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STIMULATION
OF CELLULAR SELF-RECOVERY
BY APPLICATION OF THE SIMILIA-PRINCIPLE
Self-recovery
and similia-principle
R. VAN WIJK AND F.A.C. WIEGANT
Department of Molecular Cell Biology, Utrecht
University
P.O Box 80.056, 3508 TB Utrecht, The Netherlands
2 - Stressor - specificity with Respect to Induction of
Gene Expression
3 - Cross- sensitization
4 - Discussion
The stimulation of self-recovery by applying the similia-principle is considered to be the essence of homeopathy. The similia-principle indicates that a substance which causes certain symptoms in a healthy organism, may be applied in a curative sense to a diseased organism with identical symptoms.
Previously, it has been shown that the recovery of cells that were damaged by a certain stressor, are stimulated by the application of a low dose of the same stressor. In this respect, the stimulatory effect of low doses could be observed after a heat shock (Van Wijk et al., 1994) as well as after a treatment with arsenite (Ovelgönne et al.,1995a; Wiegant et al.,-this volume-) or with cadmium (Wiegant et al., in prep).
As parameters for stimulation of self-recovery the development of tolerance is used. An important line of research deals with the molecular aspects underlying the process of recovery. In this respect a particular set of proteins, indicated as heat shock proteins (hsps) or stress proteins are supposed to play a crucial role (Welch 1992; Parsell and Lindquist 1994). This set of proteins includes different protein families with approximate molecular masses (in kD) of 28 (hsp28), 60-65 (hsp60), 68-73 (hsp70), 84-90 (hsp84) and 100-110 (hsp100). The evidence for their role in protection comes from studies of hsp synthesis under differential conditions of environmental stress (Li and Laszlo 1985; Nover 1991) as well as by transfection of hsp-genes (Landry et al., 1989; Li et al., 1991; Angelidis et al., 1991; Jaatela et al., 1992; Parsell and Lindquist 1994).
A further study of the similia-principle requires additional experiments aimed at the specificity of this low-dose stimulation of tolerance development and of hsp induction. In this paper we focus on the specificity of low doses of stressors with respect to the enhancement of hsp induction.
With respect to the specificity of the similia-principle, our working hypothesis is that the most effective low-dose stimulation of hsp-induction in damaged cells is obtained with the same stressor or a stressor which has an identical pattern of induction of the different stress proteins. With less similarity between the damaged condition and the low-dose stimulating condition, the low-dose stimulation will be less or even absent.
If for instance arsenite is applied as the disturbing agent, it can be hypothesized that stimulation of sub-optimal hsp induction as part of self-recovery will only occur by the application of a small dose of arsenite and not, for instance, by a small dose of cadmium, unless cadmium shows the induction of a similar pattern of hsps.
The present study, including arsenite, heat shock and cadmium as damaging conditions, and the same three conditions at a low dose applied afterwards, deals with the relationship between degree of similarity and efficiency of low dose stimulation. In this way the specificity of the similia-principle can be verified.
2. Stressor-specificity with Respect to Induction
of Gene Expression
In our study different stressors were used in order to identify a possible stressor-specific induction in the pattern of heat shock proteins. If a non-specific expression is observed, i.e. when all stressors induce the same set of hsps to an identical degree, then it would be impossible to study the specificity of the similia-principle.
2.1. STRESSOR-SPECIFIC INDUCTION OF HSPS
The induction of hsps by a variety of stressors including heat shock, arsenite and cadmium has been studied extensively and recently reviewed by Nover (1991) and Welch (1992). In a number of these studies the effects of two or more stressors was compared (Li 1983; Anderson et al.,. 1987; Pipkin et al.,. 1987; Lee et al.,1989; Aoki et al.,. 1990; Deaton et al., 1990; Edwards et al., 1990; Lai et al., 1993). The shortcoming of most of these studies is that the pattern of induced proteins was not studied over a range of concentrations nor were they studied under conditions that lead to similar consequences on the cellular physiology. The limited conditions in most studies might be the cause of severe biases. A reliable method to compare the effects stressors have on the stimulation of hsp synthesis, is to establish their concentration dependency and then compare the hsp patterns in these concentration ranges. To this end, Reuber H35 rat hepatoma cells were exposed to different concentrations of various environmental stressors (including heat shock, arsenite and cadmium). For a careful comparison of these stressor conditions, the stressors were all applied during one experiment in order to exclude slight differences in culturing conditions. The possible stressor-specific effect on the induction of hsps was studied as well as the effects of these stressors on the inhibition and recovery of protein synthesis and on cell survival (Wiegant et al., 1994).
A comparison of the stressor-specific induction of the major hsps was made under conditions of identical impact on cellular physiology; i.e. those stressor conditions were chosen that resulted in approximately equal percentages of cell death (iso-survival dose: 40-50%). These iso-survival doses were: 1 h at 42°C; 2 h of 200 µM arsenite and 2 h of 100 µM cadmium. The pattern of hsp synthesis of stressed cells consists of the hsp28, hsp60, hsp68, hsp70, hsp84 and hsp100 (figure 1). Cells exposed to cadmium show the highest relative synthesis of hsp28 if compared with cell cultures treated with heat shocks or arsenite. No induction of hsp60 is observed after cadmium treatment, whereas a large induction of hsp60 is observed after heat shocks and arsenite. Hsp68 and hsp70 are both induced by heat shocks as well as arsenite and to a lesser extent by cadmium. More or less the same pattern is observed with respect to hsp84 and hsp100. (Wiegant et al., 1994 for a more detailed analysis).
Each hsp induced after the treatment with a stressor is supposed to have a characteristic function with respect to the recovery of specific cellular damage induced by this stressor. Although not all the effects of the various stressors have been identified at the molecular level, it can be assumed that cellular damage in general requires a differential activity of hsps and that specific cellular damage requires the presence of specific hsps. In this respect, it can be proposed that the differential induction of the group of hsps induced by a specific stressor is exactly attuned to the precise need of the cell and that it can be taken as an indicator for the type of cellular damage which is under repair.
Figure
1. The effect of three different stressors (heat shock at 42°C for 60
minutes, 200 µM arsenite for 2h and 100 µM cadmium for 2h) on the synthesis of
the major hsps is shown (a-hsp28; b-hsp60; c-hsp68; d:-hsp70; e-hsp84;
f-hsp100). In the control situation several hsps are synthesized at a low
level. Synthesis of the major stress proteins is expressed relative to the
synthesis of actin.
2.2. STRESSOR SPECIFIC INDUCTION OF HSP mRNA
In order to determine whether the stressor-specific enhanced expression of HSP-genes is caused by a differential gene expression, experiments were performed in which both the levels of specific hsps and of hsp mRNAs were determined in parallel cultures after the application of a heat shock, arsenite or cadmium. The stressor-specificity of the synthesis of hsps was confirmed by hsp mRNA data (Ovelgönne et al., 1995b). The inability of cadmium to induce hsp60 appeared to correlate with an absence of the induction of hsp60 mRNA at the various concentrations studied.
In previous papers, the low-dose stimulation has been studied in an isopathic approach. Thus, a cell population that previously had been damaged and sensitized by a high dose of a stressor was used to study the effect of a low dose of the same stressor on stimulation of self-recovery and hsp induction. (Van Wijk et al., 1994; Ovelgönne et al., 1995a; Wiegant et al., -this volume). The application of a mild heat treatment or a low dose of arsenite immediately after a heat shock or an intoxication with arsenite respectively, stimulated the synthesis of hsps as well as the development of tolerance. Cells were highly sensitized; i.e. they responded to relatively low doses, whereas these low doses of stressors were without any effect in control cells during the time period studied. It was argued that in this way the similia-principle can be studied in its most elementary form.
In the present study it became of interest to determine whether hsp-induction can also be stimulated by low doses of other (non)-related stressors. Are sensitized cells also stimulated by low doses of non-related stressors.
Figure
2. Cross-sensitization. The
effect of a low doses of a stressor on the synthesis of the major hsps, which
was applied immediately following a pretreatment with a high doses of either a
heat shock (HS: 42°C for 30 minutes), cadmium (Cd: 100 µM for 1 h) or arsenite
(As: 100 µM for 1 h). The incorporation of [35S]-labelled amino acids into the
major hsps over an 8-h period was expressed relative to the total incorporation
into cellular protein. The results for each hsp are grouped per effect of the
high-doses stressor. Of each group of 4 histograms, the first bar shows the
induced synthesis of the indicated hsp in control conditions (0) or by a
pretreatment with the high-doses stressor (HS, As or Cd), whereas the other
three show the effect on the synthesis of hsps of the post-incubation in a low
doses of heat shock (hs: 8h in 40.5°C), cadmium (cd: 8h 10 µM) or arsenite (as:
8h 10 µM). Results are the mean of three experiments.
In this respect it was hypothesized that the specificity of the similia-principle reveals itself in the fact that for the stimulation of hsps by low doses identical or strongly similar conditions must be applied. If condition 'A' is applied as the disturbing agent, the stimulation of a suboptimal hsp-induction will only result from the application of a small dose of condition 'A' and not by a small dose of condition 'B' or 'C', unless B or C has a similar effect on the cellular physiology as condition 'A' has.
If, however, the stimulation of self-recovery by a low dose is an a-specific phenomenon, then a low dose of stressor 'B' or 'C' can also be expected to be able to stimulate the hsp-induction. Such a phenomenon, indicated as cross-sensitization, has been studied with Reuber H35 cells exposed to heat shock, arsenite or cadmium.
A number of experiments were performed in which a pretreatment was given with either a heat shock (30 min. at 41.5°C), sodium arsenite (100 µM for 1h) or cadmium chloride (100 µM for 1 h). After the pretreatment the cultures were exposed to low, mostly subliminal conditions of heat (40°C or 40.5°C), arsenite (3 or 10 µM), cadmium (3 or 10 µM) or by an incubation at control conditions (37°C) for a period of up to 8h. In this period the stimulating effect on the synthesis of various hsps was studied. The results are shown in figure 2.
Figure
3. The effect of a low doses
of a stressor (heat shock at 40.5°(hs); 10 µM cadmium (cd); or 10 µM arsenite
(as), immediately applied after a high doses of heat shock (HS: 42°C for 30
min), on the extra synthesis of the major hsps over a period of 8h. The effect
of the HS has been subtracted in order to show the stimulatory effect of the
low doses only.
The first step in the analysis of the results of these experiments was to determine which of the species of hsps was stimulated by the low-dose iso-condition.
- A small increase in temperature after a more severe heat shock enhances the induced synthesis of all studied hsps, but mainly hsps 60, 68, 70 and 100 and to a lesser extent hsp84. The extra synthesis of the selected hsps, induced by low dose stimulation after heat shock, is also presented in figure 3 (left panel).
- Of the hsps induced by a treatment of arsenite, mainly the subgroup of hsps, consisting of hsp28, hsp68, hsp70 and hsp100, was further stimulated by arsenite at a subliminal concentration (figure 2).
- Of the hsps induced by a pretreatment with cadmium, a subsequently added subliminal concentration of cadmium, stimulated the synthesis of a subgroup of hsps which consisted of hsp28, hsp68 and hsp70 (figure 2). These subgroups of hsps thus seem to be indicative for a particular role in the stimulation of self-recovery.
The second step in the analysis involved the stimulation of synthesis of the selected hsps by subliminal concentrations of other stressors than the ones used for the pretreatment.
- If heat shock was used as the disturbing pretreatment, hardly any extra stimulation of hsp-synthesis is observed upon the subsequent application of a low dose of cadmium (figure 2 and figure 3 -middle panel-). In contrast when a low dose of arsenite is applied to cells which were previously heat shocked, a large extra synthesis of hsp68, 70 and 100 occurs. Interestingly no extra enhancement at all of hsp60 and 84 is observed (figure 2 and figure 3 -right panel-). This demonstrates a rather specific effect of low doses of the used stressors on heat-treated cell cultures.
- If arsenite was the first disturbing agent, the synthesis of hsp28, hsp68, hsp70 and hsp100 was also stimulated when a mild heat treatment was applied. Application of a subliminal cadmium concentration only stimulated hsp28, whereas no effect of cadmium was found on the synthesis of hsp 68, hsp70 and hsp100 (figure 2).
- If cadmium was used as the disturbing agent for the pretreatment, stimulation of hsp28, hsp68, hsp70 occurred after a successive application of a subliminal cadmium concentration. The synthesis of these hsp protein species was also stimulated by a very mild heat shock when applied as a second treatment. The application of a subliminal arsenite concentration stimulated only hsp28 and hsp70.
This analysis of the experiments on cross-sensitization is very elementary and takes no account of the quantitative differences between the stimulatory effect of small doses of the various stressors.
Nevertheless, from the data it can be observed that even this elementary analysis demonstrates a specificity in the synergistic action between a pretreatment and a subliminal condition. A schematic presentation of the qualitative similarity between the different conditions and the cellular capacity to enhance synthesis of specific hsps upon low dose stimulation is given in Table 1.
TABLE 1: Cross-sensitization
_____________________________________________________________________
First stressor
Cadmium Heat shock Arsenite
______________________________________________________________________________________
Second
stressor
Heat shock ++++ ++++ ++++
Arsenite +++ ++++ ++
Cadmium - + ++++
______________________________________________________________________________________
The range -, +, ++, +++, ++++ is indicative
for the degree of extra synthesis of hsps by
a low dose of the second stressor.
Based on a comparison of the hsp-induction pattern of the three stressors, it was concluded that the heat shock- and the arsenite pattern resemble each other more than the heat shock- and the cadmium-pattern. This higher degree of similarity is now also reflected in a higher degree of extra synthesis of hsps upon low dose stimulation and thus confirms the aspect of specificity of the similia-principle.
The aim of our research program was to study the process of self-recovery and its stimulation by low doses of compounds applied to diseased biological systems according to the similia-principle. For the study of the similia-principle it is for the researcher of importance to be able to concentrate on the most elementary level where changes manifests themselves. Therefore, we have proposed the following question in our research program:
Which molecular processes and interactions are of importance in the process of cellular self-recovery and which molecular parameters should be used to evaluate (stimulation of) cellular self-recovery ?
In our study we have focused on the synthesis of hsps as a measure of cellular self-recovery. This choice is based on our own experimental results (Van Wijk and Wiegant, 1994) as well as on and data from the literature (for recent review see: Nover, 1991; Welch, 1992; Parsell and Lindquist, 1994). The pattern of synthesis of the individual hsps can be considered as the pattern of symptoms specifically linked to the recovery process of the cell after damage.
Our comparative study of hsp induction in H35 cells by three different stressors: -heat shock, cadmium and arsenite- have shown that the heat shock- and arsenite-induced patterns of synthesis of the major hsps resemble each other more than the heat shock- and cadmium-induced patterns of hsp-synthesis. Furthermore, the possible existence of a stressor-specific attunement to the cellular requirements of the induced hsps, has been tested in experiments in which a pretreatment was given with a heat shock, cadmium or arsenite, then followed by incubation in stressor-free conditions for 16h. This provided for a period of time in which all hsps could reach their maximal level. Subsequently, the effect on the induction of hsps of a second application of a stressor of the same dose (heat shock, arsenite or cadmium) was studied in a cross-tolerance design. The synthesis of hsps was followed over a period of 8h after application of the second stressor. A more detailed description of the results will be described elsewhere (Wiegant et al., 1995). From the results however, three aspects of interest for the present discussion can be mentioned:
1. In general, a reduced induction of hsps is observed when a stressor is applied during a tolerant period. Therefore a certain degree of attunement to the induced cellular damage appears to be present in the effect of all stressors used in this study. The degree of attunement depends on the similarity between the first and second stressor with respect to their effect on the synthesis of hsps.
2. The repeated application of the same stressor (heat shock-heat shock, arsenite-arsenite, cadmium-cadmium) provides the highest degree of attunement and therefore the highest degree of tolerance, since in these conditions the lowest induction of all hsps occurs after the application of the second stressor.
3. The application of different stressors (heat shock-arsenite, heat shock-cadmium, arsenite-heat shock, arsenite-cadmium, cadmium-heat shock, cadmium-arsenite) results in a larger induction of the different hsps. Apparently the transient insensitivity as observed in self-tolerance has not developed to a similar degree. The attunement of the various hsps to the specific damage inflicted by the first stressor does not seem to include the damage inflicted by the second stressor since the induction of (other) hsps is required. However, it is interesting to note that of the different combinations of stressors, cross-tolerance between heat shock and arsenite develops to a higher degree than the cross-tolerance between heat shock and cadmium or cadmium and arsenite. This suggests that the pattern of induced hsps by a heat shock is better attuned to the damage inflicted by arsenite than to the damage caused by cadmium. It can therefore be concluded that of the three stressors studied, heat shock and arsenite shows the largest similarity in effect.
With respect to the substantiation of the similia-principle - the following expectation has been formulated:
The specificity of the similia-principle will reveal itself in the fact that for the stimulation of self-recovery always identical conditions or conditions with a reasonable degree of similarity must be applied however in a smaller concentration or in a lighter form. If condition 'A' is applied as a disturbing agent, stimulation of suboptimal self-recovery will only happen by applying a small dose of 'A'. By applying a small dose of condition 'B', when 'B' provokes symptoms that are highly different from 'A' stimulation will not happen. Finally stimulation of suboptimal self-recovery to a lesser degree will happen with a small dose of condition 'C', when 'C' provokes symptoms partly similar to 'A'.
In this paper we have provided evidence in which the specificity of the similia-principle is substantiated. Further studies applying detailed quantitative analysis of hsp patterns will be presented elsewhere. For the interest of homeopathy it is of importance to ask the question:
5. Do Studies on the Similia-principle at
the Cellular Level Have any Significance?
It has been argued that the similia-principle has been formulated for the organismal level and therefore can not be verified or falsified by research on the cellular level alone.
There is, of course, a clear difference between research on the similia-principle as described here and the clinical practice. The first difference is the systemic level or level of organization which is studied or treated (cells versus organisms). Secondly, in our experiments cells are brought into a pathological condition, whereas patients who come to a doctor already are in this condition. Thirdly, cells are grown in cultures and treated as a group. Their behaviour and response with respect to the process of self-recovery is evaluated as the mean of the total population. There is of course a variability in self-recovery. Thus, not only cells showing a fast and spontaneous recovery, but also cells showing a slow and hampered self-recovery are evaluated. In contrast to research with patients, a single case approach or a time-series design for studies on the behaviour of individual cells is not possible or is extremely complex.
However, we expect the assumption of the similia-principle as being a general biological phenomenon to gain weight by the study at the cellular level of regulatory processes underlying this principle. The demonstration of the stimulation of self-recovery by compounds according to the similia-principle will substantiate the fundamental basis of homeopathy and will improve the acceptance of this basic point of view in bio-medicine.
Acknowledgement
The financial support of the HomInt organisation is gratefully acknowledged.
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