A Molecular Basic for Understanding the Benefits from Subharmful Doses of Toxicants

An Experimental Approach to the Concept of Hormesis and the Homeopathic Similia Law R. van Wijk, H. Ooms, F.A.C. Wiegant, J.E.M. Souren, J.H. Ovelgönne, J.M. van Aken and A.W.J.M. Bol Reprinted from Environmental Management and Health, Vol. 5, No. 1, 1994 pp. 13 15 with authorization from MCB University Press. 0956 6163 from Biomedical Therapy / Vol. XV / No. 1, 1997 This work has been supported by a grant from the Homint organization Introduction and General Outline Cells and organisms are continually exposed to physical and chemical influences that threaten their functioning and survival. Ionizing radiation can cause fragmentation of molecules into ions or radicals. Heat exposure can lead to denaturation of proteins, whereas high concentrations of toxic chemicals interact with proteins blocking receptor sites or enzyme reactions. A critical question is how much exposure and under which circumstances is harmful. In toxicology, Hart and Turrurro [1] have described the paradigm which relates dosing history of noxious agents to transient and permanent effects on cells and tissues. Operating within this framework, risk assessment strategies tend to relate the distribution of the time, until occurrence of the event, to the dose. The problem of risk assessment is thereby reduced to that of extrapolating the response from a known dose rate. An example of the presentation of a toxic or harmful effect (which refers to environmentally induced injury) is the well known "survival" curve (Figure 1). This curve can be applied to every harmful influence.

Figure 1. Hypothetical survival curve. Increasing time of application of a toxic condition results in decreased survival of cells. In contrast to harmful effects of toxic doses, beneficial effects such as stress-induced proliferation are observed when cells are exposed to low doses of a toxicant like a moderate non-lethal heat shock. A net increase in cell number occurs, and injury is manifested only at more severe exposures. Such proliferation was observed after conditions in which the cell population had previously become resistant to higher toxic doses. This stimulating effect of low doses suggests that cells are sensitized and have developed a response to counteract the possible toxic effect. Furthermore, data suggest that cells become committed to develop this stress response after a short time of exposure. Development continues even in the absence of the toxic condition. The autonomous development of this response, leading to tolerance and proliferation stimulation can be traced back to early changes in oncogene and stress protein gene expression. There is now a considerable body of evidence, which indicates that when intact organisms or cells are exposed to low levels of toxicants (ionizing radiation, pesticides, organic solvents, etc.), beneficial changes are induced and paralleled by a change in gene expression, as part of a general cellular adaptation syndrome. In toxicological literature, the term "hormesis" has been applied to various forms of "stimulation" produced by low doses of toxic substances. In biochemical and biological literature, the studies on stress-induced cellular changes are valuable to provide a molecular basis of the hormesis concept. It has been proposed that organisms and individual cells would react the most strongly to low doses in suboptimal conditions [2], but evidence is lacking. This evidence is of primary importance for any theory concerning the therapeutic effect of a low dose application after an intoxication or other injury. The latter line of thinking is followed in the similia law of homeopathy. Very few investigators have made the link between the three fields of research: hormesis, molecular stress biology, and the similia law of homeopathy. This article presents an overview of reports from these three fields without going too far in detail. Most information concerns heat shock as the harmful event, but some reports on other toxicants are included as well. After a "historical review," the article describes the present state of research, which focuses on the evaluation of the similia principle. Hormesis The hormesis concept originated in the nineteenth century, arising predominantly from the work of Hugo Schulz and his colleagues [3 7]. They investigated the effects of chemicals on yeast fermentation. In an article on the theory of drug action [3], Schulz proposed that, depending on the magnitude of a stimulus, it could either enhance or diminish physiological capacity. In 1888, Schulz published the now classic paper entitled "Ueber Hefegifte," in which he demonstrated that various toxic chemicals (mercuric chloride, iodine, bromine, arsenious acid, chromic acid, salicylic acid, and formic acid) could stimulate respiration and growth in yeast [4]. As Rudolph Arndt arrived at similar conclusions, this paradoxical mode of drug action became known as the Arndt-Schulz Law: "Weak stimuli accelerate vital activity, medium ones promote it, strong ones inhibit it, and very strong ones snuff it out." [6] Both Arndt and Schulz asserted that the paradoxical stimulation-depression phenomenon was "a fundamental characteristic of the excitability of protoplasm." [6 8] Unaware of the work on the stimulation of yeast growth, Southam and Ehrlich [9] reported in 1943 that hot-water-soluble extracts of western red cedar heartwood (possibly phenolic compounds) had a twofold effect on the growth of fungi. At higher concentrations, the extract was either fungistatic or fungicidal, whereas at low concentration, enhanced fungal growth was observed for some species. The authors speculated that the phenomenon of accelerated growth represented "an initial response, followed by progressive desensitization to subinhibitory concentrations of a toxic constituent of the extract." They proposed the term "hormesis" to designate stimulator (growth) effects induced by subinhibitory concentrations of toxic substances on organisms. Based on another series of investigations, Luckey [1] proposed the name "hormoligosis" to characterize compounds that were stimulatory at low doses, somewhat harmful at modest (therapeutic) levels, and toxic at high levels. These studies demonstrated that birds, including germ-free birds, grew more rapidly when their diets were supplemented by low doses of antibiotics [10, 11, 12]. By 1959, the term "hormesis" was defined as stimulatory action of subinhibitory amounts of a toxin and the term "hormoligosis" as stimulation by small amounts of any agent on living organisms [13]. Luckey and co-workers cited literature demonstrating that the hormoligosis concept was a generalized phenomenon producing a broad array of responses with a variety of compounds (for example, heavy metals, ionizing radiation, chemicals, head, cold, light) throughout the plant and animal kingdom. According to Luckey [13], "the stimulatory action must be explained as the protoplasmic response to an unfulfilled stress warning." Hormesis Can Occur in Every Organism At present, the hormesis concept has been applied to viability, growth, and development; cancer reduction; resistance to disease or infection; wound healing; radiation resistance; resistance to heat, respiration and enzyme induction [14, 15]. The most common form of the hormetic curve, demonstrating a stimulator action of subinhibitory amounts of toxic compounds, the  curve shown in Figure 2.

Figure 2. Hypothetical  curve. This type of curve shows a hormetic effect, i.e., a stimulatory action of subinhibitory amounts of toxic compounds.

An explanation of the concept should deal with the following question: Is hormesis a consequence of a response, which is common to all organisms, and which can be provoked by different toxicants, or is hormesis due to specific properties of the agents that cause enhanced growth? As mentioned earlier, the effects of a number of agents on a single organism strongly suggest that hormesis can occur in every organism as a result of exposure to quite different kinds of inhibitory agents. Stebbing [14, 16] suggested that, because hormesis has been induced with a plethora of toxicants across taxa, any unifying theory of hormesis must relate the phenomenon to control mechanisms that operate at the cellular level and are common to all forms of life. In other words, and evolutionary conserved mechanism. An effort to explore such a mechanism should take into account studies of stress-induced compensatory cell proliferation and tolerance mechanisms, including the synthesis and release of stress proteins. From these disciplines, it is apparent that living organisms respond to unfavorable conditions at the cellular level by a rapidly and transiently increased expression of a small number of genes. A General Cellular Adaptation Syndrome The Heat Shock or Stress Response Although heat shock has been known for more than 50 years to alter programs of gene expression in ontogeny [17], the current interest in the heat shock response has grown gradually since Ritossa's demonstration is 1962 that heat shock induced a new chromosome puffing pattern in Drosophila [18]. Initially, experiments concentrated on heat shock as a model of gene regulation. However, subsequent studies in the early 1970s associated synthesis of specific proteins with changes in the puffing pattern [19]. The heat shock proteins were initially characterized by gel electrophoresis, and their nomenclature is largely based on their relative molecular weight. The major heat shock proteins have been classified into four proteins families: heat shock proteins with molecular weight of . 97 112 . 84 90 (hsp90 family) . 68 70 (hsp70 family) . 20 28 kilodaltons (hsp 20 228 kilodaltons (hsp20 family) (for review, see [20 27]). The hsps synthesized in these major molecular weight groupings have various numbers of major and minor isomers that may be phosphorylated or methylated. In addition, closely related sets of polypeptides are sometimes synthesized, whereby synthesis of other related proteins is usually constitutive and enhanced only slightly or in non-inducible. An example of heat-induced synthesis of heat shock proteins in rat hepatoma cells is shown in Figure 3.

Inductance of heat shock protein synthesis in Reuber rat Hepasome (H35) cells. Com¬pari¬son of the polypeptide composition of control cells (1) with heat shocked cells (2). Sam¬ples were taken 4 hours after a heat shock at 42.5oC during 30 minutes (a=action). Figure 3. Interaction of Heat Shock Proteins

Inducers of the Response Besides heat shock, the induction of the stress response can be achieved by addition of sodium arsenite, ethanol, sulfhydryl reagents, hydrogen peroxide, heavy metals, amino acid analogs, 2.4 dinitrophenol, sodium azide, etc. (for reviews see [23, 28 30]). The common links between these various treatments, if any, have not been established beyond the assumption that all these experimental conditions lead to microenvironmental changes that cells or tissues recognize as conditions requiring a change in the pattern of gene expression (this will be elaborated later on). Since these treatments cause injury at high doses, they are all assumed to stress the cells. The induced sets of proteins are generally termed "stress proteins." A Universal and Highly Conserved Response The stress response has been observed in all examined organisms encompassing species of bacteria, yeasts, plants, invertebrates, and vertebrates, the latter including humans. There are only a few exceptions such as during early embryonic development [31, 32]. (For review, see [33].) Although there are small differences among the various organisms, a remarkable similarity has remained in very distant species throughout evolution. Comparison of the sequences of the respective heat shock genes from bacteria, yeasts, plants, Drosophila, vertebrates and man, as well as the use of antibodies to test similarities of stricture between heat shock proteins from a number of distance species, have indicated that the heat shock proteins are among the most highly conserved proteins in nature. The most highly conserved hsp are those in the 68 70 kd weight class [24 36]. The Beneficial Effect of the Stress Response Tolerance Development and Stress-induces Proliferation When cells or organisms are exposed to sufficiently severe stress conditions, the majority dies as expected. If, however, prior to this lethal stress condition, they undergo a milder treatment, a considerable proportion of them survive. Thermo-tolerance was first described by radiation biologists characterizing the hyperthermic biology of cultured mammalian cells [37 39]. They observed that a less lethal head treatment could lead to the development of substantial resistance to a subsequent heating, which would normally be lethal. This phenomenon has been termed "induced thermal resistance," "thermo-tolerance," and "thermo-tolerance." Nowadays, the term "thermo-tolerance" is commonly accepted. Thermo-tolerance is a common phenomenon that appears to occur in nearly all cells. An example of the induction of thermo-tolerance is shown in Figure 4.

A. In this hypothetical graph, the kinetics of induction of thermo-tolerance is shown. Cells were then returned to 37o during the time indicated on the abcissas and subsequently given a uniform second, lethal treatment. B. Survival curves at 42oC determined at various intervals at 37oC after heat conditioning at 42oC for 30 minutes. The interaction intervals are indicated in hours. Curve at 0 hr represents continuous heat treatment at 42oC (Data from Schamnart, et. al [91]. Figure 4: Induction of thermo-tolerance

Other mild-stress conditions can also lead to development of tolerance against a severe treatment with the same stressor. For example, a mild arsenite treatment results in a tolerance to the lethal effects of higher arsenite concentrations [40, 41]. In some cases, it has been reported that mild treatment with one stressor leads to tolerance towards a severe treatment with another stressor. Examples are the thermo-tolerance induced by arsenite, cadmium, etc. [42 44]. Severe heat shock can kill cells and cause a delay in cell cycle progression of surviving cells. However, the effect of heat treatment on cell cycle progression is temperature dependent and opposite effects have also been observed. In heat treated GI phase neuroblastoma N2A cells, synchronized by mitotic shake off at (non-lethal) temperatures of 41.3o and 41.9oC, an increase in the rate at which cells enter a second cell cycle is observed; while at higher temperatures, there is a mitotic delay [45]. We have further demonstrated that heat shock stimulated proliferation of previously quiescent cells and also enhanced stimulation of progression from the GI phase to the S phase of the cell cycle by epidermal growth factor (EGF) in 3T3 cells [46]. Similar stimulatory effects of heat shock have been observed with respect to prostaglandin F2a induced proliferation (Figure 5).

A. Effect of different lengths of incubation at 45.5oC. on stimulation of DNA synthesis with EFG and/or PGFa (EGF [4 ng/mi] []. PGF2A (30 ng/mi) (), EGF - PGF2a ( ). DNA synthesis was determined 24 hrs after stimulation and expressed as a percentage of labeled nuclei. B. The effect of heat treatment (20 min 43.7oC) on the fraction of cells that remain unlabeled after addition of EGF (4 ng/mi) to confluent quiescent Swiss 3T3 cells. EFG added to contract cells (0), EGF added to hyperthermia-treated cells (o). No EGF was added to contract cells () and hyperthermia-treated cells (). Heat shock was 3 hrs prior to EGF addition (data from Van Wijk et. al. [46, 47]. Figure 5: Effect of heat treatment on stimulation of DNA synthesis

These heat shock conditions leading to stimulation of proliferation can be characterized as mild treatments (20 mins. 42.0 44.0oC) and are non-lethal. A comparison of the duration and temperature of the heat shock treatments that lead to potentiation of EGF induced DNA synthesis and the treatment required for the induction of thermo-tolerance showed that both effects were induced by similar conditions [47]. In Figure 6, it is shown that the time dependence of heat shock (43.7oC) on EGF induced DNA synthesis (Figure 6c) and induction of thermo-tolerance (Figure 6b) shows a close resemblance.

A. The effect of temperature in a 20 min pre-incubation period on the percentage of quiescent Swiss 3T3 cells which are heat-resistant after a 30 min 46oC treatment as determined by the percentage of proliferating cells. The 46oC treatment follows 3 hrs after pre-incubation. B. The effect of time of pre-incubation at 43.7oC on the percentage of proliferating cells when followed by an interval of 3 hrs at 37oC before heat treatment at 46oC for 30 min was given. C. The effect of treatment at 43.7oC during various times on the percentage of labeled nuclei (o) and percentage of cells which were resistant for heat-induced cell loss from the maneuver () as determined at 24 hrs after addition of EGF (4 ng/mi) to quiescent Swiss 3T3 cells and at 27 hrs following heat treatment. (Data from Van Wijk e. al. [47]) Figure 6. Temperature and time dependency of thermo-tolerance induction and of heat-induced growth stimulation.

Although thermo-tolerance was also induced following more severe heat shocks, such treatment leads to a net inhibition of proliferation and to cell death. Similar data on stress-induced enhancement of proliferation have recently been published for chemical stress conditions. Mild treatments with arsenite [48], cadmium [49], and ischemia [50] were shown to induce cells to proliferate. Molecular Mechanisms of Beneficial Stress Response In attempting to unravel the phenomenon of stress-induced proliferation, several investigators observed that levels of some oncogene m RNAs were increased following stress. In recent years, the kinetics of induction of cell cycle regulated oncogenes has been well investigated [51]. Proto-oncogene transcripts are also among the (few) very labile mRNAs stimulated by serum growth factors. Each proto-oncogene has its own characteristic induction kinetics. Generally, increased c-fos mRNA levels precede increase of c myc mRNA levels, c ras mRNA levels increase after the increase of c myc mRNA levels, etc. [52]. Different treatments can activate this same set of genes, always showing the same sequence of events, but not necessarily the same kinetics. The transient induction of this gene is clearly not a secondary response to transit through the cell cycle. Several arguments suggest that c fos serves a role in coupling mitogens to long-term responses, leading to cell proliferation. Thus, the production of c fos antisense RNA inhibits 3T3 cell proliferation [53] and blocks renewed growth of quiescent 3T3 cells [54]. Furthermore, affinity-purified anti c fos antibodies injected in fibroblasts block serum stimulated DNA synthesis [55]. Recently, it was shown that a heat shock which induces the stress response increased c fos mRNA levels [56 61]. In Figure 7, it is shown that heat shock both induces an enhanced level of c fos mRNA as well as of HSP 84 mRNA.

mRNA levels of quiescent C3H1OT ½ cells of hso84 abd c-fos after a 30-nubyte heat shock at 42oC. Total RNA was isolated at different times after heat shock. Graph shows mRNA levels relative to GAPOH mRNA levels. Arrow shows the time period of the heat shock. ns = heat shock  = c fos,  = reso84. **** Autoracdiografic RNA was isolated before 910 or at different times after the end of heat shock: 0 min (2): 15 mins (3): 30 mins (4): 60 mins (5): 2 hrs (6). (With kind permission of M. Tuyi) Figure 7. Effect of Heat Shock on hsp84 and c-fos mRNA

Other inducers of the stress response were also shown to cause a significant elevation of c fos mRNA in HeLa cells. Thus, sodium arsenite triggered the induction of c-fox mRNA, and this stimulation was also observed following incubation with heavy metals (an equal mixture of cadmium chloride and zinc chloride [56]). These results are in favor of the hypothesis that stressors trigger c-fos expression leading to increased cell cycling and proliferation. But is it clear that verification of this hypothesis calls for additional study. Stress proteins may be involved in other growth control mechanisms. An example is the association between hsp70 and the p53 cellular phosphoprotein. The latter protein is believed to be a competence factor for DNA synthesis [62]. The potential role of stress proteins in hormesis is further suggested by studies that show that they are involved in tolerance and that they enhance survival. Several investigators observed that the increased synthesis of the major molecular weight class stress proteins roughly paralleled the development of thermo-tolerance. Direct evidence that heat shock proteins enhance survival during and after stress was recently obtained by Raibowol et. al. [63]. Affinity-purified antibodies to the hsp 70 family of heat shock proteins (the constitutive 73 kd and inducible 72 kd protein) were microinjected into rat embryo fibroblasts. The injected cells were then unable to translocate hsp 70 proteins into the nucleus after a mild beat shock pretreatment, and were unable to survive a subsequent severe (30 min 45oC) heat shock treatment. Fibroblasts injected with control antibodies survived the heat shock. There is, however, conflicting evidence on the importance of the induced synthesis of hsp families in the development of thermo-tolerance. This evidence suggests that pathways not critically dependent on hsp synthesis can also be elicited as primary or alternative mechanisms to protect cells. In fact, it suggests more than one way to induce tolerance, depending on the severity of the stressor dose [64 66]. The previous sections are intended to demonstrate some representative bodies of knowledge concerning the early subcellular changes after stressor application. Furthermore, examples are given to demonstrate that short exposure time can lead to growth stimulation and development of a tolerant cell population. In fact, these data strongly indicate a factual basis for the concept of hormesis, and that its occurrence in organisms of many taxa, as a result of exposure to different kinds of typically toxic substances, has a common explanation. For this explanation, attention must be paid to the general cellular stress response, including the very early change in chromatin structure which results in the enhanced expression of a number of conservative genes for proliferation and toxic resistance. Although many additional studies are needed for a better understanding of this response, one special question needs to be answered. This question deals with the nature of a common initial trigger for the change in chromatin structure and functioning. Biophysical as well as biochemical studies provide some information. An interesting set of data is derived from biophoton measurements of cells and organisms. Photon emission and/or the interaction with photons is supposed to give information on the state of the system. In short, this line of thought describes the living state in terms of chaos and order. An ideal chaotic system is a thermal equilibrium system, while the alternative, namely a well-ordered state, is represented by optical coherency. Evidence for the existence of a well-ordered state is derived from different experimental and theoretical approaches: . Bohm [67] and Precht et al. [68] examined the temperature response of photon emission. they observed an overshoot reaction, which in general is typical for temperature-dependent physiological processes. . Mandoli and Briggs [69], Popp [70-], and van Wijk and van Aken [71] measured transparency of disperse media as well as cell layers. They observed non-linear optical absorbance which is an argument for a high degree of coherence [72, 73]. . Non-stationary photon statistics were examined by Popp [74]. If a complete chaotic field is excited, it decays according to exponential relaxation dynamics. Excitation of coherent field results in a coherent decay, as observed in several systems [71 75]. One must further note that chaotic chemiluminescence does not react very sensitively to weak external influences, while a fully coherent field reacts to perturbation with a high sensitivity. Experimental results [76 78], as well as theoretical considerations [79 82], point to biopolymers, in particular, to exciplexes (ground states) of DNA, as the essential source of a coherent field within living tissue. Following this line of thought, there would be metabolic "feeding" of excited states, either by proton transfer or chemical "pumping." This corresponds to photon storage by the formation of excited exciplexes. All toxicants can be assumed to influence this process leading to changes in the exciplex state, which is far from equilibrium and subject to coherent vibronic and/or soliton interactions [80, 83]. In parallel, biochemical studies on protein denaturation, protein aggregation, and protein-DNA structures (reviewed recently [84]) under mild and severe stress conditions have demonstrated the variety of molecular changes during the period of critical transition of DNA and activation of a certain set of promoters. Hormesis and the Homeopathic Similia Principle: One Identical or Two Separate Concepts? In the previous paragraphs, we discussed hormesis as a general biological type of regulation by potential toxicants, distinct from their harmful effects at high doses. Some interesting new questions can now be asked. Does the positive response occur only after application of a low dose to an unstressed organism? Or does it also occur after a primary challenge that has been overwhelming and has resulted in injury? In other words, is the survivor of a high dose exposure more sensitive to a low dose in terms of its beneficial stress response? In this latter case, a beneficial effect is expected by a low dose application. At present, it is known that, after the application of a stressor, the cells show stress gene expression and cells build up tolerance to this stressor for a certain period of time. A low (sublethal) dose range of the stressor might influence the building up of tolerance in a dose-dependent way. Under these sublethal conditions, organisms or cells already show the beneficial stress response, although not optimal. Then the question can be asked as to whether they are more sensitive to low doses, and whether the low doses increase the beneficial stress response, as, for instance, in the stimulation of proliferation, development of tolerance, and activation of gene expression. A profound insight into the effect of a low dose after injury by a high does is of primary importance for proposing any therapeutic use of low dose application. Nevertheless, such a therapeutically application of low doses is proposed in homeopathic and isopathic medicine. In the next paragraphs, we will summarize the possible links between the concepts of hormesis and homeopathy. Finally, we will summarize the experimental approaches to this field. Therapeutic Application of the Low Dose Concepts: The Homeopathic Similia Principle as a Step-down Protocol Early doctrines of medicine were based upon the application of small or minute doses of drugs to cure a variety of diseases. The similia similibus curentur doctrine of Hippocrates suggested the application of drugs that gave reactions similar to disease symptoms. Hippocrates noted that, although Heilebore caused cholera-like diarrhea at high doses, it successfully treated cholera at low doses. Cantharidin tincture, used to treat cystitis, caused cystitis and hematuria at higher doses. Homeopathy, as established in 1810 by Samuel Hahnemann, has as its first premise a revival of the Hippocratic doctrine "like is healed by like." Homeo¬pathic practice rests on the proposition that disturbances (diseases) must be corrected by minute doses of remedies, which have the poser at higher doses to produce effects closely resembling the symptoms of the disease being treated (homeopathy) or by minute doses of remedies that actually caused the disease (isopathy). In other words, that which causes disease at high doses cures disease at low doses. Homeopathic and isopathic medicine is practiced in many countries. It has been severely criticized on the basis that unreal dilutions are used. Recently, however, studies of the meta-analysis type have demonstrated beneficial therapeutic effects of homeopathic remedies [85 87]. Since the primary concept is "like being healed by like" and not the magnitude of the applied dose, of which the only requirement is probably that it is not toxic, this concept needs further study at the organismal as well as at the cellular level. Luckey [2] stated that a major difference between homeopathy and hormesis is that the former adheres to special drugs for each syndrome, while the latter suggests that many agents could accomplish the same result. In fact, this point leads to the following questions: If a low-dose effect of a certain compound is found on a system in a pathological state, does this imply that the compound is related to the toxicant responsible for the pathological state, as predicted in isopathy (same substance) and homeopathy (different compounds inducing comparable toxicity symptoms)? Or could the same effect be achieved with a low dose of an arbitrary other compound? Low-dose Application During Tolerance Development after an Acute High Dose Treatment The earliest studies on mammalian cells which demonstrated that heat induces a transient state of heat resistance were those of Henie and Leeper [88] and Gerner and Schneider [89]. As shown by van Rijn et al. [90 ] and Schamhart et al. [91], thermo-tolerance can be induced in Reuber H35 cells by a short heat treatment (5 15 mins at 43oC), followed by incubation at 37oC. Resistance was demonstrated by comparing the survival curve after a second heating with the same curve after the first showing that many more cells survive a second heating than the first. Examination of the kinetics of induction and decay of tolerance showed that the cells were maximally sensitive immediately after heating. An interval of about 4 8 hours at 37oC was required before maximum thermo-tolerance manifested itself. Resistance persisted for at least 24 hours. Below approximately 42oC, tolerance can also be induced during continuous heating. Partially on the basis of the type of data discussed in the previous paragraph, Li and Hahn [92] proposed a phenomenological model of thermo-tolerance. Their basic hypothesis was that the acquisition of resistance involves at least two distinct steps: . an initial signal ("trigger"), and the . subsequent processing of the initial step ("development") Survival is further modified by the slow disappearance of thermo-tolerance ("decay") in the continuous absence of stress. At temperatures below approximately 43oC, the three processes proved on simultaneously. At higher temperatures, development and (very likely) decay of thermo-tolerance dies not take place. Development can only occur if the cells are returned to their normal growth temperature. A diagram of the model of Li and Hahn is shown in Figure 8.

Thermo-tolerance develops in two steps: a triggering event converts normal cells to the triggered state with a rate constant k1. These triggered cells are subsequently converted to thermotolerant cells with a rate constant k2. Below about 42.5oC, this occurs almost immediately (k2 = k1), whereas above 42.5oC (k2 0), the triggered cells remain sensitive. They can only become tolerant on transfer to 37oC. Finally, thermo-tolerance cells recover to their normal sensitive states at a slow rate governed by k3. (After Li and Hann [92]). Figure 8. Model of Thermo-tolerance.

The rate constant k 1 governs the conversion by heat of untreated sensitive cells to cells in an activated form (triggered cells). These cells are, however still heat sensitive. Conversion to resistant cells occurs only under conditions where the highly temperature-sensitive rate constant k 2 takes on a non-zero value. The decay constant k 3 is also temperature sensitive. Its magnitude varies with the temperature in a manner similar to that of many enzymatic processes. For example, below approximately 25oC, k 3 = 0; i.e., thermo-tolerance decays very slowly. According to this model, it is of interest to study the effect of low-dose application during the development of tolerance. In fact, the extensive experience with hyperthermia has resulted in many data of animals and cells evaluating the relative efficiency of various fractionation protocols, including that of the so-called step-down heating in terms of survival. For example, Heine and Leeper [88] have shown that incubation at 40oC was non-traumatic, and that cells grew normally up with doubling time similar to that at 37oC. There was no evidence of cell killing at 40oC. When incubated at 37oC, cells heat treated at 45oC developed thermo-tolerance to subsequent heat treatment. Cells exposed to 40oC incubation prior to hyperthermia (10 or 20 min at 45oC) also developed thermo-tolerance. However, when hyperthermia at 45oC was followed by incubation at 40oC, survival was reduced. Joshi and Jung [93] reported that hyperthermic temperature as low as 38 or 39oC modify the response of cells to elevated temperature such that pre-treatment induces thermo-tolerance whereas post-treatment enhances the response. Henle [94] studied the interaction of 45oC damage with damage at 41.5oC. He measured by fractionation of 10 minutes at 45oC and of 4 hours at 41.5oC. Survival increased exponentially with fractionation intervals up to two hours and then slowed to reach the additional level by four hours. A fractionation interval of eight hours at 37oC resulted in more than additive survival, which suggests significant development of thermo-tolerance. In their recent study, Lindegaard and Overgaard [95] mentioned 20 studies on the effect of step-down heating as observed in cultured cells in vitro, in several experimental tumors and normal tissues in vivo [94 113]. According to Lindegaard and Overgaard [95], an effect of step-down heating has been observed in all in vitro and in vivo studies where a high-to-low temperature sequence has been applied. The step-down heating response should therefore be regarded as a general biological phenomenon. A number of investigations in vitro [94, 98, 105, 109] have demonstrated that the step-down is influenced both by the time and the temperature of the initial high temperature sensitizing treatment. A reduction of the sensitizing temperature decreased the degree of thermo-sensitization being developed. The operational model of Li and Hahn [92] has been proposed to enlighten the nature of the step-down phenomenon. The explanation provided by the model for thermo-resistance (in previously unheated cells) below 43oC is that k 1, k 2, and k 3 all have non-zero values during exposure of the cells at these mildly elevated temperatures, so that thermo-tolerance continuously develops and decays. This process requires protein synthesis. The heat shock at the higher temperature, however, temporarily stops protein synthesis and thereby reduces k 2 to zero. Until the rate constant recovers to control values, the cells remain sensitive. heat-shocked cells at 40oC did not rapidly recover the ability to synthesize proteins: k 2 remained zero and the cells remained sensitive. Furthermore, the model predicts that once cells have been made thermo-tolerant, they are no longer subject to an increased rate of killing by step-down heating. This, too, has been verified. The model is, however, obviously oversimplified. Several reports indicate that the step-down response is a complex process that involves more than just the inhibition of thermo-tolerance [95, 98, 99, 101, 108, 109, 113, 114]. Jung [99] has advocated an alternative model where the cell killing by heat is described by a two-step production of non-lethal lesions and conversion of these lesions into lethal ones. The step-down effect is then explained by a differential time-temperature relationship for the production and the conversion rate. Thus, because the conversion rate changes much less with temperature than the production rate, non-lethal lesions produced during the high-temperature sensitizing treatment can readily become lethal during the following low-temperature treatment. According to Lindegaard and Overgaard [95], the basic heat-killing mechanism for both step-down heating and single-heating involves a two-step process, and the thermo-tolerance acts on top of these basic mechanisms, thereby reducing the effect of single heating at low temperature and step-down heating induced with low sensitizing temperatures. In view of all these studies, which definitively demonstrate a sensitizing effect following severe stressor application, the attention can now be focused on any sensitizing effect of mild, non-lethal stress treatments. In particular, the question can be asked whether, after mild (non-lethal) stressor application, a sensitizing effect is observed leading to an increased beneficial stress response. Recently, a few investigations have demonstrated that sensitization to heat is not limited to lethal conditions, but should be regarded as a more general biological phenomenon, influencing, among others, development of tolerance and the expression of stress genes. After mild pre-treatment conditions, the expression of stress genes is increased by subsequent incubation at 40oC, although incubation at 40oC alone had no effect [115]. The development of tolerance is also enhanced when mild step-down heating is applied [115, 116]. Concluding Remarks In this article, a survey is given of the studies concerning low dose effects. The effects of low dose of toxicants have been studied in the field of hormesis, the cell biology of the stress response (tolerance and step-down studies), and the therapeutic application of homeopathy and isopathy. These disciplines are concerned both with the induction of a beneficial effect by a low dose which can counteract the toxic or lethal effects when the harmful principle is present for a longer period of time. The system counteracts by (a) the acquirement of a state of resistance or tolerance, and b increasing the proliferative fraction of the cell population by stimulating the G0 G1 S transition. At the molecular level, such events can be partially explained by the recent findings with respect to stress-induced alterations of gene expression which lead to increased levels of c-fos mRNA and stress protein mRNAs. These mRNAs code for proteins whose function in cell proliferation and tolerance development is now, at least partially, solved. Most of the studies presented in this survey deal with heat shock as the toxic principle. Although many identical observations have been made at the cellular and molecular levels using other, especially chemical, toxins, further studies are necessary to establish the generality of the presented model. The early events in the process by which many toxic chemicals induce well-defined changes in DNA are still to be considered as a black box. We discussed the model of a coherent photon field as a possible explanation, but it should be noted that experimental studies, carefully designed using the appropriate culturing conditions, still have to be carried out. The statement that low doses of toxicants have beneficial effects is one of the major principles of homeopathy. Homeopathic practice rests on the presumption that bodily disturbances (diseases) must be corrected by low doses of remedies, as higher doses produce effects closely resembling the symptoms of the disease being treated. 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