In vitro study of the stress response of human skin cells to GSM-1800 mobile phone signals compared to UVB radiation and heat shock

The evolution of mobile phone technology is toward an increase of the carrier frequency up to 2.45 GHz. Absorption of radiofrequency (RF) radiation becomes more superficial as the frequency increases. This increasingly superficial absorption of RF radiation by the skin, which is the first organ exposed to RF radiation, may lead to stress responses in skin cells. We thus investigated the expression of three heat-shock proteins (HSP70, HSC70, HSP27) using immunohistochemistry and induction of apoptosis by flow cytometry on human primary keratinocytes and fibroblasts. A well-characterized exposure system, SXC 1800, built by the IT'IS foundation was used at 1800 MHz, with a 217 Hz modulation. We tested a 48-h exposure at an SAR of 2 W/kg (ICNIRP local exposure limit). Skin cells were also irradiated with a 600 mJ/cm2 single dose of UVB radiation and subjected to heat shock (45 degrees C, 20 min) as positive controls for apoptosis and HSP expression, respectively. The results showed no effect of a 48-h GSM-1800 exposure at 2 W/kg on either keratinocytes or fibroblasts, in contrast to UVB-radiation or heat-shock treatments, which injured cells. We thus conclude that the GSM-1800 signal does not act as a stress factor on human primary skin cells in vitro.     

Cytogenetic studies in human cells exposed in vitro to GSM-900 MHz radiofrequency radiation using R-banded karyotyping

It is important to determine the possible effects of exposure to radiofrequency (RF) radiation on the genetic material of cells since damage to the DNA of somatic cells may be linked to cancer development or cell death and damage to germ cells may lead to genetic damage in next and subsequent generations. The objective of this study was to investigate whether exposure to radiofrequency radiation similar to that emitted by mobile phones of second-generation standard Global System for Mobile Communication (GSM) induces genotoxic effects in cultured human cells. The cytogenetic effects of GSM-900 MHz (GSM-900) RF radiation were investigated using R-banded karyotyping after in vitro exposure of human cells (amniotic cells) for 24 h. The average specific absorption rate (SAR) was 0.25 W/kg. The exposures were carried out in wire-patch cells (WPCs) under strictly controlled conditions of temperature. The genotoxic effect was assessed immediately or 24 h after exposure using four different samples. One hundred metaphase cells were analyzed per assay. Positive controls were provided by using bleomycin. We found no direct cytogenetic effects of GSM-900 either 0 h or 24 h after exposure. To the best of our knowledge, our work is the first to study genotoxicity using complete R-banded karyotyping, which allows visualizing all the chromosomal rearrangements, either numerical or structural.     

Characterization of two heat shock proteins (Hsp70/Hsc70) from grass carp (Ctenopharyngodon idella): evidence for their differential gene expression, protein synthesis and secretion in LPS-challenged peripheral blood lymphocytes

Two cDNAs, encoding the stress-inducible 70-kDa heat shock protein (Hsp70) and the constitutively expressed 70-kDa heat shock cognate protein (Hsc70), were isolated from grass carp. The Hsp70 and Hsc70 cDNAs were 2250 bp and 2449 bp in length and contained 1932 bp and 1953 bp open reading frames, respectively. Tissue distribution results showed that Hsp70/Hsc70 was highly expressed in gill, kidney, head kidney and peripheral blood lymphocytes (PBLs). Using grass carp PBLs as a cell model, effects of lipopolysaccharide (LPS) on the mRNA and protein levels of Hsp70/Hsc70 were examined. In this case, LPS increased the mRNA expression of Hsp70 in a time- and dose-dependent manner, but had no effect on Hsc70 mRNA expression. In agreement with this, LPS elevated the intracellular Hsp70 markedly, but not the Hsc70 protein levels in parallel experiments. Furthermore, Hsp70 protein was also detected in culture medium. Moreover, inhibition of LPS on Hsp70 release in a time-dependent manner was observed, indicating that there may be a dynamic balance between Hsp70 stores and Hsp70 release in grass carp PBLs following exposure to LPS. Taken together, these results not only shed new insights into the different regulations of LPS on Hsp70/Hsc70 gene expression, protein synthesis and release, but also provide a basis for further study on the functional role of Hsp70 in fish immune response.     

Cognitive impairment in rats after long-term exposure to GSM-900 mobile phone radiation

Considering the frequent use of mobile phones, we have directed attention to possible implications on cognitive functions. In this study we investigated in a rat model the long-term effects of protracted exposure to Global System for Mobile Communication-900 MHz (GSM-900) radiation. Out of a total of 56 rats, 32 were exposed for 2 h each week for 55 weeks to radio-frequency electromagnetic radiation at different SAR levels (0.6 and 60 mW/kg at the initiation of the experimental period) emitted by a (GSM-900) test phone. Sixteen animals were sham exposed and eight animals were cage controls, which never left the animal house. After this protracted exposure, GSM-900 exposed rats were compared to sham exposed controls. Effects on exploratory behaviour were evaluated in the open-field test, in which no difference was seen. Effects on cognitive functions were evaluated in the episodic-like memory test. In our study, GSM exposed rats had impaired memory for objects and their temporal order of presentation, compared to sham exposed controls (P = 0.02). Detecting the place in which an object was presented was not affected by GSM exposure. Our results suggest significantly reduced memory functions in rats after GSM microwave exposure (P = 0.02).     

70-kDa heat shock protein expression in cultured rat astrocytes after hypoxia: regulatory effect of almitrine

Induction of heat shock proteins (Hsps), especially the 70-kDa family, is well observed in nervous tissues in response to various stressful conditions. By using rat astrocytes in primary culture, the expression of the inducible (Hsp70) and the constitutive (Hsc70) 70-kDa Hsps immunoreactivity of cells exposed to hypoxic conditions has been investigated. We observed that exposure of astroglial cells to an hypoxic-normoxic sequence induces a significant decrease of Hsc70 immunoreactivity. The presence of the heat inducible stress protein Hsp70 is never observed in hypoxic cells not in control. Hsc 70 lowering is associated with ultrastructural alterations characterized by mitochondria swelling, formation of vacuoles and accumulation of dense material in the cell cytoplasm. The effects of addition of almitrine to the culture medium before and during hypoxia on Hsps immunoreactivity have been examined. The presence of the drug prevents the decrease of Hsc 70 immunoreactivity induced by hypoxia. Furthermore, some ultrastructural improvement is observed in astroglial cells treated with almitrine suggesting some protecting role of Hsc70 on cell damage induced by hypoxia.     

Hsp105alpha suppresses Hsc70 chaperone activity by inhibiting Hsc70 ATPase activity

Hsp105alpha is a mammalian member of the HSP105/110 family, a diverged subgroup of the HSP70 family. Hsp105alpha associates with Hsp70/Hsc70 as complexes in vivo and regulates the chaperone activity of Hsp70/Hsc70 negatively in vitro and in vivo. In this study, we examined the mechanisms by which Hsp105alpha regulates Hsc70 chaperone activity. Using a series of deletion mutants of Hsp105alpha and Hsc70, we found that the interaction between Hsp105alpha and Hsc70 was necessary for the suppression of Hsc70 chaperone activity by Hsp105alpha. Furthermore, Hsp105alpha and deletion mutants of Hsp105alpha that interacted with Hsc70 suppressed the ATPase activity of Hsc70, with the concomitant appearance of ATPase activity of Hsp105alpha. As the ATPase activity of Hsp70/Hsc70 is essential for the efficient folding of nonnative protein substrates, Hsp105alpha is suggested to regulate the substrate binding cycle of Hsp70/Hsc70 by inhibiting the ATPase activity of Hsp70/Hsc70, thereby functioning as a negative regulator of the Hsp70/Hsc70 chaperone system.     

Modulation of HSP70 GlcNAc-directed lectin activity by glucose availability and utilization

It is well-accepted that protein quality control (occurring either after protein synthesis or after cell damage) is mainly ensured by HSP, but the mechanism by which HSP decides whether the protein will be degraded or not is poorly understood. Within this framework, it has been hypothesized that O-GlcNAc, a cytosolic and nuclear-specific glycosylation whose functions remain unclear, could take a part in the protection of proteins against degradation by modifying both the proteins themselves and the proteasome. Because the synthesis of O-GlcNAc is tightly correlated to glucose metabolism and Hsp70 was endowed with GlcNAc-binding property, we studied the relationship between GlcNAc-binding activity of both Hsp70 and Hsc70 (the nucleocytoplasmic forms of HSP70 family) and glucose availability and utilization. We thus demonstrated that low glucose concentration, inhibition of glucose utilization with 2DG, or inhibition of glucose transport with CytB led to an increase of Hsp70 and Hsc70 lectin activities. Interestingly, the response of Hsp70 and Hsc70 lectin activities toward variations of glucose concentration appeared different: Hsp70 lost its lectin activity when glucose concentration was >5 mM (i.e., physiological glucose concentration) in contrast to Hsc70 that exhibited a maximal lectin activity for glucose concentration approximately 5 mM and at high glucose concentrations. This work also demonstrates that HSP70 does not regulate its GlcNAc-binding properties through its own O-GlcNAc glycosylation.     

Modulation of heat shock protein response in SH-SY5Y by mobile phone microwaves

AIM: To investigate putative biological damage caused by GSM mobile phone frequencies by assessing electromagnetic fields during mobile phone working. METHODS: Neuron-like cells, obtained by retinoicacid-induced differentiation of human neuroblastoma SH-SY5Y cells, were exposed for 2 h and 4 h to microwaves at 1800 MHz frequency bands. RESULTS: Cell stress response was evaluated by MTT assay as well as changes in the heat shock protein expression (Hsp20, Hsp27 and Hsp70) and caspase-3 activity levels, as biomarkers of apoptotic pathway. Under our experimental conditions, neither cell viability nor Hsp27 expression nor caspase-3 activity was significantly changed. Interestingly, a significant decrease in Hsp20 expression was observed at both times of exposure, whereas Hsp70 levels were significantly increased only after 4 h exposure. CONCLUSION: The modulation of the expression of Hsps in neuronal cells can be an early response to radiofrequency microwaves.     

Differential acquisition of antigenic peptides by Hsp70 and Hsc70 under oxidative conditions

Hsp70 and Hsc70 are two chaperones of high homology expressed under contrasting situations. Hsc70 is constitutively expressed and poorly stress-inducible, whereas Hsp70 is unabundant in normal physiological situations and strongly induced under oxidative stress. In the present study we show that the chaperoning activity of purified Hsp70 and Hsc70 is minimal under reducing conditions and increases in environments that mimic oxidative stress. Association with peptides is more pronounced for Hsp70 than for Hsc70 in every condition tested and is accompanied with a gradual change in secondary structure during oxidation. The binding of peptides to Hsp70 and Hsc70 under oxidative conditions is not reversible by treatment with a reducing agent, confirming that other chaperone-associated factors are required for substrate release. These findings support the idea that formation of HSP70-peptide complexes and possibly their immunogenicity is enhanced in conditions of stress.     

Heteromeric complexes of heat shock protein 70 (HSP70) family members,including Hsp70B′, in differentiated human neuronal cells

Human neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis have been termed “protein misfolding disorders.” Upregulation of heat shock proteins that target misfolded aggregation-prone proteins has been proposed as a potential therapeutic strategy to counter neurodegenerative disorders. The heat shock protein 70 (HSP70) family is well characterized for its cytoprotective effects against cell death and has been implicated in neuroprotection by overexpression studies. HSP70 family members exhibit sequence and structural conservation. The significance of the multiplicity of HSP70 proteins is unknown. In this study, coimmunoprecipitation was employed to determine if association of HSP70 family members occurs, including Hsp70B′ which is present in the human genome but not in mouse and rat. Heteromeric complexes of Hsp70B′, Hsp70, and Hsc70 were detected in differentiated human SH-SY5Y neuronal cells. Hsp70B′ also formed complexes with Hsp40 suggesting a common co-chaperone for HSP70 family members.     

Salicylic acid influences Hsp70/Hsc70 expression in Lycopersicon esculentum: dose- and time-dependent induction or potentiation

Overexpression of heat-shock (HS) proteins (HSP) is often sufficient to protect against lethal environmental stresses. Anti-inflammatory salicylates potentiate the induction of the 70 kDa HSP (Hsp70) in mammals in response to HS and enhance thermotolerance. In plants, salicylic acid (SA) is a natural signalling molecule, mediating resistance in response to avirulent pathogens. The influence of SA on the HS response in plants is, however, unknown. We investigated the effect of SA, alone or with HS, on Hsp70/Hsc70 expression in tomato cells using biometabolic labelling and Western blotting. A dose- and time-dependent influence on Hsp70/Hsc70 accumulation was observed: SA at 1.0 mM (3 h) potentiated heat-induced accumulation, while 1.0 mM (5 h) and 0.5 mM (8 h) induced expression, the latter preceded by increased membrane permeability. These results suggest that in plants, as in mammals, low SA concentrations do not induce Hsp70/Hsc70 expression but potentiate HS induction and confer membrane protection, while cytotoxic levels induce Hsp70/Hsc70.     

Bag-1M accelerates nucleotide release for human Hsc70 and Hsp70 and can act concentration-dependent as positive and negative cofactor.

The cytosol of mammalian cells contains several Hsp70 chaperones and an arsenal of cochaperones, including the anti-apoptotic Bag-1M protein, which regulate the activities of Hsp70s by controlling their ATPase cycles. To elucidate the regulatory function of Bag-1M, we determined its influence on nucleotide exchange, substrate release, ATPase rate, and chaperone activity of the housekeeping Hsc70 and stress-inducible Hsp70 homologs of humans. Bag-1M and a C-terminal fragment of it are potent nucleotide exchange factors as they stimulated the ADP dissociation rate of Hsc70 and Hsp70 up to 900-fold. The N-terminal domain of Bag-1M decreased the affinity of Bag-1M for Hsc70/Hsp70 by 4-fold, indicating a modulating role of the N terminus in Bag-1M action as nucleotide exchange factor. Bag-1M inhibited Hsc70/Hsp70-dependent refolding of luciferase in the absence of P(i). Surprisingly, under physiological conditions, i.e. low Bag-1M concentrations and presence of P(i), Bag-1M activates the chaperone action of Hsc70/Hsp70 in luciferase refolding. Bag-1M accelerated ATP-triggered substrate release by Hsc70/Hsp70. We propose that Bag-1M acts as substrate discharging factor for Hsc70 and Hsp70.     

Hsp70 localizes differently from chaperone Hsc70 in mouse mesoangioblasts under physiological growth conditions

Mouse A6 mesoangioblasts express Hsp70 even in the absence of cellular stress. Its expression and its intracellular localization were investigated under normal growth conditions and under hyperthermic stress. Immunofluorescence assays indicated that without any stress a fraction of Hsp70 co-localized with actin microfilaments, in the cell cortex and in the contractile ring of dividing cells, while the Hsc70 chaperone did not. Hsp70 immunoprecipitation assays confirmed that a portion of Hsp70 binds actin. Immunoblot assays showed that both proteins were present in the nucleus. After heat treatment Hsp70 and actin continued to co-localize in the leading edge of A6 cells but not on microfilaments. Although Hsp70 and Hsc70 are both basally synthesized they showed different cellular distribution, suggesting an Hsp70 different activity respect to the Hsc70 chaperone. Moreover, we found Hsp70 in the culture medium as it has been described in other cell types.     

Reactions of keratinocytes to in vitro millimeter wave exposure.

The effects of millimeter waves (MW) on human keratinocytes were studied in vitro using the HaCaT keratinocyte cell line. MW-induced modulation of keratinocyte function was studied in proliferation, adhesion, chemotaxis, and interleukin-1beta (IL-1beta) production assays. Spontaneous proliferation, adhesion to tissue culture plate, random migration, and IL-8- and RANTES induced chemotaxis were not affected by exposure of cells to millimeter waves under the following conditions: frequency, 61.22 GHz; SAR, 770 W/kg; duration of exposure, 15-30 min. However, MW irradiation resulted in a modest but statistically significant increase in the intracellular level of IL-1beta. These data suggest that exposure of human skin (with keratinocytes being the major component of epidermis) to MW can cause activation of basal keratinocytes resulting in an elevated level of IL-1beta production.     

Identification of CHIP, a Novel Tetratricopeptide Repeat-Containing Protein That Interacts with Heat Shock Proteins and Negatively Regulates Chaperone Functions

The chaperone function of the mammalian 70-kDa heat shock proteins Hsc70 and Hsp70 is modulated by physical interactions with four previously identified chaperone cofactors: Hsp40, BAG-1, the Hsc70-interacting protein Hip, and the Hsc70-Hsp90-organizing protein Hop. Hip and Hop interact with Hsc70 via a tetratricopeptide repeat domain. In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture. Hsc70 and Hsp70 were identified as potential interaction partners for this protein in a yeast two-hybrid screen. In vitro binding assays demonstrated direct interactions between CHIP and both Hsc70 and Hsp70, and complexes containing CHIP and Hsc70 were identified in immunoprecipitates of human skeletal muscle cells in vivo. Using glutathione S-transferase fusions, we found that CHIP interacted with the carboxy-terminal residues 540 to 650 of Hsc70, whereas Hsc70 interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of CHIP. Recombinant CHIP inhibited Hsp40-stimulated ATPase activity of Hsc70 and Hsp70, suggesting that CHIP blocks the forward reaction of the Hsc70-Hsp70 substrate-binding cycle. Consistent with this observation, both luciferase refolding and substrate binding in the presence of Hsp40 and Hsp70 were inhibited by CHIP. Taken together, these results indicate that CHIP decreases net ATPase activity and reduces chaperone efficiency, and they implicate CHIP in the negative regulation of the forward reaction of the Hsc70-Hsp70 substrate-binding cycle.     

Nuclear-mitochondrial cross-talk during heat shock in Arabidopsis cell culture

Apart from energy generation, mitochondria perform a signalling function determining the life and death of a cell under stress exposure. In the present study we have explored patterns of heat-induced synthesis of Hsp101, Hsp70, Hsp17.6 (class I), Hsp17.6 (class II) and Hsp60, and the development of induced thermotolerance in Arabidopsis thaliana cell culture under conditions of mitochondrial dysfunction. It was shown that treatment by mitochondrial inhibitors and uncouplers at the time of mild heat shock downregulates HSP synthesis, which is important for induced thermotolerance in plants. The exposure to elevated temperature induced an increase in cell oxygen consumption and hyperpolarization of the inner mitochondrial membrane. Taken together, these facts suggest that mitochondrial functions are essential for heat-induced HSP synthesis and development of induced thermotolerance in A. thaliana cell culture, suggesting that mitochondrial-nuclear cross-talk is activated under stress conditions. Treatment of Arabidopsis cell culture at 50 degrees C initiates a programmed cell death determined by the time course of viability decrease, DNA fragmentation and cytochrome c release from mitochondria. As treatment at 37 degrees C protected Arabidopsis cells from heat-induced cell death, it may be suggested that Hsp101, Hsp70 and small heat-shock proteins, the synthesis of which is induced under these conditions, are playing an anti-apoptotic role in the plant cell. On the other hand, drastic heat shock upregulated mitochondrial Hsp60 synthesis and induced its release from mitochondria to the cytosol, indicating a pro-apoptotic role of plant Hsp60.     

Human keratinocytes in culture exhibit no response when exposed to short duration, low amplitude, high frequency (900 MHz) electromagnetic fields in a reverberation chamber

We exposed normal human epidermal keratinocytes to short duration, high frequency, and low amplitude electromagnetic fields, similar to that used by mobile phone technologies. We paid particular attention to the control of the characteristics of the electromagnetic environment generated within a mode stirred reverberation chamber (statistical homogeneity and isotropy of the field and SAR distribution). Two non‐thermal exposure conditions were tested on the epidermal cells: 10‐min exposure with a field amplitude of 8 V/m, and 30 min with 41 V/m. Corresponding specific absorption rates ranged from 2.6 to 73 mW/kg (continuous wave, 900 MHz carrier frequency). We collected RNA from cells subjected to these conditions and used it for a large‐scale microarray screening of over 47000 human genes. Under these conditions, exposure of keratinocytes to the electromagnetic field had little effect; only 20 genes displayed significant modulation. The expression ratios were very small (close to 1.5‐fold change), and none of them were shared by the two tested conditions. Furthermore, those assayed using polymerase chain reaction did not display significant expression modulation (overall mean of the exposed samples: 1.20 ± 0.18). In conclusion, the data presented here show that cultured keratinocytes are not significantly affected by EMF exposure. Bioelectromagnetics 32:302–311, 2011. © 2010 Wiley‐Liss, Inc.     

Quantitative mRNA expression profiling of heat-shock protein families in rainbow trout cells

We isolated multiple HSPs from rainbow trout Oncorhynchus mykiss RTG-2 cells and quantitatively compared their mRNA levels between unstressed and heat-shocked cells using real-time RT-PCR analysis. Consequently, we isolated nine cDNAs encoding HSPs from heat-shocked RTG-2 cells, namely, Hsp90betaa, Hsp90betab, Grp78, Hsp70a, Hsc70a, Hsc70b, Cct8, Hsp47, and DnaJ homolog. Quantitative RT-PCR analyses, in which Hsp70b isolated previously was included, showed that the mRNA accumulation levels of Hsp70a, Hsp70b, Hsc70a, Hsc70b, and Hsp47 were significantly increased after heat shock, and the increased levels of two Hsp70s, Hsp70a, and Hsp70b, were most conspicuous. In the case of Hsc70s, the increased level of Hsc70b was more remarkable than that of Hsc70a. These results demonstrate the importance of a comprehensive expression analysis of HSPs for better understanding of the cellular stress response in fish, especially in tetraploid species such as rainbow trout.     

Hsp70 Architecture: The Formation of Novel Polymeric Structures of Hsp70.1 and Hsc70 after Proteotoxic Stress

Heat induces Hsp70.1 (HSPA1) and Hsc70 (HSPA8) to form complex detergent insoluble cytoplasmic and nuclear structures that are distinct from the cytoskeleton and internal cell membranes. These novel structures have not been observed by earlier immunofluorescence studies as they are obscured by the abundance of soluble Hsp70.1/Hsc70 present in cells. While resistant to detergents, these Hsp70 structures display complex intracellular dynamics and are efficiently disaggregated by ATP, indicating that this pool of Hsp70.1/Hsc70 retains native function and regulation. Hsp70.1 promotes the repair of proteotoxic damage and cell survival after stress. In heated fibroblasts expressing Hsp70.1, Hsp70.1 and Hsc70 complexes are efficiently disaggregated before the cells undergo-heat induced apoptosis. In the absence of Hsp70.1, fibroblasts have increased rates of heat-induced apoptosis and maintain stable insoluble Hsc70 structures. The differences in the intracellular distribution of Hsp70.1 and Hsc70, combined with the ability of Hsp70.1, but not Hsc70, to promote the disaggregation of insoluble Hsp70.1/Hsc70 complexes, indicate that these two closely related proteins perform distinctly different cellular functions in heated cells.