Induction of a cell-survival adaptive response in MRC-5 cells by hydroquinone

Although it is known that some cell types exhibit an adaptive response to low levels of cytotoxic agents, its molecular mechanism is still unclear and it has yet to be established whether this is a universal phenomenon that occurs in all cell types in response to exposure to every chemical. Hydroquinone is a synthetically produced as well as naturally occurring chemical. Human exposure to hydroquinone is predominantly through diet, cigarette smoke and occupational contact. Here, we asked whether exposure of human lung embryonic MRC-5 fibroblasts to low doses of hydroquinone leads to a cell-survival adaptive response. We further examined the possible mechanisms of an adaptive response using proteomics. We found that exposure of MRC-5 cells to low levels of hydroquinone resulted in adaptation to further exposure to lethal doses of hydroquinone at the cell-survival level, measured using the alamarBlue assay, lactate dehydrogenase leakage assay and Annexin V-FITC/PI staining. To determine the polypeptide products involved in the adaptive response, two-dimensional electrophoresis combined with mass spectrometry was performed. Twenty-three protein spots were significantly changed during the adaptive response. Among them, 21 protein spots were identified by peptide mass fingerprinting and/or peptide sequence analysis by MALDI-TOF-TOF. The identified proteins included proteins involved in energy metabolism, protein folding, redox regulation, cell structure and cell signaling. Our data suggest that the hydroquinone-induced adaptive response is a complex process involving in a modulation of diverse cellular functions, and that the redox regulation might be a common mechanism during the adaptive response.     

An investigation of hormesis of trichloroethylene in L-02 liver cells by differential proteomic analysis

Hormesis is the dose–response pattern of the biological responses to toxic chemicals, characterized by low-dose stimulation and high-dose inhibition. Although it is known that some cell types exhibit an adaptive response to low levels of cytotoxic agents, its molecular mechanism is still unclear and it has yet to be established whether this is a universal phenomenon that occurs in all cell types in response to exposure to every chemical. Trichloroethylene (TCE) is an organic solvent widely used and is released into the atmosphere from industrial degreasing operations. Acute (short-term) and chronic (long-term) inhalation exposure to trichloroethylene can affect the human health. In order to elucidate a cell-survival adaptive response of L-02 liver cells exposed to low dose of TCE, CCK-8 assay was used to assess cytotoxicity, and examined the possible mechanisms of hormesis by proteomics technology. We found that exposure of L-02 liver cells to low level of TCE resulted in adaptation to further exposure to higher level, about 1,000 protein-spots were obtained by two-dimensional electrophoresis (2-DE) and five protein spots were identified by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry sequencing of tryptic peptides. Our results suggest that a relationship may exist between identified proteins and TCE-induced hormesis, which are very useful for further study of the mechanism and risk assessment of TCE.     

Adaptive response and induced resistance

Cellular stress responses are upregulated following exposure to radiation and other DNA-damaging agents. Therefore radiation response can be dose dependent so that small acute exposures (and possibly exposures at very low dose rates?) are more lethal per unit dose than larger exposures above a threshold (typically 10-40 cGy) where induced radioprotection is triggered. We have termed these interlinked phenomena low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases. HRS/IRR has been recorded in cell-survival studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal normal-tissue models in vivo. There is indirect evidence that cell survival-related HRS/IRR in response to single doses is a manifestation of the same underlying mechanism that determines the well-known adaptive response in the two-dose case and that it can be triggered by high- and low-LET radiations as well as a variety of other stress-inducing agents such as hydrogen peroxide and chemotherapeutic agents. Little is currently known about the precise nature of this underlying mechanism, but there is evidence that it operates by increasing the amount and rate of DNA repair, rather than by indirect mechanisms such as modulation of cell-cycle progression or apoptosis. Changed expression of some genes, only in response to low and not high doses, may occur within a few hours of irradiation and this would be rapid enough to explain the phenomenon of induced radioresistance although its specific molecular components have yet to be identified. Net cancer risk is a balance between cell transformation and cell kill. Our known low-dose cell-survival responses suggest that lethality may more than compensate for transformation at low radiation doses. However, adaptive reduction in sensitivity to radio-mutation has also been reported, which implies the existence also of enhanced mutation following very low single doses. So far this has not been confirmed, but provided the trigger dose for mutational protection was lower than the trigger dose for protection against cytotoxicity, cell killing would still dominate over at least the first 10 cGy of low-LET exposure. This would lead to a non-linear, threshold, dose-risk relationship and even provide some explanation for anecdotal reports of apparent 'health promoting' effects and lowered cancer risk from very low exposure to ionising radiation.     

Reduced levels of ADP-ribosylatable elongation factor-2 in aged and SV40-transformed human cell cultures

The elongation step is involved in the regulation of protein synthesis during the cell cycle, environmental stress, ageing and transformation. Using a diphtheria toxin-mediated assay for measuring the levels of ADP-ribosylatable elongation factor EF-2, we have observed an irreversible decrease of up to 64% in the amount of ADP-ribosylatable EF-2 in normal diploid human fibroblasts MRC-5 undergoing ageing in vitro. However, a similar decrease in low serum-associated G0/G1-arrested cells is reversible both in MRC-5 cells and in their SV40-transformed counterparts. Reduced levels of ADP-ribosylatable EF-2 could account for the slowing-down of protein synthesis during cell cycle arrest and during cellular ageing in culture.     

IR-inducible clusterin gene expression: a protein with potential roles in ionizing radiation-induced adaptive responses, genomic instability, and bystander effects

Clusterin (CLU) plays numerous roles in mammalian cells after stress. A review of the recent literature strongly suggests potential roles for CLU proteins in low dose ionizing radiation (IR)-inducible adaptive responses, bystander effects, and delayed death and genomic instability. Its most striking and evident feature is the inducibility of the CLU promoter after low, as well as high, doses of IR. Two major forms of CLU, secreted (sCLU) and nuclear (nCLU), possess opposite functions in cellular responses to IR: sCLU is cytoprotective, whereas nCLU (a byproduct of alternative splicing) is a pro-death factor. Recent studies from our laboratory and others demonstrated that down-regulation of sCLU by specific siRNA increased cytotoxic responses to chemotherapy and IR. sCLU was induced after low non-toxic doses of IR (0.02-0.5 Gy) in human cultured cells and in mice in vivo. The low dose inducibility of this survival protein suggests a possible role for sCLU in radiation adaptive responses, characterized by increased cell radioresistance after exposure to low adapting IR doses. Although it is still unclear whether the adaptive response is beneficial or not to cells, survival of damaged cells after IR may lead to genomic instability in the descendants of surviving cells. Recent studies indicate a link between sCLU accumulation and cancer incidence, as well as aging, supporting involvement of the protein in the development of genomic instability. Secreted after IR, sCLU may also alter intracellular communication due to its ability to bind cell surface receptors, such as the TGF-beta receptors (types I and II). This interference with signaling pathways may contribute to IR-induced bystander effects. We hypothesize that activation of the TGF-beta signaling pathway, which often occurs after IR exposure, can in turn activate the CLU promoter. TGF-beta and IR-inducible de novo synthesized sCLU may then bind the TGF-beta receptors and suppress downstream growth arrest signaling. This complicated negative feedback regulation most certainly depends on the cellular microenvironment, but undoubtedly represents a potential link between IR-induced adaptive responses, genomic instability and bystander effects. Further elucidation of clusterin protein functions in IR responses are clearly warranted.     

Comparative proteomic analysis of Arabidopsis thaliana roots between wild type and its salt-tolerant mutant

Two-dimensional electrophoresis (2-DE) showed the variation expression of Arabidopsis thaliana root proteins between wild type and its salt-tolerant mutant obtained from cobalt-60 γ ray radiation. Forty-six differential root protein spots were reproducibly presented on 2-DE maps, and 29 spots were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MS). Fifteen protein spots corresponding to 10 proteins, and 14 protein spots corresponding to 9 proteins were constitutively up-regulated and down-regulated in the salt-tolerant mutant root. Bioinformatic analysis indicated that those differential proteins might be involved in the regulation of redox homeostasis, nucleotide metabolism, signal transduction, stress response and defense, carbohydrate metabolism, and cell wall metabolism. Peroxidase 22 might be a versatile enzyme and might play dual roles in both cell wall metabolism and regulation of redox homeostasis. Our work provides not only new insights into salt-responsive proteins in root, but also the potential salt-tolerant targets for further dissection of molecular mechanism adapted by plants during salt stress.     

Redox control and the evolution of multicellularity

Redox chemistry, involving the transfer of electrons and hydrogen atoms, is central to energy conversion in respiration; in addition, control of gene expression by redox state commonly occurs in bacteria, allowing a rapid response to environmental changes, such as altered food supply. Colonial metazoans often encrust surfaces over which the food supply varies in time or space; hence, in these organisms redox control of the development of feeding structures and gastrovascular connections could be similarly adaptive, allowing colonies to adjust the timing of development and spacing of structures in response to a variable food supply and other environmental factors. Experimental perturbations of redox state in colonial hydroids support this notion of adaptive redox control, and redox signaling in metazoans may have evolved in this ecological context. At the same time, redox signaling has important consequences for the evolutionary transition from unicellular to multicellular organisms. Unlike protein or peptide signaling, redox signaling acting in concert with programmed cell death may automatically inflict a cost on those cells that "defect," that is, selfishly favor their own replication rate over that of the multicellular group. In this way, redox signaling may have allowed multicellular individuality to evolve and more easily be maintained.     

Radiation-induced bystander effects and adaptive responses--the Yin and Yang of low dose radiobiology?

Our current knowledge of the mechanisms underlying the induction of bystander effects by low doses of high or low LET ionizing radiation is reviewed. The question of what actually constitutes a protective effect is discussed in the context of adaptive (often referred to as hormetic or protective) responses. Finally the review considers critically, how bystander effects may be related to observed adaptive responses or other seemingly protective effects of low doses exposures. Bystander effects induce responses at the tissue level, which are similar to generalized stress responses. Most of the work involving low LET radiation exposure discussed in the existing literature measures a death response. Since many cell populations carry damaged cells without being exposed to radiation (so-called "background damage"), it is possible that low doses exposures cause removal of cells carrying potentially problematic lesions, prior to exposure to radiation. This mechanism could lead to the production of "U-shaped" or hormetic dose-response curves. The level of adverse, adaptive or apparently beneficial response will be related to the background damage carried by the original cell population, the level of organization at which damage or harm are scored and the precise definition of "harm". This model may be important when attempting to predict the consequences of mixed exposures involving low doses of radiation and other environmental stressors.     

Effect of low-dose radiation on repair of DNA and chromosome damage

In this report results of studies on the effect of different doses of low LET (linear energy transfer) radiations on the unscheduled DNA synthesis (UDS) and DNA polymerase activity as well as the induction of adaptive response in bone marrow cells (BMC) by low dose radiation were presented. It was found that whole-body irradiation (WBI) with X-ray doses above 0.5 Gy caused a dose-dependent depression of both UD5 and DNA polymerase activity, while low dose radiation below 250 mGy could stimulate the DNA repair synthesis and the enzyme activity. WBI of mice with low doses of X-rays in the range of 2-100 mGy at a dose rate of 57.3 mGy per minute induced an adaptive response in the BMC expressed as a reduction of chromosome aberrations following a second exposure to a larger dose (0.65 mGy). It was demonstrated that the magnitude of the adaptive response seemed to be inversely related to the induction dose. The possibility of induction of adaptive response in GO phase of the cell cycle and the possibility of a second induction of the adaptive response were discussed.     

Proteomics of Arabidopsis redox proteins in response to methyl jasmonate

Protein redox regulation is increasingly recognized as an important switch of protein activity in yeast, bacteria, mammals and plants. In this study, we identified proteins with potential thiol switches involved in jasmonate signaling, which is essential for plant defense. Methyl jasmonate (MeJA) treatment led to enhanced production of hydrogen peroxide in Arabidopsis leaves and roots, indicating in vivo oxidative stress. With monobromobimane (mBBr) labeling to capture oxidized sulfhydryl groups and 2D gel separation, a total of 35 protein spots that displayed significant redox and/or total protein expression changes were isolated. Using LC–MS/MS, the proteins in 33 spots were identified in both control and MeJA-treated samples. By comparative analysis of mBBr and SyproRuby gel images, we were able to determine many proteins that were redox responsive and proteins that displayed abundance changes in response to MeJA. Interestingly, stress and defense proteins constitute a large group that responded to MeJA. In addition, many cysteine residues involved in the disulfide dynamics were mapped based on tandem MS data. Identification of redox proteins and their cysteine residues involved in the redox regulation allows for a deeper understanding of the jasmonate signaling networks.     

Low doses of reactive oxygen species protect endothelial cells from apoptosis by increasing thioredoxin-1 expression

The redox regulator thioredoxin-1 (Trx-1) is required for the redox potential of the cell and exerts important functions in cell growth and apoptosis. Severe oxidative stress has been implicated in the oxidation of proteins and cell death. However, the role of low doses of reactive oxygen species (ROS) is poorly understood. Here, we show that 10 and 50 microM H2O2 and short-term exposure to shear stress significantly increased Trx-1 mRNA and protein levels in endothelial cells. Since it is known that Trx-1 exerts anti-apoptotic functions, we next investigated whether low doses of ROS can inhibit basal and serum-depletion induced endothelial cell apoptosis. Indeed, treatment of endothelial cells with 10 and 50 microM H2O2 significantly reduced apoptosis induction. Reduction of Trx-1 expression using an antisense oligonucleotide approach resulted in the induction of apoptosis and abolished the inhibitory effect of low doses of H2O2. Taken together, our results demonstrate that low doses of ROS act as signaling molecules and exert anti-apoptotic functions in endothelial cells via upregulation of the redox-regulator Trx-1.     

Reciprocal adaptive response of human peripheral lymphocytes induced by bleomycine or gamma rays.

The adaptive response and reciprocal adaptive response induced in vitro by exposure to low doses of gamma rays (0.05 Gy) or bleomycin (0.05 microg/ml) in human peripheral blood lymphocytes were assessed by the frequency of chromosome aberrations. Gamma rays (1.5 Gy) or bleomycin (1.5 microg/ml) were used as the challenge doses. In the experiments, blood samples from 5 healthy donors were investigated. It has been found that low doses of bleomycin and gamma rays induced a reciprocal adaptive response to high doses of gamma rays or bleomycin. Moreover, the results confirmed that the adaptive response did not correlate with the radiosensitivity of the peripheral blood lymphocytes.     

Effect of low-level radiation on the death of male germ cells

Hormetic and adaptive responses induced by low-level radiation in hematopoietic and immune systems have been observed, as shown by stimulatory effects on cell growth and resistance to subsequent radiation-induced cytogenetic damage. However, in terms of cell death by apoptosis, the effects of low-level radiation are controversial: Some studies showed decreased apoptosis in response to low-level radiation while others showed increased apoptosis. This controversy may be related to the radiation doses or dose rates and also, more importantly, to the cell types. Testes are one of the most radiosensitive organs. The loss of male germ cells after exposure to ionizing radiation has been attributed to apoptosis. In the present study, the effects of low-level radiation at doses up to 200 mGy on mouse male germ cells in terms of apoptosis and the expression of apoptosis-related proteins were examined at different times after whole-body exposure of mice to low-level radiation. In addition, the effect of pre-exposure to low-level radiation on subsequent cell death induced by high doses of radiation was examined to explore the possibility of low-level radiation-induced adaptive response. The results showed that low-level radiation in the dose range of 25-200 mGy induced significant increases in apoptosis in both spermatogonia and spermatocytes, with the maximal effect at 75 mGy. The increased apoptosis is most likely associated with Trp53 protein expression. Furthermore, 75 mGy low-level radiation given pre-irradiation led to an adaptive response of seminiferous germ cells to subsequent high-level radiation-induced apoptosis. These results suggest that low-level radiation induces increased apoptosis in male germ cells but also induces a significant adaptive response that decreases cell death after a subsequent high-dose irradiation.     

Extremely low priming doses of X radiation induce an adaptive response for chromosomal inversions in pKZ1 mouse prostate

An adaptive response is a response to a stress such as radiation exposure that results in a lower than expected biological response. We describe an adaptive response to X radiation in mouse prostate using the pKZ1 chromosomal inversion assay. pKZ1 mice were treated with a priming dose of 0.001, 0.01, 1 or 10 mGy followed 4 h later by a 1000-mGy challenge dose. All priming doses caused a similar reduction in inversions compared to the 1000-mGy group, supporting the hypothesis that the adaptive response is the result of an on/off mechanism. The adaptive response was induced by a priming dose of 0.001 mGy, which is three orders of magnitude lower than has been reported previously. The adaptive responses completely protected against the inversions that would have been induced by a single 1000-mGy dose as well as against a proportion of spontaneous background inversions. The distribution of inversions across prostate gland cross sections after priming plus challenge irradiation suggested that adaptive responses were predominantly due to reduced low-dose radiation-induced inversions rather than to reduced high-dose radiation-induced inversions. This study used radiation doses relevant to human exposure.     

Features of action of low doses of gamma-radiation on yeast cells

Lethal effect of low doses and adaptive response to low doses of prolonged irradiation were investigated in experiments on yeast cells. The phenomenon of hypersensitivity at low dozes was not found in yeast cells at gamma-irradiation. The adaptive response was observed after exposure to low doses of prolonged irradiation, the degree of the reaction depends on a dose rate. The adaptive reaction was kept for some time after the termination of adaptive irradiation then the sensitivity of cells increased even in comparison with unirradiated ones.     

Effect of low doses of ionizing radiation on thymus-dependent humoral immune response and the polyclonal activation of B lymphocytes

The exposure of mice to low dose of gamma-rays (10 cGy, 1 cGy/min) increased thymus-dependent humoral immune response and polyclonal activation of B-cells. Injection of hydroquinone eliminated radiation-induced augmentation of polyclonal response of B-lymphocyte. Thymogen decreased significantly the radiation-induced immunostimulation. The study of the dynamics of primary immune response showed that the period of radiation-induced elevation was followed by the phase of profound reduction of antibody formation. Possible negative consequences of action of low doses of ionizing radiation on immune system is discussed.