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STIMULATION OF SELF-RECOVERY BY LOW DOSES OF ARSENITE IN ARSENITE-INTOXICATED CELLS
Self-recovery
by low doses of arsenite
F.A.C. WIEGANT, J.H. OVELGÖNNE, J.E.M. SOUREN, AND R. VAN WIJK
Department of Molecular Cellular Biology, Utrecht University, PO Box 80.056, 3508 TB Utrecht, The Netherlands
1 - Introduction
2 - Optimal Conditions to Study the stilulation of the Recovery
of Damaged Cells
3 - Do Low Doses of Arsenite Stimulate Self-recovery in
Arsenite-intoxicated Cells?
4 - Discussion
The aim of our studies is to understand the stimulation of self-recovery processes at the cellular level by compounds which are applied according to the similia-principle.
In order to induce self-recovery, cells are exposed to mildly damaging conditions. In our research a non-lethal concentration of sodium arsenite was used which induces the so-called heat shock- or stress-response (Nover, 1991; Welch, 1992). This response includes the synthesis of a specific class of proteins, the heat shock proteins (hsps) and the development of tolerance. It has been shown that self-recovery can be evaluated in terms of the development of tolerance and the enhanced production of hsps (Van Wijk and Wiegant, 1994). Hsps facilitate repair and refolding of stress-damaged proteins and also prevent irreversible denaturation and aggregation of cellular proteins (for review see Hightower, 1991; Welch, 1992; Parsell and Lindquist, 1994). In this latter function they may act as 'protector proteins', which may be one of the underlying molecular mechanisms by which cells acquire tolerance.
With respect to the aim of our studies, it is of interest to determine whether, after an exposure to a damaging condition, cells can be stimulated in their development of tolerance and the production of hsps, by stressor doses that are not effective in undamaged (control) cells. Surprisingly, hardly any studies have been performed to determine the effect on the synthesis of hsps or the development of tolerance of an exposure of cells to these so-called step-down protocols; a transient incubation with a high dose of a compound, which is followed by the application of a low dose of the same or a similar compound.
In the present paper, experiments are described which were performed to study the effect of low doses of arsenite on the synthesis of hsps and on the development of tolerance in arsenite-sensitized cell cultures. In first instance, however, the conditions for a cell model are summarized as far as it is required for the study of self-recovery and its stimulation with low doses.
2. Optimal Conditions to Study the Stimulation
of the Recovery of Damaged Cells
A variety of factors influence the effect of stressors and the process of hsp-activation. For the experiments described in this paper, mild stressor conditions are used in order to prevent the occurrence of too much irreversible damage. Cell populations should be obtained that are sensitized and are able to express self-recovery (as evaluated by the synthesis of hsps and the development of tolerance).
2.1. THE USE OF CELLS SHOWING SUB-OPTIMAL SELF-RECOVERY
In order to be able to stimulate recovery processes, a (sub-)population of cells is required which shows sub-optimal self-recovery. Using mild stressor conditions, a variability exists in reaction pattern and consequently in the speed of tolerance development in the different cells of the population. There will be a subpopulation of cells showing a fast recovery, whereas another subpopulation of cells will show a slow recovery and hence a slow development of tolerance. Our studies are focused on the sub-population of cells showing a slow recovery. This sub-population has been threatened in such a way that they are damaged but are relatively weakly activated in their production of hsps. The replenishment of the pool of protector proteins, i.e. the new production of these hsps, is hampered causing the occurrence of a sensitive state. The subpopulation of cells being in this state has the highest probability of becoming irreversibly damaged and of dying. Our research is aimed on stimulating this part of the cell population which shows a sub-optimal self-recovery, by applying low doses of the stressor. The hypothesis is formulated that a low doses is effective when cells are damage-sensitized. The effect on self-recovery will be evaluated by studying the mean reaction pattern of hsp synthesis and tolerance development as measured in the total cell population.
2.2. SELF-RECOVERY AND THE SIMILIA PRINCIPLE
According to the similia-principle, self-recovery may be stimulated by low doses of a specific substance that is capable of inducing similar symptoms in a healthy system. The stimulation of self-recovery should not occur with low doses of substances that induce different symptoms in the healthy system.
In essence, the isopathic-treatment is an example of the similia-principle in its most elementary form. The same substance which has caused a dis-balance or a pathological condition will support the recovery from this dis-balance (provided that an adequate doses is given). The application of this principle in clinical practice is difficult since in many cases the cause of pathology is unknown. Then a substance should be chosen which is able to induce symptoms in a healthy organism optimally resembling the symptoms expressed by the organism in the pathological state. With respect to the treatment of patients, one is entering here into the specialism of homeopathy. Homeopathic physicians claim self-recovery processes to be stimulated when an adequate homeopathic remedy is administered to the patient.
At the cellular level, it can be expected that - in an isopathic approach - self-recovery can be stimulated with a small doses of the substance which was responsible for the disturbance of the system. This stimulation will show itself in an increased activation of the expression of the cellular gene program for protective proteins, leading to a stimulated supplementation of protective proteins and in an increased resistance for the disturbing agent.
2.3. NUTRITIONAL CONDITIONS
When the effect of low doses are studied, the medium is changed various times during the experiments, resulting in the possibility of a stimulation of the self-recovery when nutritional components are introduced. However, when the medium is refreshed several times before and during the experiment the possibility that self-recovery is stimulated by other factors than the application of low doses of the stressor, can be excluded. For this reason, Reuber H35 hepatoma cells were grown in complete media, given fresh media 12 h before the experiment and incubated in a stressor-containing fresh medium again. After the application of the stressor, cells are rinsed extensively with phosphate buffered saline (PBS) and incubated again in fresh medium containing the low doses.
Summarising, to be able to demonstrate the stimulation of the recovery process and to avoid a cell population with an increasing number of cells with irreversible damage it is essential to expose cells to mild damaging conditions. Furthermore, the metabolic capacity of the cells to perform self-recovery processes should be optimal in order to prevent changes to be due to the nutritional condition of the cells. For this reason, the cell culture is provided with optimal nutritional conditions. The model developed can then be used to test the similia-principle and to determine the specificity of this principle.
3. Do Low Doses of Arsenite Stimulate Self-recovery in Arsenite-Intoxicated Cells?
This study has been focused on the regulation of cellular sensitivity to arsenite-intoxication. There is evidence for the existence of a state of tolerance to arsenite (Lee and Hahn, 1988; Lee et al. 1989) and recently the phenomenon of an arsenite-induced transition from enhanced (sensitization) to reduced (tolerance) sensitivity to arsenite has been demonstrated (Wiegant et al., 1993). Previously, arsenite has been shown to be an effective activator of hsp gene expression in a variety of mammalian cell types (Johnston et al., 1980; Levinson et al., 1980; Li, 1983; Wiegant et al., 1987). We have investigated whether a treatment of H35 rat hepatoma cells with arsenite sensitizes cells to arsenite. It can be argued that when a threat is applied which is just strong enough to stimulate cells to start the replenishment of hsps, that this replenishment can be stimulated by a lower doses of the stressor, provided that it is given directly following the first threat. For this reason we incubated arsenite-treated cells with low doses of arsenite, according to the step-down protocol, which could enhance the synthesis of the levels of hsp68-mRNA as well as the induction of the major hsps. In addition, the effect of low concentrations of arsenite on the induced development of arsenite-tolerance has been studied in arsenite-sensitized cells. According to previous definitions, 'tolerance' is described as a stress-induced cellular resistance to a lethal stress treatment, whereas 'sensitization' is defined as an increase in cellular susceptibility for a stress treatment (Wiegant et al., 1993).
3.1. LEVELS OF HSP68-mRNA
In first instance, the effect of the step-down (arsenite) protocol on the level of hsp68-mRNA was studied. H35 hepatoma cells were exposed to various concentrations of arsenite (10, 30, 100 or 300 µM) for 1 h. Samples were taken immediately after the exposure to arsenite and at 2 h intervals up to 8 h during an incubation in an arsenite-free medium. Increased hsp68-mRNA levels could be detected immediately after arsenite treatment, the increase continuing during the incubation in arsenite-free medium. An exposure to 10 µM for 1 h did not have any effect on the induction of hsp68-mRNA. As shown in figure 1, the magnitude and the kinetics of the induction depended on the arsenite concentration. In general the response was stronger at the higher arsenite concentrations. Hsp68-mRNA levels reached the highest levels later and also decayed more slowly when the concentration of arsenite was raised.
To study the effect on the stimulation of hsp68-mRNA levels in arsenite-pretreated cells of an exposure to low concentrations of arsenite, exposures of 1 h to 100 µM were chosen as the sensitizing treatments. As shown above, an exposure to 100 µM arsenite is considered to be a mild treatment, appropriate for the study of the stimulatory effect of a low doses.
Figure
3. The influence of a
step-down arsenite treatment on the induction of hsp68-mRNA. Pretreatment with
0 or 100 µM arsenite from 1 to 0 h, was followed by a subsequent
incubation in 0, 1, 3 or 10 µM arsenite. The means and standard deviation are
shown after quantification of five experiments.
In order to study the synthesis of hsp68 as well as of other hsps, the effect of the step-down (arsenite) protocol was studied on the induction of specific heat shock proteins.
3.2. HEAT SHOCK PROTEINS
A 1 h treatment with 0 or 100 µM arsenite was either or not followed by a continuous exposure to low concentrations of arsenite (1, 3 or 10 µM). At 3 h intervals samples were incubated with [35S]-methionine for 1 h, andsubsequently prepared for one-dimensional gel-electrophoresis. In figure 4 an example is given of a 1 hour treatment with 100 µM followed by either 0, 1, 3 or 10 µM arsenite. The relative rate of synthesis of each hsp was quantified and the time course of the different hsps, expressed relative to actin synthesis, was determined and presented in figure 5. A treatment with 100 µM, followed by an incubation in an arsenite-free medium shows a transient increase in the synthesis of hsp60, 68, 70, 84 and 100. This condition is considered to be a 'mild' treatment. The increase in relative synthesis of most hsps reaches a maximum at 3 h after the pretreatment.
Figure
5. The relative synthesis of
the major hsps (hsp60, 68, 70, 84 and 100) after a treatment with 0 µM (A) or
100 µM (B) arsenite for 1 h, subsequently followed by an incubation in 0 µM
(circle), 1 µM (triangle-up), 3 µM (square) or 10 µM (triangle-down) arsenite
during the indicated time (h). The data are expressed as the ratio of the
optical density between the indicated hsp and actin.
When cells pretreated with 100 µM are incubated in media containing a low concentration of arsenite (1, 3 or 10 µM), an enhanced and prolonged induction of several hsps can be observed. Incubation of cultures in low concentrations of arsenite only, did not induce the synthesis of hsps. As the treatment with 3 or 10 µM arsenite alone (without pretreatment) did not cause any induction of hsps, it is concluded that a step-down arsenite treatment enhances the relative synthesis of hsps. Apparently, the sensitizing effect of an arsenite pretreatment can be determined in terms of an enhanced synthesis of specific hsps.
3.3. SURVIVAL CHARACTERISTICS AND DEVELOPMENT OF TOLERANCE IN ARSENITE-PRETREATED CELLS
It has been demonstrated that hsps facilitate repair and refolding of stress-damaged proteins and also prevent irreversible denaturation and aggregation of cellular proteins (for review see Hightower, 1991; Welch, 1992; Parsell and Lindquist, 1994). In this latter function they may act as 'protector proteins'.This may be one of the underlying molecular mechanisms by which cells acquire tolerance; i.e. tolerance to an otherwise lethal stress after exposure to a sublethal stress which is sufficient to induce stress protein synthesis.
We investigated whether the induction of arsenite-tolerance in arsenite-pretreated H35 cell cultures could be modulated by a post-incubation with low concentrations of arsenite. First, the H35 cell population was incubated for 1 h with 100 µM arsenite. Subsequently the culture was incubated with a lower concentration of arsenite (up to 10 µM) for 4 h. Then the sensitivity or the degree of tolerance was tested by establishing the survival after an exposure for 2 h to 300 µM arsenite. In Figure 6A it is shown that when cells have been pre-treated with 100 µM arsenite and post-incubated at 1, 3 or 10 µM, a decreased sensitivity is observed in comparison with cells that have been post-incubated in the absence of a low doses of arsenite. Apparently, pre-treated cells are able to develop a higher level of tolerance in the presence of low concentrations of arsenite. In cultures without pretreatment the presence of low concentrations of arsenite for up to 4 h did not induce tolerance (data not shown).
Figures
6.
6A: The percentage survival after a step-down
arsenite treatment, indicating the level of tolerance obtained after a
pretreatment with 100 µM arsenite, followed by a 4 h post-treatment with either
0, 1, 3 or 10 µM arsenite and finally a test treatment with 300 µM arsenite for
2 h.
6B: Survival curves of H35 cells subjected to
a step-down arsenite treatment. Cell cultures were incubated during 1 h in 100
µM arsenite, followed by a post-treatment in either 0 µM (open circle) or 10 µM
(closed circle) arsenite for 4 h, subsequently followed by a test-treatment
with 300 µM during a period of up to 4 h. Data were corrected for the effect of
pretreatment and presented as the surviving fraction of pre-treated cells.
In order to investigate whether the rate of cell killing and/or the capacity to withstand a stress treatment was affected (represented by D0 and Dq respectively); a pretreatment with 100 µM arsenite was followed by an incubation with either 0 or 10 µM for 4 h. After this incubation a test-treatment with 300 µM over a period of up to 4 h was given. As can be observed in figure 6B the survival curve of the cell culture which is post-treated with 10 µM, shows a slope which is less steep than found in cultures which were not post-treated and starts to decline at a later moment in time. Consequently, a larger D0-value is found (51.4 min in comparison with 34.7 min in post-treated and non-posttreated cultures respectively) as well as a larger Dq value (89.0 min in post-treated in comparison with 65.2 min in non-post-treated cultures), indicating that cells die at a lower rate when post-treated with 10 µM. The larger Dq value indicates that cells start to die later in time when post-treated with 10 µM, which means that the cell population has obtained a larger capacity to withstand a stress treatment.
From the experimental data presented in this paper, it can be concluded that an exposure of Reuber H35 rat hepatoma cells to arsenite concentrations of 100 µM for 1 hour, rapidly changes the sensitivity to a second arsenite treatment with respect to the induction of the heat shock proteins and the development of tolerance.
The present experiments demonstrate that after a step-down arsenite treatment and under appropriate conditions, an increase of the level of hsp68-mRNA and an increased synthesis of arsenite-induced heat-shock proteins could be observed when compared with the rate of hsp68-mRNA and of hsp synthesis in cultures which received arsenite pretreatment only. An incubation of cell cultures in low concentrations of arsenite only (without arsenite-pretreatment), did not result in any detectable increase of the hsp68-mRNA level nor in a detectable induction of hsp-synthesis. It was also observed that under arsenite step-down conditions, the development of tolerance was enhanced in comparison with the level of tolerance obtained by a single treatment with arsenite.
These results are in agreement with our previous experiments using heat as the stressor condition. It has been shown that during step-down heating under appropriate conditions, the relative rate of hsp-synthesis as well as of hsp68-mRNA was increased to a greater extent in comparison with the values observed in cultures to which only the heat pretreatment was applied (Schamhart et al., 1992; Van Wijk et al., 1994). In addition, an enhanced development of tolerance during step-down heating conditions was described (Delpino et al., 1992; Van Wijk et al., 1994).
In conclusion, a stimulation of the self-recovery has been demonstrated upon the application of the isopathic principle (the similia principle in its most elementary form). For the next step in our research program, the model presented here can be used for the study of the specificity of the similia-principle. The results of the experiments aimed to identify the specificity of the similia-principle are described elsewhere in this volume.
Acknowledgement
The financial support of the HomInt organisation is gratefully acknowledged.
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