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Perspectives on Hormesis and Implications for Pesticides Edward J. Calabrese* Department of Environmental Health Sciences, School of Public Health and Health Sciences, Morrill I, N344, University of Massachusetts, Amherst, Massachusetts 01003, United States *E-mail: [email protected]. Phone: 413-545-3164. Fax: 413-545-4692.

This paper addresses three areas relating to the topic of hormesis: (1) a retrospective reflection of how I came to be involved in hormesis research, (2) a summarization of developments in 13 areas of hormesis research and evaluation (i.e., terminology, relationship to homeopathy, objective criteria to assess hormesis, hormesis as a dose-time relationship, control group variation and hormesis detection, hormesis as manifested by biologically integrated endpoints, the frequency and quantitative features of hormesis, hormesis generality, beneficial and harmful effects, preconditioning and adaptive responses as manifestations of hormesis, dose-response model validity and epidemiology and hormesis) and (3) an evaluation of the hormesis data base for pesticides and how their dose response features compare with those of other chemical classes.

Introduction The concept of biphasic dose responses is now nearly 150 years old, having being first being observed by Hugo Schulz and his assistant in a crude laboratory in the early 1880s at the University of Greifswald in northern Germany. For Schulz this observation seemed as surprising as many modern accounts by researchers in numerous fields of biological research who likewise encountered biphasic dose responses when none were expected. As a result of his initial unexpected © 2017 American Chemical Society Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


observation, Schulz thought that it was an experimental artifact, the result of some error in experimental design, procedure or some other unknown aspect of the experiment. He therefore repeated the experiment with the same biphasic dose response occurring. Numerous other replications were undertaken with the same result. At some point in this process Schulz would come to the conclusion that what he and his assistant were observing was highly reproducible, that is, a real biological phenomenon, neither an error nor an unexpected chance observation due to normal variability. In an autobiographic remembrance during his 70th year Schulz (1923, translated by Crump) (1) would recount the experience of what has become a seminal observation in experimental biology, reflecting both the excitement of seeing a novel phenomenon and the uncertainty of whether what was observed was reliably reproducible. The reflections of Schulz (as translated by Crump (1)) are given in the quote below and reveal a common experience within the research community, that biphasic dose responses were unexpected and probably due to a mistake or simply chance/normal variability. Schulz Quote (1): “Since it could be foreseen that experiments on fermentation and putrescence in an institute of pathology would offer particularly good prospects for vigorous growth, I occupied myself as well as possible, in accordance with the state of our knowledge at the time, with this area. Sometimes, when working with substances that needed to be examined for their effectiveness in comparison to the inducers of yeast fermentation, initially working together with my assistant, Gottfried Hoffmann, I found in formic acid and also in other substances the marvelous occurrence that if I got below their indifference point, i.e., if, for example, I worked with less formic acid than was required in order to halt the appearance of its anti-fermentive property, that all at once the carbon dioxide production became distinctly higher than in the controls processed without the formic acid addition. I first thought, as is obvious, that here had been some kind of experimental or observation error. But the appearance of the overproduction continually repeated itself under the same conditions. First I did not know how to deal with it, and in any event at that time still did not realize that I had experimentally proved the first theorem of Arndt’s fundamental law of biology.”

Personal Perspective If one can fast forward from about 1883-1884 to 1966, some 82 years, the same general story was one that I experienced, as an undergraduate student, taking a laboratory and greenhouse based plant physiology course in which one of the many exercises was to assess the effects of a synthetic plant growth retardant (i.e. phosfon) on the growth of peppermint plants. The results of an initial experiment revealed that the phosfon appeared to stimulate plant growth, making the professor raise the question whether this observation was due to a mistake by his students or perhaps was something real and reproducible, with the later possibility being the least likely. I was the only student who took up the professor’s challenge to further explore this question. Nearly 40 experiments later, using soil and hydroponic media, multiple plant species and a range of other synthetic plant growth retardants 84 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


we came to the conclusion that phosfon could indeed stimulate the growth of the peppermint plant but that it did so in a manner that reflected a biphasic dose response relationship. During this period I was not aware that the phenomenon that we were studying and observing had a specific name (i.e., hormesis). We simply called the findings a low dose stimulation and a high dose inhibition (2–4). Like Schulz, doubt was a dominating concern for my professor as he was unrelenting in his demand that I replicate and strengthen the original experimental design and findings. In fact, when the series of peppermint-phosfon replications in soil were completed, it numbered about ten. In the case of the hydroponic experiments, the consistency of the biphasic dose response with the findings in soil was such that he required a few less replications. During this first experience as a developing scientist I did not fully appreciate the focus of my professor on the need to so extensively replicate the findings. An appreciation of this perspective within the context of low dose research came much later (1985) as my scientific journey would lead full circle back to where things all started, with a desire to understand better the biphasic dose response. Throughout the course of this research on low dose effects I became aware of concepts that would precondition me to better understand the hormesis phenomenon decades later that I did not understand this at that time. For example, I came to appreciate: the dominant role of control group variation in assessing low dose effects and how to evaluate its quantitative significance via statistical simulation exercises; sample size and statistical power concepts for study designs, especially in low dose experiments; dynamic changes in treatment responses over time with repeat measures; the value of large numbers of doses, properly spaced in the study designs; and the need for dose range studies to identify threshold concentrations to assist in dose spacing in larger and more powerful experiments. I also came to appreciate that replication was key to this process. I was learning why it took so long to be become a scientist. It was not all about classroom knowledge and laboratory techniques but also the development of subtle intuitive insights borne of many positive and negative experiments, all integrated to yield reliable evidence about specific scientific questions/hypotheses. In many respects, at least on the experimental level, it appeared that much had not changed from the time of Schulz’s autobiographical reflection to the mid-1960s, in terms of how to deal with the assessment of biphasic dose responses. That is, biphasic dose responses remained unexpected, problematic and little understood with respect to occurrence, generality, mechanism and biological, medical and evolutionary significance. On the positive side, as in Schulz’s era, we were sufficiently intrigued with the initial observations that it was worth follow up experimentation. Similar to Schulz, at some point in this process, both my professor and me came to the conclusion that we found something that was reproducible. This introduction to scientific research in the area of low dose effects was a bit overwhelming to this undergraduate student as I found it difficult to grasp the need to so substantially replicate findings that we had continuously observed. It was true that no experiment was exactly like the others, as they were done in a greenhouse at differing times of the year, that the nature of doses, the number of plants per dose and the dose ranges could vary by experiment, typically becoming progressively 85 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


more extensive and demanding as the scientific stakes got higher. Yet, I sensed unspoken doubt in the mind of my professor such that he was reluctant to submit our research to peer-review unless he was certain beyond any reasonable doubt that the low dose stimulation findings was the correct conclusion. My problem was not so much him and his demanding altitude but that I was the one doing the experiments and that I had been convinced much earlier. However, he was the one in charge and the replications and other experiments would have to be done, prior to our attempt to share the findings with the scientific community. At some point I graduated and decided that I simply could not pursue this type of research with its extreme demands for replication along with the apparent lack of confirming mechanism. It was quite a relief when my next step on the academic ladder found me working with a professor whose views on the need for replication were considerably different and the linkage to mechanism more realistic. His approach was once your preliminary experiments were completed, you designed an experiment with very strong statistical power, obtained your findings and then replicated the experiment. If these two studies were in agreement then it was time to move on to the next set of questions. The questions that I was pursuing at this time were not dose response per se but physiological, but still required strong confirmation of reliability. However, what appeared to be unrealistic demands for replication by my first advisor were replaced with a procedure that seemed more reasonable to me at the time. For the next two decades I had moved on to questions in the field of toxicology that involved research in a relatively high dose realm, addressing regulatory agency questions as to what might be a safe level of exposure to harmful agents. Thus, my concerns were toxicities and their mechanisms, not effects that might be occurring below traditional toxicological thresholds, that is, in what is now called the hormesis zone. My return to research biphasic responses was as unexpected as when first it occurred. This time I received a conference brochure about a Radiation Hormesis conference in Oakland, California in August, 1985. Upon reading the brochure I wondered whether my earlier work with peppermint and the growth inhibitor might be an example of the hormesis phenomenon described in the brochure. I called Dr. Leonard Sagan, the conference director, and told him my peppermint-phosfon story. He questioned me at great length about experimental design and the capacity for the findings to be replicated and how general I thought this response was in plants. He also asked me if I had observed bias against the publication of hormetic findings. While I expected to be questioned on the quality of the research I was surprised with his publication bias question. I told him that this manuscript was my first scientific paper that I was publishing on my own (i.e. without the assistance of an advisor) and as such I received substantial criticisms from reviewers and that the manuscript had been rejected by several journals. However, I persisted and kept trying to improve the manuscript and address specific criticisms. I therefore told him that I had not seen any bias with my paper and that the review process was helpful despite my several rejections. At the end of the conversation Sagan invited me to make a presentation at the conference on the topic of chemical hormesis. It was this conference that refocused my attention on hormesis, with a peerreviewed conference proceedings being published two year later in 1987 within Health Physics (5). However, another two years would pass until the next catalytic 86 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


moment which was a debate in the journal Science by Sagan (6) and Shelly Wolff (7), the radiation geneticist whose team discovered adaptive response in radiation in 1984 (8). This debate in Science would spark the creation of my long term focus on hormesis that has continued until the present. The status of hormesis in 1990 was still marginalized at best, with the same set of scientific issues and uncertainties hovering over it. The most visible current leadership in the area came from the writings of two people, Donald Luckey, who had published a detailed book on ionizing radiation and hormesis in 1980 (9), and the work of Tony Stebbing, a marine toxicologist at the Plymouth Research Station in the UK, who was focused principally on chemical examples of hormesis since the mid to late 1970s. In fact, as told to me in a conversation with S. Hattori of the Japanese Electric Power Research Institute, it was the book by Luckey that inspired him to contact Leonard Sagan, leading to the creation of the 1985 conference on Radiation Hormesis. In the cases of Luckey and Stebbing, both laboratory oriented scientists, it was also unsuspected laboratory observations similar to Schulz’s experience that would activate their scientific curiosities to follow up their own unexpected low dose stimulatory responses. For Luckey this would occur in the 1940s while with Stebbing it was some 30 years later. While Luckey would make progress in his own laboratory research on hormesis for the next three decades, he made several efforts to summarize some aspects of the hormesis story in the mid-1970s, although none of these (10, 11) were able to provide a conceptual break through. However, his book entitled “Ionizing Radiation and Hormesis” in 1980 did finally get widespread attention on hormesis, and affecting a swirl of follow up activities. While an assessment of the 1987 conference proceedings in Health Physics reveals a useful complement to the individual activities of Luckey and Stebbing, a careful review of these proceedings indicates that the knowledge of hormesis that exists today strikingly exposes how limited the field of knowledge on hormesis was at that time. Consistent with this view is how limited the interest in hormesis was within the research community during this earlier era. For example, there were only 5 citations (median) in the Web of Science data base per year in the 1980s for the terms hormesis or hormetic whereas by 2015 this number had increased to more than 7,300 (Figure 1). During the decade of the 1980s, therefore, the concept of hormesis was neither well recognized nor broadly accepted. It was not incorporated into major textbooks in pharmacology, toxicology or risk assessment, suggesting that the efforts of Luckey and Stebbing to develop integrative and detailed assimilations of the hormesis literature were limited in their impact. Of interest is that the key 1982 article of Stebbing (12) on hormesis, which now has over 500 citations in the Web of Science, averaged about 11 citations per year in its first decade since publication. However, over the past decade this article has been cited over 300 times with two years over 50 citations each year. This remarkable reactivation of interest in the Stebbing paper is due to the major initiatives taken on hormesis since the late 1990s. A similar reactivation of interest occurred for the 1943 article of Southam and Ehrlich (13) which provided the term hormesis. While it was only cited 20 times from 1943 to 1987, over the past 30 years it has been cited 183 times. Figure 1 provides the citation frequency by year in the Web of 87 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Science. Thus, the field of hormesis has shown a strong growth since the 1985 conference, but a close look at Figure 1 reveals that this progressive increase started in the late 1990s, with the onset of consistent research funding and with the widespread use of in vitro experimentation which facilitated the assessment of a larger number of concentrations that could be efficiently evaluated.

Figure 1. The number of citations in the Web of Science data base using the term ‘hormesis or hormetic’.

Hormesis: Scientific and Related Developments The following section summarizes developments in some key areas over the past several decades relating to the topic of hormesis. This section is intended to provide both an historical and a biological-based contextual framework to evaluate hormesis as a biological concept and dose-response model. Terminology The study of biphasic dose responses has been made much more difficult because of the use of a wide range of terms to describe what often appears to be biologically similar dose response relationships. This is seen in the use of the Arndt-Schulz Law, Hueppe’s Rule, hormesis, U-shaped dose responses, inverted U-shaped dose responses, J-shaped dose responses, biphasic dose responses, diphasic dose responses, non-monotonic shaped dose responses, bitonic dose responses, low dose stimulation, rebound effects, Yerkes-Dodson Law, hormolegosis, repeat bout effect, and others. The use of many terms for what appears to be the same general concept can be problematic, creating discipline-specific terms further truncating the hormesis concept, affecting the capacity to evaluate its generality. In addition, other concepts, such as adaptive response in radiation and preconditioning in the biological and biomedical research domains, are manifestations of the hormesis concept based on their 88 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

dose-response characteristics (14, 15) and yet are usually not discussed within the hormetic dose response context. While there has been significant progress toward developing an integrated conceptual framework and language/terminologies (16), this represents an area where considerable progress needs to be made.


Differentiating Hormesis from Homeopathy The principal reason why hormesis has been linked very closely with the medical practice of homeopathy is because when Hugo Schulz made the discovery of the hormetic concept he quickly made it known that he believed that he had discovered the explanatory principle of homeopathy (17–19). This made Schulz the object of considerable criticism from leaders within traditional medicine, especially leaders in the area pharmacology, such as Alfred J. Clark, a leading professor at Edinburgh and author of several highly influential text books which included significant criticism of Schulz (20–22). In fact, the principal reason why the concept of hormesis had such a difficult time getting established was, in part, due to the intense criticism directed toward it by leaders in the traditional medicine community, individuals who tended to strongly influence government funding of research, textbook development and have major leadership roles within society. Given the extreme historical antipathies between traditional medicine and homeopathy, Schulz should have presented the hormesis concept solely as a biological hypothesis rather than linking it to a controversial medical practice within the context of a social and economic dispute. If he had done so, it is likely that traditional medicine would have been welcoming toward the hormesis concept rather than being hostile, effectively blocking its capacity to become integrated into the field of medicine for the remainder of the 19th century and essentially for the entire 20th century as well. Development of Objective Criteria To Assess Hormesis The question of how does one know when there is hormesis can be quite complex and draped in uncertainty. There is not an exact test for hormesis as there are for specific cellular processes or specific types of damage or disease syndromes. This is because hormesis is not a specific cellular process but dose response phenomenon that depends on the integration of complex biological processes and is limited with respect to the magnitude of response. One can make judgments about the presence of hormesis based on experiments that are properly designed and with adequate statistical power. Such judgements also the need to adequately be tested via replication of findings, all within the context of having sufficient knowledge of control group variation. It is also of considerable value, when making judgements on hormetic dose responses, to clarify essential aspects of mechanistic pathways that mediate the hormetic dose response, including identifying receptors and cell signaling pathways and confirming the presence of various types of molecular switches that affect hormetic dose response expression. Thus, multiple factors contribute to the development of objective criteria for the evaluation of whether a biphasic dose response may be considered hormetic or not. 89 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Recognition That Hormesis Is a Dose-Time-Relationship Hormesis based experimentation can be challenging since this dose response relationship involves both dose and time considerations. This insight became apparent in my peppermint research as I was treating the plants with up to ten doses of phosfon and following their growth on a weekly basis for up to two months. Thus, my experiments were dose-time relationships. Of importance in this respect was that I learned that during the initial two weeks of my peppermint/phosfon experiments that the phosfon typically suppressed the growth of the plants in a dose dependent manner. It was only in the later weeks that I observed accelerated recovery growth at the lower concentrations as a type of overcompensation stimulation (23). I would eventually learn that this was central to the mechanistic thinking of Tony Stebbing (12) and by multiple researchers as far back as the late 1890s and early decades of the 20th century (24–29). Ironically, many scientists refused to recognize overcompensation as being biologically significant because it did not involve a so-called direct stimulation. Such attitudes and judgments profoundly diminished the acceptance of the hormesis concept in the first half of the 20th century especially in the area of radiation hormesis. As research progressed and became better integrated it was found that hormetic stimulations could involve either a direct or overcompensation, with each type of stimulatory response displaying the same quantitative features of the dose response (30). Control Group Variation Since hormetic responses are modest, being usually at most only 30-60% greater than the control group, one has to be very knowledgeable about control group variability. It is especially important to have a good understanding of the historical control group variability, not simply the control group values of the specific experiment. Computer simulation exercises can be very useful since they offer the possibility to estimate how often hormetic-like effects may occur due entirely to assumed variation, and not be a real treatment effect. Not only does control group variation have the capacity to lead to false positive hormetic responses but this process may also work in the reverse, leading to an underestimate in hormetic responses. As a general rule, use of control groups with low variability would reduce the likelihood of both false positive and false negative findings for hormesis. Integrated Endpoints Since hormesis is an adaptive response, these responses are those that can contribute to health and survival. These are seen with key integrative endpoints such as growth, maintenance functions, disease incidence, tissue repair, fecundity, memory and cognition enhancement, reproductive proclivities, survival and lifespan. In each of these types of integrative endpoints there are multiple pathways, interactive processes and mechanisms that result in the selected endpoint response. The assessment of hormesis therefore reveals that hormetic endpoints are the result of complex and highly integrated biological processes. 90 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Attempts to evaluate hormesis outside of biological context can be problematic and misleading. For example, the assessment of cell free systems for possible hormetic dose responses may yield examples of apparent hormetic-like biphasic dose responses. However, such responses should not be confused with being actual hormetic endpoints as understood within an evolutionary/adaptation based context. The use of in vitro experimentation as noted above, created vast opportunities to evaluate hormesis dose response hypotheses since it became efficient to assess large numbers of doses/concentrations in an inexpensive manner as compared to in vivo experiments. In fact, the tracking of hormetic dose responses over time within the hormesis data base (31) reveals that more than 60% of the hormetic dose responses reported since the mid 1990’s have been via in vitro experimentation. Despite such substantial progress on hormesis hypotheses using in vitro methods, it is important to realize that hormetic endpoints are typically those that reflect biologically integrative responses and occur via the interactions of biological processes across multiple biological systems/organs. The Limits of Epidemiology The use of epidemiology in the assessment of hormesis is affected by its own limits. Epidemiology is significantly influenced by numerous uncertainties that affect its capacity to detect low risks and benefits. These limits are borne from difficulties in obtaining appropriate and accurate information about study subjects. Such limits of epidemiology have long been known and have been incorporated into legal systems and toxic tort evaluations. For example, in the U.S. epidemiological studies showing an odds ratio less than 2.0 cannot be used to establish causality. This judgment is significant within the framework of an hormetic dose response since most maximum hormetic dose responses are less than double the control group value. It is for this reason that research findings that have been incorporated into the various hormesis data bases are experimental (31), rather than epidemiological, in nature. While this general view should not be interpreted to mean that epidemiological evidence is excluded in the evaluation of hormetic dose responses, it does reflect the need for caution in such judgments. It would also suggest that efforts should be made to undertake meta-analyses in order to overcome some of the limits of epidemiology, especially when addressing exposures in the low dose zone. Frequency of Hormesis In 2003 Calabrese and Baldwin (32) published an estimate of the frequency of hormesis in the toxicological and pharmacological literature based on a review of the entire set of papers published in three journals from their inception in the mid-1960s to the present. The total number of articles screened exceeded 21,000. Each paper was subjected to rigorous a priori entry and evaluative criteria. The results of that process yielded an estimate of 37% for hormesis frequency. This was the first attempt to derive such an insight, revealing that when experiments are designed with an adequate number of doses, proper dose spacing and appropriate 91 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


sample size for adequate statistical power that hormetic effects are commonly observed. However, it was felt that this estimate may have been falsely low since most studies using multiple doses often measure responses at only one time point whereas the hormetic response could result from an overcompensation to a disruption on homeostasis as well as via a direct stimulation. That is, since hormesis is a dose time response, it is likely that hormetic effects are under reported to some extent due to such experimental design limitations. Furthermore, the evaluative criteria used in the Calabrese and Baldwin (32) study were very rigorous. If these criteria were slightly relaxed, the hormetic frequency estimate would have been significantly higher. The findings of this study were especially important because the studies were very broadly based in terms of biological models and endpoints measured, suggesting that hormesis had a relatively high frequency with very broad capacity for generalization. Follow up studies with more restrictive biological models (e.g. 12 strains of yeast) revealed significantly higher frequency of hormetic responses (33). While these studies have been valuable in providing insights on the frequency of hormesis, it is possible that frequency estimates may vary somewhat by biological model and endpoint. Quantitative Features of Hormesis The most unique characteristic of the hormetic dose response is that the stimulatory response is modest, with 80% or more of the cases in the Hormesis Data Base showing a maximum response that is less than twice the control group value (31, 34, 35). In fact, the maximum response is typically only in the 30-60% zone above the control group. This is the case regardless of biological model, endpoint, inducing agent, mechanism or whether the hormesis phenomenon is a direct stimulation or overcompensation response. This observation was unexpected and suggested that the hormesis stimulation may describe the maximum biological gain in the system and define the limits of biological plasticity (36, 37). If this were the case, the hormetic dose response would be of considerable biological and evolutionary significance, being a fundamental survival strategy, managing and regulating biological resources, as well as mediating adaptive and reparative processes. Generality of Hormesis When I became refocused on hormesis there was little thought given to how general this concept was. The focus was on establishing reliable evaluative criteria by which dose responses could be assessed with respect to whether or not specific dose responses reflected likely hormetic dose responses. Over time thousands of such dose responses were identified and entered into the Hormesis Data Base. Once analyses were conducted it became clear that hormetic dose responses were very widespread, being reported in all major classes of plants, microorganisms and animals. This was a striking discovery since it suggested that hormesis was evolutionarily-based and long preserved. Evidence continues to be generated on hormetic dose responses on phylogenetically diverse biological models. Such hormetic dose responses also are observed throughout the lifecycle and within 92 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

males and females, making the hormetic dose response highly generalizable. In addition to evaluating the generality of hormesis via its phylogenetic framework, hormetic dose responses are also very generalizable with respect to specific cell types and organs, and is seen across all levels of biological organization. Thus, by essentially all biological standards the concept of hormesis should be considered to be evolutionary based and highly generalizable.


Beneficial and Harmful Effects The hormesis trait was selected for early during the course of evolution. While this is generally accepted, it is also the case that what is beneficial for one individual may be harmful for another and must be understood with its biological context. There are also two basic types of hormetic dose responses, one that reflects a direct stimulatory response and the other an overcompensation response following a disruption in homeostasis or the occurrence of low to modest toxicity. Both cases of hormesis have been selected for, even though they are fundamentally different biological phenomena. What integrates them under the same classification is that the quantitative features of their respective dose responses are similar. Nonetheless, they represent two different types of hormetic dose responses. When hormetic dose responses occur for cancer cells (i.e., enhancing tumor cell proliferation), humans may readily ascribe a harmful/undesirable effect to that type of hormesis. This may also be the case if this were applied to harmful bacteria or to weed growth or to the effects of endocrine disruption agents or to various types of auto-immune responses. In past review articles on major topic areas such as tumor cells (38) and immune responses (39) such harmful effects have been well documented. Despite the obvious possibility for hormesis mediated adverse effects, considerable attention has been directed to its capacity to induce beneficial outcomes such as disease incidence reduction, enhanced longevity, neuroprotection, enhanced crop productivity, enhanced memory, and proconditioning to reduce a plethora of adverse health effects amongst many other beneficial effects. The key with hormesis is that it is important to understand when it can be beneficial and when it can be harmful and when such hormetic effects are likely not to be particularly practically significant. Preconditioning and Adaptive Response Are Hormesis A prior low dose of a stressor often can protect against a subsequent toxic dose of the same or similar agent. When such studies include sufficient conditioning doses analyses of the dose response typically reveal an hormetic-like biphasic dose response, with the same quantitative features of the hormetic dose response (14, 15). Based on an extensive assessment of this area of research it was proposed that both preconditioning and adaptive responses to chemicals or radiation should be considered as manifestations of the hormetic dose response. This conclusion has important implications as it suggests that the magnitude of the protective effects as seen within the Hormesis Data Base also apply to the preconditioning and adaptive response phenomena. 93 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Model Validation and Lack Thereof The initial decades of the 20th century saw the rejection of the hormetic dose response model by the biomedical community and the acceptance of the threshold dose response model (17, 18). By the 1930s efforts by some leaders in the radiation genetics community challenged the capacity of the threshold dose response to account for the capacity of ionizing radiation to account for the occurrence of germ cell mutation at low doses (40). Within two decades governmental agencies had become persuaded to this perspective and adopted the linear dose response model for radiation induced genetic damage, and soon generalized it to radiation induced cancer. Two decades later the U.S. EPA would generalize this position further, applying the dose response findings with ionizing radiation to chemically induced cancer. However, in this process the scientific, medical and regulatory communities failed to validate or even attempt to validate the capacity of the threshold dose response. They simply assumed it to be accurate. In fact, the entire 20th century would pass with no one in these respective research/regulatory communities attempting to evaluate the capacity of the threshold dose response model to make accurate predictions in the low dose zone. However, with the re-emergence of the hormetic dose response concept in the later decades of the 20th century the question was raised as to how to evaluate the capacity of this model to make correct predictions in the low dose zone. It was thought that one could learn from how the scientific, medical and regulatory communities must have validated the threshold model and then make some accommodations and applications when evaluating the hormetic model. However, after extensive searching no evidence was found of an attempt to validate the threshold dose response model. That is, the entire regulatory program of governments that used the threshold dose response as its default dose-response assumption had never been validated to make accurate predictions in the zone where people live, that is, the low dose zone. Recognition of this failure to validate regulatory agencies dose response model resulted in a series of studies to compare the capacity of the threshold, linear and hormetic models to make accurate predictions in the low dose zone. Using three independently derived large data bases, including a 57,000 dose response public data base of the U.S. NCI, and employing rigorous a prior entry and evaluative criteria, it was found that only the hormetic dose response was able to make accurate predictions in the low dose zone (32, 33, 41–43). The findings were striking, challenging actions of regulatory agencies worldwide in their use of the threshold and linear models. These studies also raised the key question as to why the hormetic dose response model was never given serious consideration throughout the entire 20th century. Despite the serious multiple failings of the regulatory agency models to make accurate predictions in the low dose zone, these models continue to control and dictate the regulatory approach for assessing chemicals and radioactivity.

94 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Hormetic Data Base and Pesticides The hormetic dose response data base has > 9,000 dose responses, from the broad range of biological and biomedical literature. Of this total, 597 were classified as pesticidial agents. Of the 597 dose responses 60.1% of those were derived from in vivo studies. Approximately 80% of the hormetic dose responses have at least two doses below the estimated threshold or zero equivalent point (ZEP) (Figure 2). About 30% of the dose responses for in vivo (29.5%) and in vitro (33.1%) studies had four or more doses below the ZEP (Figure 2). The maximum stimulatory responses for inverted U-shaped dose responses were generally modest with 82% for both in vivo/in vitro studies having a stimulatory response less than twice the control group values (Figure 3). The width of the stimulatory range was less than 100-fold below the ZEP for 87.5% (in vivo) and 75.3% (in vitro) of the studies (Figure 4). These overall results are consistent with findings reported in the Hormetic Data Base for a broad range of chemical agents, demonstrating the broad generality of the hormesis dose-response characteristics and that pesticide responses, are consistent with the dose response pattern of all other chemical classes as well as ionizing radiation.

Figure 2. Percent in vivo and in vitro experiments with pesticides for the number of doses below the zero equivalent point (ZEP) (each total added to 100%). 95 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 3. Percent of experiments in the Hormesis Data Base by maximum stimulatory response relative to the control group of pesticide studies.

Figure 4. Percent of experiments in the Hormesis Data Base by width (dosage) of stimulatory range of pesticides studies. 96 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Discussion Hormesis is a fundamental biological concept that represents how cells, organs and organisms adapt to exogenous and endogenous stressor agents and how it is mediated in dose-response patterns. The hormetic dose response integrates both evolutionary “strategies” of adaptation and biological resource allocation “tactics” to achieve biological performance goals that enhance survival. The past three decades of research in this area have revealed that the biological and biomedical communities overlooked this fundamental concept by which adaptive responses are mediated due to an unusual clustering of circumstances involving historical conflicts between homeopathy and traditional medicine that affected the capacity to objectively evaluate biphasic dose responses, the use of only a few very high doses for hazard assessment, the failure of regulatory communities to validate dose-response models (e.g., threshold) in regulation and the unique quantitative features of the hormetic dose response that requires rigorous study designs, heightened statistical power for proper evaluation, and a commitment to replicate findings. This clustering condition led to the failure to recognize a key biological principle having profound implications for medicine, agriculture, pharmaceutics, environmental risk assessment and numerous other areas dependent on dose-response relationships. That hormesis would be widely reported for pesticides, therefore, would not be surprising. While the past has witnessed the adoption of research strategies that have ignored or even denied the possibility of the hormetic dose response, there is now convincing evidence that hormesis needs to become an integral component in the education of biomedical scientists but also part of their functional experimental strategies and tactics that can lead to improved identification of biological responses in the low dose zone for both risk assessment and response efficacy purposes. Failure to integrate the hormesis concept within the hazard and risk assessment process has been a serious and long-term failure of the scientific and regulatory communities, compromising environmental and public health goals and resulting in invalid cost/benefit analyses that result in flawed governmental policies as well as poor decisions by individuals on personal health matters.

Acknowledgments Research activities in the area of dose response have been funded by the United States Air Force and ExxonMobil Foundation over a number of years. However, such funding support has not been used for the present manuscript. The views and conclusions contained herein are those of the author and should not be interpreted as necessarily representing policies or endorsement, either expressed or implied. Sponsors had no involvement in study design, collection, analysis, interpretation, writing and decision to submit.


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