately 9,000 Bq/sec, and when external exposure is included, it totals about 20,000 Bq/sec [3, 4]. The primary effect of low-dose radiation is its reaction with water to produce reactive oxygen species (ROS), which means that the biological effects of low-dose radiation are mediated through ROS as chemical agents. ROS act as signaling molecules, and the Keap1-Nrf2 pathway is capable of detecting them [5]. This pathway contributes to biological defense against oxidative stress derived from ROS and electrophilic foreign substances. The NF-κB/IκB system is also involved in ROS regulation but is broadly associated with inflammation, immunity, cell division, differentiation, development, and apoptosis [6]. Organisms that have evolved alongside radiation naturally exhibit genetic responses to it [7]. Luckey's comprehensive work, which cites 1,269 references, includes numerous examples of radiation hormesis across various life forms, including bacteria, protozoa, plants, and animals [8].\u003c/p\u003e \u003cp\u003eOn the other hand, it is estimated that we are exposed to tens of thousands of chemical substances. There are numerous examples of cells and organisms exhibiting hormesis in response to these chemicals, showing stimulation at low doses and inhibition at high doses. Southam and Ehrlich discovered that an extract from wood showed growth stimulation at low doses and growth inhibition at high doses for certain types of fungi, and they named this phenomenon \"hormesis\" [9]. Subsequently, Townsend and Luckey searched pharmaceutical literature and compiled a list of over 100 substances that showed biphasic responses with stimulation at low doses and inhibition at high doses [10]. Calabrese and Blain expanded on this work, documenting over 900 hormetic responses for radiation, radioactive substances, inorganic compounds, and organic compounds [11]. Based on these extensive studies, hormesis is now considered to be a ubiquitous phenomenon in the biological world.\u003c/p\u003e \u003cp\u003eOur collaborative study group within the Mammalian Mutagenicity Study Group (MMS) of the Japanese Environmental Mutagen and Genome Society (JEMS) investigated thresholds for mutagens. Using cell viability and proliferation as endpoints, we observed reverse U-shaped hormetic responses [12, 13]. However, when examining hormesis in micronucleus tests, typical J-shaped hormetic responses were difficult to obtain due to the low natural frequency of micronuclei (around 1%). To address this, we conducted challenge tests involving low-dose pre-treatment followed by high-dose post-treatment. We also performed cross-challenge tests using different substances for pre- and post-treatment. Both approaches demonstrated suppression of micronucleus induction by post-treatments. We submitted a manuscript claiming hormetic responses in micronucleus tests, which was published as a preprint [14]. However, the paper was rejected, with reviewers suggesting we were observing adaptive responses rather than hormesis. We subsequently revised the terminology from \"hormetic\" to \"adaptive\" and successfully published in a different journal [15]. Given our view that hormesis and adaptive responses are essentially equivalent, this distinction seemed arbitrary. We therefore reexamined raw micronucleus test data obtained in the previous study [15] using the δ plot, which quantifies the Yonezawa effect―a type of radiation adaptive response. We propose that other adaptive responses fitting the δ plot could also be classified as hormesis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eI. Rationale: the scheme of the Yonezawa Effect\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. 1 illustrates the schematic representation of the Yonezawa effect, also known as the priming dose effect [17, 18].\u003c/p\u003e\n\u003cp\u003eFig. 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eII. Difficulty in detecting hormesis with a typical biphasic, J-shaped dose response curve\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore conducting the micronucleus tests, dose-finding experiments are typically performed. Fig. 2 illustrates dose-finding tests with MMC (A) and EMS (B). Both compounds exhibited a slight reduction in micronuclei at low doses, suggesting a J-shaped dose-response curve, which is characteristic of hormesis. However, this reduction was not statistically significant, and hormesis could not be confirmed with certainty. Given that the background level of micronuclei is around 1%, detecting a further reduction due to a hormetic response proves challenging. Additionally, high-dose treatments with EMS led to the appearance of numerous pycnotic cells and cells with amorphous nuclei, especially at 2,000 \u0026mu;g/mL, complicating the examination of micronuclei. A similar result, suggesting a J-shaped dose-response curve, was obtained in a dose-finding test with colchicine (see Supplementary Fig. 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIII. Challenge tests with AF-2 and EMS\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDetecting hormetic responses directly in the micronucleus test, where the background incidence is low, proved challenging (Fig. 2). Therefore, challenge tests were conducted in which cells initially exposed to low doses (priming doses) insufficient to induce micronuclei were subsequently exposed to a high dose (challenging dose) capable of inducing micronuclei (Fig. 3). Both AF-2 (Fig. 3A) and EMS (Fig. 3B) demonstrated that micronucleus induction by the challenging dose was suppressed by the priming doses. It is noteworthy that higher priming doses resulted in greater suppression of micronuclei. Similar results were observed in a challenge test with MMC (see Supplementary Fig. 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIV. Cross-challenge tests with AF-2 and MMC\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition to the challenge tests (Fig. 3), cross-challenge tests were conducted using AF-2 and MMC, where cells were first primed with AF-2 and subsequently challenged with MMC (Fig. 4A). The results of these cross-challenge tests exhibited similar response patterns to those observed in the challenge tests (Fig. 3). Likewise, when cells were initially primed with MMC and then challenged with AF-2, comparable results were obtained (Fig. 4B). In both cases, higher priming doses led to greater suppression of micronuclei under the current experimental conditions. Similar results were observed in cross-challenge tests involving EMS and MMC (see Supplementary Fig. 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 4\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eYonezawa et al. demonstrated a radiation-induced adaptive response in mice, where an initial low-dose radiation (priming dose, D\u003csub\u003e1\u003c/sub\u003e in Fig. 1) reduced acute lethality from a subsequent high-dose radiation (challenging dose, D\u003csub\u003e2\u003c/sub\u003e in Fig. 1). The survival rates of mice that received both D\u003csub\u003e1\u003c/sub\u003e and D\u003csub\u003e2\u003c/sub\u003e were higher than those that received only D\u003csub\u003e2\u003c/sub\u003e. Key factors in the Yonezawa effect include D\u003csub\u003e1\u003c/sub\u003e, D\u003csub\u003e2\u003c/sub\u003e, the time interval between D\u003csub\u003e1\u003c/sub\u003e and D\u003csub\u003e2\u003c/sub\u003e, and the endpoint (mouse survival). The framework of the Yonezawa Effect, as shown in Fig. 1, can essentially be applied to the micronucleus test (Figs. 3 and 4). Therefore, the present micronucleus test can be considered a chemically induced adaptive response.\u003c/p\u003e\n\u003cp\u003eHowever, the micronucleus test described here differs from the mouse survival test in several key ways. (1) The appearance of micronuclei is time- and dose-dependent\u0026mdash;micronuclei tend to appear earlier with low-dose treatments, later with high-dose treatments, and disappear after a prolonged period. This necessitates determining the optimal expression time. The micronucleus test using suspended cells is particularly convenient, as samples can be collected consecutively (e.g., 18, 24, 30, and 36 h after treatment) from the same culture to estimate the optimal expression time. (2) The continuous presence of chemicals in cell cultures may affect both cell division and micronucleus formation, as demonstrated in the EMS experiment (Fig. 2B), especially at higher doses. Some chemicals, such as microtubule disruptors like colchicine, complicate the micronucleus test due to their specific effects on cell division. In such cases, the chemicals can be removed by washing, as shown in the MMC experiment (Fig. 2A). (3) When the toxicity of a test chemical reduces cell numbers, the cells can be concentrated through brief centrifugation, as described in the \u0026quot;Preparation of AO-Coated Slides for Supravital Staining\u0026quot; section in Materials and Methods. (4) Supravitally stained cells can be stored at -80\u0026deg;C for a month or longer until examination.\u003c/p\u003e\n\u003cp\u003eDose-response relationships in biological processes are often non-linear, and dose-response curves have been described as biphasic, bell-shaped, U-shaped, inverted U-shaped, J-shaped, diphasic, bimodal, bidirectional, sinusoidal, and more [20]. Over 50 researchers studying these diverse dose-response patterns have recommended adopting more consistent terminology. They proposed the common use of the term \u0026quot;hormesis,\u0026quot; including specific categories like physiological conditioning hormesis, radiation conditioning hormesis, and chemical conditioning hormesis when preconditioning is involved [21]. Later, Calabrese, the lead author of the referenced paper, argued that \u0026quot;Preconditioning is hormesis\u0026quot; [22, 23]. The \u0026quot;Yonezawa Effect\u0026quot; is an example of preconditioning, where a small preconditioning dose administered before a high (challenging) dose protects against the harmful effects of the latter, and this can be considered a hormetic response. Since the \u0026delta; plot shown in Fig. 1 is essentially applicable to the adaptive response observed in the micronucleus test, this test can also be considered a form of hormesis.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe \u0026delta; plot can also be effectively applied to cross-adaptive responses between radiation and chemicals [24, 25, 26]. These adaptive responses can similarly be referred to as hormesis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse lymphoma cells (L5178Y) were obtained from the JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition, Japan.\u0026nbsp;Cells were maintained in RPMI 1640 medium supplemented with penicillin and streptomycin (25 \u0026mu;g/mL) and enriched with 10% fetal calf serum. Cells were kept in a 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eincubator at 37\u0026deg;C. Cell counts were performed using a hemocytometer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemicals\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAcridine orange (AO, CAS: 494-38-2), Mitomycin C (MMC, CAS: 1950-07-7), and (Z)-2-(2-furyl)-3-(5-nitro-2-furyl) prop-2-enamide (AF-2, CAS: 3688-53-7) 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) was supplied by Sigma-Aldrich Co. LLC, St. Louis, MO. To administer chemicals to the cells, solutions were prepared by either directly dissolving them in the culture medium or creating a 10-fold concentrated chemical solution. The latter was then used to generate serial dilutions with a dilution factor of 2. Subsequently, the prepared solutions were added to the cell culture medium at a ratio of 1:10.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of AO-coated slides for supravital staining method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn 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 \u0026mu;L) of the solution was placed between two glass slides, which were then 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 \u0026mu;L) 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 pieces of paper tissue. In cases where cell numbers were expected to be low due to toxic treatments, a larger volume of the cell suspension (e.g., 100 \u0026mu;L) was transferred 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 \u0026mu;L). Code numbers were sealed with a \u0026quot;Post-it\u0026quot; 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate statistical differences, a table developed by Kastenbaum and Bowman was used [16]. For easy comparison, a computer-generated table was created, displaying significance levels of 0.01 and 0.05, P-values ranging from 0.01 to 0.99, along with the corresponding number of micronuclei from 1 to 1000.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eOn July 1, 2023, the Asia Research Awards, an organization headquartered in India, awarded SS the \u0026ldquo;International Distinguished Scientist Award\u0026rdquo; among scientists for his research on \u0026ldquo;biological effects of low-dose radiation.\u0026apos;\u0026apos; On October 1, 2023, the organization awarded the \u0026ldquo;Asia\u0026apos;s Outstanding Researcher Award\u0026rdquo; to SS among scientists, researchers, doctors, and professors for his work on \u0026quot;research on radiation hormesis.\u0026quot; As a year-round recipient, the organization awarded SS \u0026ldquo;the International Innovative Scientist of the Year Award\u0026rdquo; on March 16, 2024 for his research on \u0026quot;radiation and chemical hormesis.\u0026quot;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003eAlthough the micronucleus test data using L5178Y cells were collected by SS, these raw data were presented in a previous paper and are shared by all the authors of reference [15]. The original manuscript was prepared by SS, and all authors reviewed and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e: Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This work is not supported by third parties.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKaram, P.A. \u0026amp; Leslie, S.A. Calculations of background beta-gamma radiation dose through geologic time. \u003cem\u003eHealth Phys\u003c/em\u003e. 77, 662-667 (1999). \u003c/li\u003e\n\u003cli\u003eSutou, S. Low-dose radiation effects. \u003cem\u003eCurr. Opin. Toxicol\u003c/em\u003e. 2022;30:100329.\u003c/li\u003e\n\u003cli\u003eSutou, S. Hormetic effects of low-dose radiation in \u003cem\u003eLow dose radiation in health and disease \u003c/em\u003e(eds. Pandy, B.N. \u0026amp; Huilgol, N.G.) 1-16 (Narosa Publishing House, 2024).\u003c/li\u003e\n\u003cli\u003eScott, B.R. et al. Radiation-stimulated epigenetic reprogramming of adaptive-response genes in the lung: an evolutionary gift for mounting adaptive protection against lung cancer. \u003cem\u003eDose-Response\u003c/em\u003e 7, 104-131 (2009).\u003c/li\u003e\n\u003cli\u003eSuzuki, M., Otsuki, A., Keleku-Lukwete, N. \u0026amp; Yamamoto, M. Overview of redox regulation by Keap1\u0026ndash;Nrf2 system in toxicology and cancer. \u003cem\u003eCurr. Opin. Toxicol.\u003c/em\u003e 1, 29\u0026ndash;36 (2016).\u003c/li\u003e\n\u003cli\u003eMorgan, M.J. \u0026amp; Liu Z.G. Crosstalk of reactive oxygen species and NF-\u0026kappa;B signaling. Cell Res. 21, 103-1015 (2011). \u003c/li\u003e\n\u003cli\u003eJargin, S.V. Hormesis and radiation safety norms: Comments for an update. \u003cem\u003eHum. Exp. Toxicol\u003c/em\u003e. 37, 1233-1243 (2018).\u003c/li\u003e\n\u003cli\u003eLuckey, T.D. Hormesis with ionizing radiation. 1-222 (CRC Press, 2019). \u003c/li\u003e\n\u003cli\u003eSoutham, C.M. \u0026amp; Ehrlich, J. Effects of extract of western red-cedar heartwood on certain wood decaying fungi in culture.\u003cem\u003e Phytopathol.\u003c/em\u003e 33, 517\u0026ndash;524 (1943).\u003c/li\u003e\n\u003cli\u003eTownsend, J.F. \u0026amp; Luckey ,T.D. Hormoligosis in pharmacology. \u003cem\u003eJ. Amer. Med. Assoc\u003c/em\u003e. 173, 1960, 44-48 (1960).\u003c/li\u003e\n\u003cli\u003eCalabrese, E.J, \u0026amp; Blain ,R. 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Res. 5, 401-405 (2009).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Adaptive response, Challenging dose, Cross-adaptive response, Hormetic, Preconditioning, Priming dose","lastPublishedDoi":"10.21203/rs.3.rs-5109349/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5109349/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCells and organisms respond dynamically to environmental factors like radiation and chemicals. These responses vary based on detection systems, leading to terms such as adaptive response, biphasic response, and hormesis. In micronucleus tests using cultured cells, obtaining a typical J-shaped dose-response curve, a hallmark of hormesis, was challenging due to low background micronucleus frequency. We conducted challenge and cross-challenge tests. In challenge tests, cells were pre-treated with low priming doses and then post-treated with a high challenging dose. In cross-challenge tests, cells were pre-treated with one chemical at low doses and then post-treated with a high dose of another chemical. Both tests showed clear suppression of micronucleus induction by high doses following pre-treatments. Our paper reporting hormesis in the micronucleus test was initially rejected, with reviewers claiming we detected an adaptive response rather than hormesis. Believing these concepts to be equivalent, we re-analyzed our data using the δ plot, which quantifies the Yonezawa effect, a type of radiation adaptive response. The analysis showed our results fit effectively with the δ plot. Since the Yonezawa effect aligns with the definition of hormesis, our findings could be termed as such. Other adaptive responses fitting the δ plot could also be considered hormesis.\u003c/p\u003e","manuscriptTitle":"Hormesis redefined: Insights from application of δ plot quantification of the Yonezawa effect to dose responses in the micronucleus test","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-17 02:36:55","doi":"10.21203/rs.3.rs-5109349/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a3bde656-6dd7-47f4-8a14-e936f6592310","owner":[],"postedDate":"March 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45750882,"name":"Biological sciences/Genetics"},{"id":45750883,"name":"Earth and environmental sciences/Environmental sciences"},{"id":45750884,"name":"Health sciences/Health care"}],"tags":[],"updatedAt":"2025-03-17T02:36:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-17 02:36:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5109349","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5109349","identity":"rs-5109349","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}