F. A. C. Wiegant

Thermotolerance in Cultured Hepatoma Cells: Cell Viability, Cell Morphology, Protein Synthesis, and Heat-Shock Proteins

Heat treatment at 42 degrees C of cultured Reuber H35 rat hepatoma cells induced both a rapid decrease of the rate of protein synthesis and the rounding up of the cells. Reincubation at 37 degrees C resulted in a gradual flattening of the cells, resumption of protein synthesis, and the synthesis of heat-shock proteins. During the recovery period cells developed a resistance toward a treatment which otherwise should lead to heat-induced cell death. Thermotolerance measured in terms of cell survival was paralleled by thermal resistance of protein synthesis and the cellular ability to refrain from rounding up under heat stress.

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.

Stressor-specific induction of heat shock proteins in rat hepatoma cells

In order to determine whether induction of specific stress proteins is dependent on a given stressor and whether induction of these proteins is linked to survival, Reuber H35 rat hepatoma cells were exposed to five different environmental stressors (heat shock, arsenite, cadmium, dinitrophenol and ethanol). The effect of these stressors was studied on cell survival as well as on inhibition and recovery of protein synthesis and on induction of heat shock proteins (hsps). In this article, we present evidence that several well-known hsp-inducers fail to stimulate specific hsps in a degree that is comparable to the induction of these hsps by heat shock. Most evidently, hsp60 is not induced by cadmium-treatment, whereas hsp100 is hardly induced by sodium arsenite. Treatment with DNP only slightly induces hsp68 and hsp84, whereas no detectable induction of hsps is observed after treatment with ethanol. In contrast, treatment with cadmium raises the amount of hsp28 to a higher level as compared to heat shock. A comparison of the stressor-specific induction of major hsps was also made under conditions of similar impact on cellular physiology: (a) stressor conditions up to the critical point that cell death starts to occur, and (b) conditions of iso-survival (50%). We conclude that hsps cannot be simply used as a general risk-assessment tool, and that the validation of stressor-specific risk-assessment warrants further research with larger groups of proteins.

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.

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.

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

1 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. 2 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. 3 The degree of stimulation appears to be stressorspecific, 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. 4 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. 5 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. 6 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. 7 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

Abstract. 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.

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.

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

In the past many scientists have published papers on hormesis, on molecular stress responses, and on the similia principle in homoeopathy. Very few, however, have stressed a common base of interdependence of these fields. Reviews the most important of these studies to demonstrate their evolution and their mutual importance. Furthermore, a multidisciplinary approach is chosen to demonstrate research into the beneficial effects of subharmful doses of toxicants administered in suboptimal conditions (such as in stressed or injured organisms and cells).