Hormesis and Cellular Quality Control: A Possible Explanation for the Molecular Mechanisms that Underlie the Benefits of Mild Stress.

In contrast to the detrimental action of severe stress conditions, the beneficial effects of mild stress, known as hormesis, is increasingly discussed and studied. A variety of applications for hormesis in risk assessment processes, anti-ageing strategies and clinical therapies have been proposed. The molecular mechanisms underlying the phenomenon of hormesis, however, are not yet fully understood. A possible mechanism that has been proposed for hormesis, the homoeostasis overshoot hypothesis, assumes that an overshoot of repair- and self-recovery mechanisms in response to mild damage can be held responsible for the beneficial effects of hormesis. The present paper proposes 'cellular quality control' as a further explanation of the molecular mechanisms underlying the benefits observed after exposure to mild stress. The most important quality control mechanisms are outlined and their known and hypothesised actions in hormesis are discussed. As an example, different aspects of protein quality control will be described in more detail, which includes the reaction of the cell upon stress-induced protein damage and -aggregation. The regulation of Heat Shock Proteins and components from the ubiquitin proteasome system as part of cellular quality control is described in relation to its beneficial role in hormesis.

Postconditioning hormesis put in perspective: an overview of experimental and clinical studies.

A beneficial effect of applying mild stress to cells or organisms, that were initially exposed to a high dose of stress, has been referred to as 'postconditioning hormesis'. The initial high dose of stress activates intrinsic self-recovery mechanisms. Modulation of these endogenous adaptation strategies by administration of a subsequent low dose of stress can confer effects that are beneficial to the biological system. Owing to its potentially therapeutic applications, postconditioning hormesis is subject to research in various scientific disciplines. This paper presents an overview of the dynamics of postconditioning hormesis and illustrates this phenomenon with a number of examples in experimental and clinical research.

Postconditioning hormesis and the homeopathic Similia principle: molecular aspects.

Postexposure conditioning, as a part of hormesis, involves the application of a low dose of stress following exposure to a severe stress condition. Depending on whether the low-dose stress is of the same type of stress or is different from the initial high-dose stress causing the diseased state, postconditioning can be classified as homologous or heterologous, respectively. In clinical homeopathy, the same distinction is found between isopathic and homeopathic application of low-dose substances. Homeopathy is unique for its Similia principle, which implies that substances causing symptoms in healthy biological systems can be used to treat similar symptoms in diseased biological systems. The evaluation of the Similia principle in an experimental set-up requires the analysis of a complex sequence of 'damage-disease-treatment-effect' events. The process of recovery from an insult is then monitored and a possible beneficial effect on this recovery process, upon application of a range of substances in low dose, can subsequently be analyzed using molecular and functional parameters. It is then possible to compare the effect of treatment with the degree of similarity between the diseased state and the effects caused by homologous and/or different heterologous substances. Beneficial effects of postconditioning mild stress conditions have been described in terms of an increase of the synthesis of stress proteins. In this commentary paper, we present additional information on this aspect. The experimental data suggest that the beneficial effect of the low-dose stress condition used as heterologous postconditioning is related to similarity in molecular stress response.

The influence of adaptogens on ultraweak biophoton emission: a pilot-experiment.

In the present study, the effect of plant adaptogens (Rhodiola rosea and ADAPT-232) on human photon emission has been determined. In a randomized double blind placebo-controlled study, 30 subjects were randomly assigned to three groups: one group (n = 10) taking placebo pills, one group (n = 10) taking Rhodiola rosea (SHR-5) pills and one group (n = 10) taking ADAPT-232 supplements (the latter being a fixed combination of the following three adaptogens: Eleutherococcus senticosus, Rhodiola rosea and Schisandra chinensis). All subjects underwent measurements to determine ultra-weak photon emission (UPE) of the dorsal side of their hands using a photon-counting device, both before and after a week of taking the supplements. In addition, the experienced levels of stress and fatigue (tiredness) were evaluated. After 1 week of supplementation, the Rhodiola group showed a significant decrease (p = 0.027) in photon emission in comparison with the placebo group. Furthermore, after supplementation, a significant decrease (p = 0.049) concerning the experienced level of fatigue in the Rhodiola group was observed compared with the placebo group. No significant changes were observed between the ADAPT-232 and the placebo group.

Plant adaptogens increase lifespan and stress resistance in C. elegans.

Extracts of plant adaptogens such as Eleutherococcus senticosus (or Acanthopanax senticosus) and Rhodiola rosea can increase stress resistance in several model systems. We now show that both extracts also increase the mean lifespan of the nematode C. elegans in a dose-dependent way. In at least four independent experiments, 250 microg/ml Eleutherococcus (SHE-3) and 10-25 microg/ml Rhodiola (SHR-5) significantly increased life span between 10 and 20% (P < 0.001), increased the maximum lifespan with 2-3 days and postponed the moment when the first individuals in a population die, suggesting a modulation of the ageing process. With higher concentrations, less effect was observed, whereas at the highest concentrations tested (2500 microg/ml Eleutherococcus and 250 microg/ml Rhodiola) a lifespan shortening effect was observed of 15-25% (P < 0.001). Both adaptogen extracts were also able to increase stress resistance in C. elegans: against a relatively short heat shock (35 degrees C during 3 h) as well as chronic heat treatment at 26 degrees C. An increase against chronic oxidative stress conditions was observed in mev-1 mutants, and during exposure of the wild type nematode to paraquat (10 mM) or UV stress, be it less efficiently. Concerning the mode of action: both adaptogens induce translocation of the DAF-16 transcription factor from the cytoplasm into the nucleus, suggesting a reprogramming of transcriptional activities favoring the synthesis of proteins involved in stress resistance (such as the chaperone HSP-16) and longevity. Based on these observations, it is suggested that adaptogens are experienced as mild stressors at the lifespan-enhancing concentrations and thereby induce increased stress resistance and a longer lifespan.

Histamine dilutions modulate basophil activation.

BACKGROUND: In order to demonstrate that high dilutions of histamine are able to inhibit basophil activation in a reproducible fashion, several techniques were used in different research laboratories. OBJECTIVE: The aim of the study was to investigate the action of histamine dilutions on basophil activation. METHODS: Basophil activation was assessed by alcian blue staining, measurement of histamine release and CD63 expression. Study 1 used a blinded multi-centre approach in 4 centres. Study 2, related to the confirmation of the multi-centre study by flow cytometry, was performed independently in 3 laboratories. Study 3 examined the histamine release (one laboratory) and the activity of H(2) receptor antagonists and structural analogues (two laboratories). RESULTS: High dilutions of histamine (10(-30)-10(-38) M) influence the activation of human basophils measured by alcian blue staining. The degree of inhibition depends on the initial level of anti-IgE induced stimulation, with the greatest inhibitory effects seen at lower levels of stimulation. This multicentre study was confirmed in the three laboratories by using flow cytometry and in one laboratory by histamine release. Inhibition of CD63 expression by histamine high dilutions was reversed by cimetidine (effect observed in two laboratories) and not by ranitidine (one laboratory). Histidine tested in parallel with histamine showed no activity on this model. CONCLUSIONS: In 3 different types of experiment, it has been shown that high dilutions of histamine may indeed exert an effect on basophil activity. This activity observed by staining basophils with alcian blue was confirmed by flow cytometry. Inhibition by histamine was reversed by anti-H2 and was not observed with histidine these results being in favour of the specificity of this effect We are however unable to explain our findings and are reporting them to encourage others to investigate this phenomenon.

Phyto-adaptogens protect against environmental stress-induced death of embryos from the freshwater snail Lymnaea stagnalis.

The main purpose of the studies presented in this paper is twofold: 1) to evaluate whether phyto-adaptogens (Acanthopanax senticosus and Rhodiola rosea) are able to exert a protective action against stress-induced death of embryos of the pond snail Lymnaea stagnalis; and 2) whether a possible protective action by phyto-adaptogens can be explained by the induction of heat shock proteins. Enhancement in resistance by phyto-adaptogens was studied by applying plant extracts for a period of 20 hours to 3-day old larvae of the pond snail Lymnaea stagnalis. Subsequently they were exposed to a high and toxic dose of different environmental stressors. The following stress conditions were selected: a physical stress condition (heat shock: 43 degrees C for 4 minutes), an oxidative stress condition (superoxide radicals induced by menadione (600 microM for 2 hours)) and heavy metal-induced stress (copper (150 microM for 1 hour) or cadmium (20 microM during 1 hour)). Both Acanthopanax and Rhodiola exert a strong protective action against a lethal heat shock. These adaptogens also significantly protect against the negative effect of superoxide radicals as induced by menadione. With respect to the protective action against exposure to heavy metals a small but significant protection was observed against intoxication with copper or cadmium by the phyto-adaptogens. In summary, there appears to be a difference in efficiency in enhancing resistance to the various stress conditions used (heat shock>menadione>copper>cadmium). Based on the results presented in this paper, we can conclude that phyto-adaptogens are able to enhance the resistance against the different stress conditions tested in developing individuals of Lymnaea. Although the degree to which resistance is enhanced appears to depend on the type of stressor applied, our results confirm the definition of phyto-adaptogens as being universal enhancers of non-specific resistance against different kinds of stress conditions. With respect to the mechanism of enhanced resistance, the question was asked whether this protective action is caused by an induction of heat shock proteins (hsps), which are known to be involved in tolerance and adaptation. The phyto-adaptogens did not induce the synthesis of any of the hsps, nor did they modulate the normal heat shock induced synthesis of these stress proteins. We conclude that it is unlikely that hsps play a major role in obtaining an enhanced state of resistance provided by phyto-adaptogens.

Heat shock response by cells treated with azetidine-2-carboxylic acid.

The purpose of this study is to reinvestigate the heat shock response in cells treated with the antimetabolite azetidine-2-carboxylic acid (azetidine), an analogue of proline. Previous studies could not clearly discriminate between the progressive thermosensitization caused by amino acid analogues and a parallel induction of thermotolerance by heat shock. Incubation of H35 cells with 2.5 mm azetidine causes an increasing thermosensitization which achieves a maximum after approximately 18-22 h. At this point, azetidine does not prevent the development of acute thermotolerance following a heat shock at 42.5 degrees C, or the simultaneous induction of chronic thermotolerance during mild hyperthermia at 38-41 degrees C. However, for both the acute and chronic heating conditions thermotolerance levels are reduced in proportion with azetidine-thermosensitization. Incorporation of azetidine causes an apparent downward temperature shift of approximately 1 degree C relative to the time-temperature relationships for normal, or following heat shocks, for thermotolerant cells. After 18 h of incubation with azetidine, protein synthesis is reduced by a factor of 4 and cells show a preferential synthesis of heat shock proteins (hsp). A heat shock then, although inducing thermotolerance, is not followed by any noticeable effect on the synthesis of hsps. It is shown that the combination of prolonged azetidine treatment and heat shock causes a persistent inhibition of protein synthesis. This is hypothesized to result in the development of hsp synthesis independent thermotolerance. Additional treatment following heat shock in azetidine-treated cells with the protein synthesis inhibitor cycloheximide does not affect the induction of thermotolerance. In contrast to the heat shock response, no thermotolerance induction is observed in azetidine-treated cells after an exposure to sodium arsenite.

The effect of temperature and protein synthesis on the renaturation of firefly luciferase in intact H9c2 cells.

A mild increase in temperature that does not exert an effect on tolerance development or synthesis of heat shock proteins (Hsps) in control cells can stimulate these processes when applied to cells that have previously been heat shocked. To study the underlying mechanism of this effect, H9c2 cells were stably transfected with the gene encoding firefly luciferase (Luc). Heat-shock-induced inactivation of Luc and its subsequent reactivation is frequently used as a model for cellular protein denaturation and renaturation. Luc reactivation was determined following a damaging heat shock (43 or 44 degrees C for 30 min) in cells that were subsequently exposed to either control temperatures (37 degrees C) or various mild hyperthermic conditions (from 38.5 to 41.5 degrees C for 1 h). To prevent changes in Luc activity consequent to new synthesis of Luc, Luc reactivation was monitored in the presence of cycloheximide, an inhibitor of protein synthesis. The results showed that reactivation of Luc was inhibited when heat-treated cells were post-treated under mild hyperthermic conditions. The observed increase in Hsp synthesis under mild hyperthermic post-heat shock conditions therefore appears to be the result of an increase in the period during which denatured proteins are present. In addition, we studied Luc reactivation in the absence of protein synthesis inhibitors. This condition led to much higher Luc activity. By estimating half-life times of Luc, the contribution of new Luc synthesis in this recovery could be determined, and only partially explained the observed increase in Luc reactivation after heat shock. Thus the synthesis of other proteins must be important for the renaturation of heat-damaged proteins.

Stimulation of survival capacity in heat shocked cells by subsequent exposure to minute amounts of chemical stressors; role of similarity in hsp-inducing effects.

A brief and moderate heat shock to Reuber H35 hepatoma cells causes a rapid increase in the synthesis of heat shock proteins (hsp) and initiates the development of thermotolerance, which results in an increased ability to survive exposure to otherwise lethal temperatures. We now demonstrate that low doses of various chemical stressors (arsenite, cadmium, mercury, lead, copper, menadione and diethyldithiocarbamate (ddtc)), at concentrations that do not exert any effect in control cultures, are able to enhance the synthesis of hsps and to stimulate the development of thermotolerance when applied to cultures which were pretreated with a mild heat shock. The degree of stimulation appears to be stressor-specific, which is not only observed in the ensuing development of thermotolerance but also in the enhancement of the heat shock-induced synthesis of stress proteins. The different hsps that show an enhanced induction when heat shocked cultures are exposed to the various secondary applied low doses of chemical stressors, were found to resemble the hsp pattern that is characteristic for the secondary stressor and not for the initial heat shock. In other words, the nature of the post-treatment determines the observed pattern of enhanced synthesis of hsps. In order to analyze the origin of the stimulation of survival capacity by low doses of the mentioned stressors, we studied whether the degree of stimulation is determined by the degree of similarity between the overall stress response to heat shock and to the second stress condition when applied singly. The degree in which low doses of chemical stressors stimulate tolerance development and enhance the synthesis of hsps in cells that were previously heat shocked, appears to be related to the degree of similarity in the hsp pattern induced by both stressors. Our results support the notion that low doses of toxic compounds may, under certain conditions, have beneficial effects related to a stimulation of endogenous cytoprotective mechanisms.

Dinitrosyl iron complexes with thiol-containing ligands and S-nitroso-D,L-penicillamine as inductors of heat shock protein synthesis in H35 hepatoma cells.

The concentration-dependent effect of various nitric oxide donors on synthesis of different heat shock proteins was evaluated in Reuber H35 hepatoma cells and their heat shock protein-inducing ability was compared with the effect of a heat shock. A 6 h incubation of H35 cells with the dimeric (diamagnetic) form of dinitrosyl iron complex with glutathione or N-acetyl-L-cysteine activated synthesis of various heat shock proteins, heat shock protein 28, 32, 60, 70, 90 and 100. Synthesis of these proteins was evaluated by [35S]methionine and [35S]cysteine labelling with subsequent separation of proteins by polyacrylamide gel electrophoresis. The dinitrosyl iron complex with glutathione appeared to be the most efficient inductor of heat shock protein synthesis and initiated the synthesis of heat shock protein 28 even more efficiently than a 30 min heating of cells. In the same experiments, S-nitroso-D,L-penicillamine exerted a considerably lesser effect on the synthesis of heat shock proteins. It was suggested that the active moiety of dinitrosyl iron complexes as inductors of heat shock protein synthesis is represented by their Fe+(NO+)2 groups which move to thiol groups of the proteins participating in the initiation of heat shock protein synthesis.

The role of hsp70 in protection and repair of luciferase activity in vivo; experimental data and mathematical modelling.

The stably transfected rat cell line HR24 expressing high levels of the inducible human hsp70 and its parental cell line Rat-1 were used for in vivo studies to analyse the role of hsp70 during thermal protein denaturation and the subsequent renaturation. In order to monitor denaturation and renaturation of a cellular protein in vivo, both cell lines were transiently transfected with firefly luciferase (Luc). The continuous monitoring of Luc activity during and after heat stress allowed a detailed analysis of the inactivation and reactivation kinetics in cells grown in monolayers. The aim of these studies was to distinguish a protective effect of increased hsp70 levels during heat shock-induced protein inactivation from a stimulation of reactivation. In this paper we show that in cells that are stably transfected with hsp70, thermal Luc inactivation decreased, and subsequent reactivation yielded higher activity levels, compared with the parental cells. The difference in early inactivation kinetics observed in the two cell lines suggests an immediate effect of the presence of an extra amount of hsp70 on enzyme inactivation. Using different mathematical models, the heat-induced inactivation and reactivation kinetics was compared with simulations of denaturation and renaturation. It is concluded that the model in which it is assumed that hsp70 is able to interact with partially denatured proteins, which did not yet lose their enzymatic activity, most optimally explains the experimental observations.

Stressor-specific enhancement of hsp induction by low doses of stressors in conditions of self- and cross-sensitization.

In this paper, the pattern of induction of heat shock proteins (hsps) was studied in cultured Reuber H35 rat hepatoma cells by sequential application of different stressors. We analyzed whether a specific stress condition is able to induce an enhanced sensitivity to a subsequent application of a low dose of either the same or another stressor (self-sensitization and cross-sensitization, respectively). As a measure of sensitization, the stimulation of hsp induction was employed. Three different stressor conditions (heat shock, sodium arsenite and cadmium chloride) were used in doses which exerted a similar impact on overall protein synthesis. A synergistic effect in induction of the synthesis of various hsps was observed when a high stressor dose was followed by an 8-h incubation in a lower stressor dose in both self- and cross-sensitization experiments. The low-dose conditions used as second treatments did not induce any responses in non-pretreated cells. Studies in cultured cells have demonstrated stressor-specific hsp induction patterns. In this study we analyzed whether the pattern of hsps induced by the low-dose condition is characteristic for the first sensitizing stressor or for the secondary stressor applied in a low dose. The pattern of hsps which was induced above the level of the high-dose effect, due to the incubation with the secondary applied low-dose condition, was found to be characteristic for the secondary stressor and not for the sensitizing primary treatment. These results are of importance for an improved understanding of the regulation of heat shock protein synthesis in conditions of self- and cross-sensitization, as well as for a proper use of hsps as biomarkers of exposure to environmental stress.

The similia principle as a therapeutic strategy: a research program on stimulation of self-defense in disordered mammalian cells.

The similia principle is considered to be the essence of homeopathy. This article describes a research program for study of the similia principle in cultured mammalian cells. This systematic program with its rather simple research model was set up ultimately to contribute to the design of studies of the similia principle with more complex organisms such as humans. With respect to application of the similia principle, the concepts of self-defense and self-recovery are central. At the cellular level, self-defense and recovery largely depend on the availability of proteins with a cell-protective function, most notably, stress or heat shock proteins. To study the similia principle, we use four lines of research to examine the processes of self-defense. First, stimulation of self-defense in disturbed and disordered cells is studied by using low doses of an agent homologous or identical to the disturbing agent. The second line of research deals with the specificity of this stimulation: Is cellular self-defense after exposure to toxicant A also effectively stimulated in an analogous or heterologous way by low doses of other toxicants such as B or C? The third line of research involves the duration of low-dose sensitivity of disordered cells for homologous stimulations, in particular, the desensitization of cells toward these homologous stimulations. The fourth line of research deals with whether-according to the similia principle-the state of desensitization can be overruled by heterologous condition(s) that induce an analogous pattern of protector proteins (ie, a pattern closely resembling the damage-induced pattern) and thus effectively stimulate cellular defense and recovery.

Enhancement of the stress response by minute amounts of cadmium in sensitized Reuber H35 hepatoma cells.

The aim of this study was to determine whether the cadmium-induced cellular stress response can be modulated by the subsequent application of low concentrations of the same ion. It is shown that exposure of Reuber H35 rat hepatoma cells to cadmium concentrations of 10 or 30 microM for 1 h leads to a biphasic change in their sensitivity towards a second exposure to cadmium, an initial sensitization is followed by development of tolerance towards the secondary treatment with cadmium. Furthermore, incubations for 1 h in the presence of 10 microM of cadmium induce the synthesis of the major heat shock proteins except for hsp60. A step-down cadmium regime, i.e. a pretreatment of 1 h with 10 or 30 microM immediately followed by incubations with lower concentrations of cadmium (ranging from 0.03 to 1 microM), leads to additional increases in hsp synthesis. Since no effect of these low concentrations was observed on hsp synthesis in non-pretreated cells, the effect of a step-down treatment thus results in a higher effect on hsp synthesis than could be expected based on their summation. The sensitized cells also develop a higher level of tolerance in the presence of the above mentioned low concentrations of cadmium. It can be concluded that during the transient period of enhanced sensitivity, low concentrations of the original stressor enhance the synthesis of hsps and thus induce higher levels of tolerance in comparison with cells which only received the primary cadmium treatment.

Is heat shock protein re-induction during tolerance related to the stressor-specific induction of heat shock proteins?

The existence of stressor-specific induction programs of heat shock proteins (hsps) leads us to analyze the possible occurrence of a stressor-specific tolerance induced by either heat shock, arsenite, or cadmium. As a measure of this tolerance re-induction of hsps was studied. In this paper, we tested whether the refractory state is either valid for each specific hsp (implying independent regulation of every member of the heat shock protein family) or extends from small subsets of the hsp-family to even larger groups of proteins (indicating a more common denominator in their regulation). (re-)induction of hsps does not seem to be regulated at the level of each individual hsp since differences in induced synthesis of hsps between two stressor conditions are not supplemented systematically upon the sequential application of the two stressors. The most notable example in this respect is hsp60. A pretreatment with cadmium, which hardly induces synthesis of this hsp, does induce a tolerance to (re)-induction by heat shock, which normally induces hsp60. This suggests the existence of a more common denominator regulating the coordinate expression of at least some hsps. From our data we conclude that the degree, but not the pattern, of hsp re-induction is influenced by the type of stressor used in the pretreatment. The pattern of hsps induced by a secondary applied stressor still shows most of its stressor-specificity and seems to be independent of any pretreatment. The possible implications of stressor-specificity are discussed.

Sensitization to x-rays by sodium arsenite or heat in normal cells and in cells with an induced tolerance for heat and arsenite.

In this study we compared sensitization to x-rays by heat or sodium arsenite and the effect of an induced heat or arsenite resistance on radiosensitization. Treatment of Reuber H35 hepatoma cells with either heat or arsenite causes a dose-dependent radiosensitization. Based on a comparison of isosurvival doses for arsenite and heat, arsenite causes a stronger enhancement of the radiosensitivity. Radiosensitization increases exponentially with increasing sensitizer dose. It is gradually lost when the time interval between irradiation and treatment with heat or arsenite increases, depending on the treatment sequence. For x-rays prior to heat, radiosensitization disappears approximately twice as fast as in the reverse case. Arsenite radiosensitization shows approximately the same kinetics for an isoeffective combination, but slightly longer times are needed for the complete clearance of the interaction. As with heat, an exposure to arsenite induces a stress response in cultured cells which results in the development of an increased tolerance towards a second exposure. Heat and arsenite induce self- as well as cross-tolerance. The reduction in arsenite or heat toxicity in tolerant cells is correlated with a reduction in radiosensitization. The mechanisms for heat and arsenite cytotoxicity appear to be different. A combination of non-toxic doses of heat and arsenite has a synergistic effect on the cytotoxicity. One hour incubation with 0.02 mM arsenite at 41 degrees C has the same cytotoxicity as 0.2 mM after 3 h incubation at 37 degrees C, and the amount of radiosensitization induced by these treatments is approximately the same.

Relationship between cadmium-induced expression of heatshock genes, inhibition of protein synthesis and cell death.

Stress proteins (heat shock proteins, HSPs) have been proposed as markers for toxicity. This study has focussed on the pattern of HSP synthesis in relation to cytotoxicity and their dependence on doses of cadmium chloride. We investigated the relationship between cadmium-induced expression of heatshock genes, inhibition of protein synthesis and cell death in a well-differentiated hepatoma cell line, Reuber H35, under exposure conditions ranging to full (> 98%) lethality. We find a non-linearity in the responses of these cells when the duration of cadmium exposure is varied. The results indicate that sublethal concentrations of cadmium can inhibit protein synthesis and also increase the synthesis of certain HSPs. The pattern of heat shock protein induction changes when exposure conditions become more severe. The most strongly inducible heat shock protein, HSP68, is, surprisingly, only synthesized under conditions which lead to severe inhibition of protein synthesis. The comparison of HSP68 mRNA levels and HSP68 synthesis showed that HSP68 mRNA is already induced under conditions where the synthesis of HSP68 protein cannot yet be traced. From these data we conclude that a differential HSP expression takes place. The translational control of HSP synthesis might be explained by the preferential translation of this mRNA under conditions of severe shut-off of general protein synthesis.