50 Hz magnetic fields of varying flux intensity affect cell shape changes in invertebrate immunocytes: the role of potassium ion channels

The effect induced by exposure to 50 Hz magnetic fields (MFs) in immunocytes from the mussel Mytilus galloprovincialis is evaluated. The whole animal was exposed for 15 and 30 min to MF intensities ranging from 200 to 1,000 microT. The changes in the cellular shape of immunocytes, expressed as shape factor (SF), were studied at different times after addition of the chemotacting substance N-formyl-Meth-Leu-Phe (fMLP). Results show that MFs provoke differing delays in fMLP-induced cellular shape changes: 200 microT are ineffective, while levels from 300 microT upwards cause a significant increase in immunocyte SF values compared to controls. Reactivation of the cells is possible up to an intensity of 600 microT. The use of PCO 400, an opener of ATP-sensitive K+ channels, shows that potassium channels are involved in the effect of MFs on M. galloprovincialis immunocytes.     

A comparison of heat shock protein genes from cultured cells of the cabbage armyworm, Mamestra brassicae, in response to heavy metals

Heat shock protein (HSP) genes, hsp90, hsp70, hsc70, hsp20.7, and hsp19.7, were cloned and sequenced from cultured cells of the cabbage armyworm, Mamestra brassicae. Analyses of the cDNA sequences revealed open reading frames of 2,151, 1,914, 1,962, 540, and 465 bp in lengths, which encode proteins with calculated molecular weights of 82.5, 69.9, 71.6, 20.7, and 19.7 kDa, respectively. An increased expression was observed in all five genes after exposure to a high temperature. The induction of gene expression was not observed during a low temperature exposure, but was observed when the cells were recovered at ambient temperature. Expression of hsp90, hsp70, and hsp20.7 was induced after exposure to 2 microM of cadmium, while the minimum cadmium concentration for induction of hsp19.7 was 5 microM. The induction of hsp90 expression was somewhat masked by basal levels of expression. Only hsp20.7 expression was induced by exposure to copper. Lead did not induce expression of any of the HSP genes tested. Cadmium-induced up-regulation of hsp70 expression was lasted longer than heat-induced one. These results suggest that hsp70 could be useful to assess the cellular distress or injury induced by cadmium.     

Regulation of the synthesis of heat-shock proteins in heat-resistant variants of Chinese hamster fibroblasts

The synthesis of the major heat-shock proteins (hsp) was compared in normal and heat-resistant Chinese hamster fibroblasts which express higher levels of the 70 kDa heat-shock protein (hsp70). Following exposure to a variety of experimental conditions that induce the elevated synthesis of the hsp, higher relative levels of hsp70 and lower relative levels of hsp89 and hsp110 were found in the heat-resistant variants. This effect was observed with all inducers tested. The relatively greater synthesis of hsp70 and relatively lower synthesis of hsp89 occurred at all temperatures tested and was found to be independent of cell culture conditions. The relatively greater increase in the levels of hsp70 in the heat-resistant variants after a mild heat shock was found to be a reflection of elevated levels of messenger RNA coding for this polypeptide. These results indicate that the heat-shock response in mammalian cells displays coordinate regulatory features and that the alteration of the expression of one of the hsp may affect the expression of the others.     

Comparative toxic potential of market formulation of two organophosphate pesticides in transgenic Drosophila melanogaster (hsp70-lacZ)

This study investigated the working hypothesis that two widely used organophosphate pesticides; Nuvan and Dimecron, exert toxic effects in Drosophila. Transgenic D. melanogaster (hsp70-lacZ) was used as a model for assaying stress gene expression and AchE activity as an endpoint for toxicity and also to evaluate whether stress gene expression is sufficient to protect against toxic insult of the chemicals and to prevent tissue damage. The study was extended to investigate the effect of the pesticides on the life cycle and reproduction of the organism. The study showed that Nuvan affected emergence of the exposed flies more drastically than Dimecron and the effect was lethal at the highest tested concentration (0.075 ppm). While Nuvan at 0.0075 and 0.015 ppm concentrations affected reproduction of the flies significantly, the effect of Dimecron was significant only at 0.015 and 0.075 ppm. Nuvan-exposed third-instar larvae exhibited a 1.2-fold to 1.5-fold greater hsp70 expression compared to Dimecron at concentrations ranging from 0.0075 to 0.075 ppm following 12 and 18 h exposure. While maximum expression of hsp70 was observed in Nuvan-exposed third-instar larval tissues following 18 h exposure at 0.075 ppm, Dimecron at the same dietary concentration induced a maximum expression of hsp70 following 24 h exposure. Further, concomitant with a significant induction of hsp70, significant inhibition of AchE was observed following chemical exposure and temperature shock. Concurrent with a significant decline in hsp70 expression in Nuvan-exposed larvae after 48 h at 0.075 ppm, tissue damage was evident. Dimecron-exposed larvae exhibited a plateau in hsp70 induction even after 48 h exposure and moderate tissue damage was observed in these larvae. The present study suggests that Nuvan is more cytotoxic than Dimecron in transgenic Drosophila melanogaster.     

Effects of 50 Hz magnetic fields on fMLP-induced shape changes in invertebrate immunocytes: The role of calcium ion channels

Plasma membrane Ca(2+) channels in immunocytes from the mussel Mytilus galloprovincialis exposed to 50 Hz sine wave magnetic fields (MFs) of various strengths were studied. At levels of 300 microT and above, MFs reduce shape changes in immunocytes induced by the chemotactic substance N-formyl-Meth-Leu-Phe, and this effect involves L-type Ca(2+) channels. Upon the addition of the Ca(2+) blocker verapamil to molluscan immunocytes exposed to MFs results in a synergistic cytotoxic action, while in the presence of the Ca(2+) opener SDZ-202, 791, a reactivation of the cells is observed. This suggests that, as previously reported for potassium channels, the damage to Ca(2+) channels induced by short exposure to MF at appropriate intensities is not permanent.     

Heat shock proteins hsp32 and hsp70 as biomarkers of an early response? In vitro induction of heat shock proteins after exposure of cell culture to carcinogenic compounds and their real mixtures

Heat shock proteins (hsp) are a highly conserved group of proteins that are synthesized as a response to different forms of stress (heat, toxic chemicals, diseases, non-physiological pH changes). Because of their high sensitivity to changes in the environment, these proteins were suggested as possible early biomarkers of exposure in ecotoxicological studies. The purpose of the present study was to check the suitability of hsp 32 and hsp70 as biomarkers of in vitro exposure to environmentally relevant carcinogens: polycyclic aromatic hydrocarbons (PAHs), their nitro-derivates, aromatic amines, acrylonitrile (ACN) and the mixture of organic compounds adsorbed onto ambient airborne particles (extractable organic matter, EOM).The expression of hsp 32 and hsp70 was studied in human diploid lung fibroblasts (HEL cells) and human monocytic leukaemia cells (THP-1 cells) incubated in vitro with different concentrations of dibenzo[a,l]pyrene (DB[a,l]P), 1-nitropyrene, (NP), 4-aminobiphenyl (ABP), ACN and EOM for different periods of time. The incubation of cells with DB[a,l]P, NP, ABP and EOM did not result in increased levels of hsp 32 or hsp70, either in dose- or time-dependent manner. ACN induced the expression of hsp 32 as well as hsp70 in HEL and THP-1 cells, which probably reflects its ability to induce oxidative stress. We conclude that hsp 32 and hsp70 are not suitable biomarkers of an early exposure to PAHs, their nitro-derivates, aromatic amines or EOM under the conditions used.     

Expression analysis of heat shock genes in the skin, spleen and blood of common carp (Cyprinus carpio) after cadmium exposure and hypothermia

Heat shock proteins are chaperones that play a pivotal role in controling multiple regulatory pathways such as stress defense, hormone signaling, cell cycle control, cell proliferation and differentiation, and apoptosis. In this study, the expression patterns of four well-known heat shock genes (hsp70, hsc70-1, hsc70-2 and hsp90α) were characterized in the skin, spleen and blood cells of the common carp, under unstressed conditions and after Cd2+ treatment or hypothermia. The examined genes were expressed in a tissue-specific manner: hsc70-2 was expressed constitutively, and was at best only slightly inducible; hsp90α exhibited a high basic expression in all three tissues, whereas hsc70-1 did so only in the blood cells, the expression of hsp70 proved to be below the level of detection in unstressed fish. Cold shock induced the expression of hsp genes in the spleen (hsp90α) and blood cells (hsp70, hsc70-1 and hsp90α), while Cd2+ treatment has no effect on the expression pattern. The highest inducibilities were detected in the skin: for hsp70 an induction of at least 20-fold after cadmium exposure, for hsc70-1 of at least 30-fold and for hsp90α of 3-fold after hypothermia.     

嗜热四膜虫五个hsp70基因的表达分析

鉴定得到嗜热四膜虫13个含有完整保守结构域的hsp70基因,对其中5个高度相似且无内含子的hsp70基因进行表达分析。在37、39和41℃热激条件下,实时荧光定量PCR结果表明,hsp70-2基因对热激响应最敏感。在四膜虫生长、饥饿和接合生殖这3种生理或发育状态下,Microarray结果显示,hsp70-4基因恒定且高表达;在热激条件下,hsp70-4基因的表达水平随着温度的升高而略微增加,证实hsp70-4基因为热休克相关蛋白hsc70基因;克隆的hsp70-4基因全长2208bp,开放阅读框长1959bp,编码653个氨基酸。Microarray结果提示,hsp70-3可能参与四膜虫饥饿早期(0～12h)的耐受和接合生殖后期(6～10h)的新大小核形成,老大核凋亡等事件;hsp70-5可能参与四膜虫饥饿晚期(12～15h)的耐受和接合生殖早期(0～6h)的小核减数分裂、小核交换和原核(pronuclear)融合事件。Blast2GO分析表明,与hsp70-3和hsp70-5共表达的基因分别参与不同的生物学过程,进一步反映了hsp70-3和hsp70-5这两个基因在功能上是存在差异的。     

Heat shock proteins hsp32 and hsp70 as biomarkers of an early response?: In vitro induction of heat shock proteins after exposure of cell culture to carcinogenic compounds and their real mixtures

Heat shock proteins (hsp) are a highly conserved group of proteins that are synthesized as a response to different forms of stress (heat, toxic chemicals, diseases, non-physiological pH changes). Because of their high sensitivity to changes in the environment, these proteins were suggested as possible early biomarkers of exposure in ecotoxicological studies. The purpose of the present study was to check the suitability of hsp32 and hsp70 as biomarkers of in vitro exposure to environmentally relevant carcinogens: polycyclic aromatic hydrocarbons (PAHs), their nitro-derivates, aromatic amines, acrylonitrile (ACN) and the mixture of organic compounds adsorbed onto ambient airborne particles (extractable organic matter, EOM).The expression of hsp32 and hsp70 was studied in human diploid lung fibroblasts (HEL cells) and human monocytic leukaemia cells (THP-1 cells) incubated in vitro with different concentrations of dibenzo[a,l]pyrene (DB[a,l]P), 1-nitropyrene, (NP), 4-aminobiphenyl (ABP), ACN and EOM for different periods of time. The incubation of cells with DB[a,l]P, NP, ABP and EOM did not result in increased levels of hsp32 or hsp70, either in dose- or time-dependent manner. ACN induced the expression of hsp32 as well as hsp70 in HEL and THP-1 cells, which probably reflects its ability to induce oxidative stress. We conclude that hsp32 and hsp70 are not suitable biomarkers of an early exposure to PAHs, their nitro-derivates, aromatic amines or EOM under the conditions used.     

hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos

We have examined differences in the spatial and temporal regulation of stress-induced hsp47 and hsp70 gene expression following exposure of zebrafish embryos to heat shock or ethanol. Using Northern blot analysis, we found that levels of hsp47 and hsp70 mRNA were dramatically elevated during heat shock in 2-day-old embryos. In contrast, ethanol exposure resulted in strong upregulation of the hsp47 gene whereas hsp70 mRNA levels increased only slightly following the same treatment. Whole-mount in situ hybridization analysis revealed that hsp47 mRNA was expressed predominantly in precartilagenous cells, as well as several other connective tissue cell populations within the embryo following exposure to either stress. hsp70 mRNA displayed a very different cell-specific distribution. For example, neither stress induced hsp70 mRNA accumulation in precartilagenous cells. However, high levels of hsp70 mRNA were detectable in epithelial cells of the developing epidermis following exposure to heat shock, but not to ethanol. These cells did not express the hsp47 gene following exposure to either of these stresses. The results suggest the presence of different inducible regulatory mechanisms for these genes which operate in a cell- and stress-specific manner in zebrafish embryos. Dev. Genet. 21:123–133, 1997. © 1997 Wiley-Liss, Inc.     

Algal toxin yessotoxin signalling pathways involve immunocyte mussel calcium channels

A fragment of a putative L-type Ca(2+) channel has been identified by molecular biology experiments in immunocytes from the mussel Mytilus galloprovincialis. Using the cell permeable and Ca(2+)-specific fluorochrome FURA 2-AM, we have demonstrated that the algal toxin yessotoxin (YTX) is able to increase intracellular Ca(2+) concentration in M. galloprovincialis immunocytes. The YTX effect on Ca(2+) increase is inhibited by the L-type Ca(2+) channel inhibitor, verapamil, which is cAMP- and cGMP-dependent, but PKA- and nitric oxide-independent. On the basis of these observations, a possible role for YTX as a potential disturber of mussel immune efficiency is suggested.     

Contaminant loading in remote Arctic lakes affects cellular stress-related proteins expression in feral charr

The remote Arctic lakes on Bj?rn?ya Island, Norway, offer a unique opportunity to study possible affect of lifelong contaminant exposure in wild populations of landlocked Arctic charr (Salvelinus alpinus). This is because Lake Ellasj?en has persistent organic pollutant (POP) levels that are significantly greater than in the nearby Lake ?yangen. We examined whether this differential contaminant loading was reflected in the expression of protein markers of exposure and effect in the native fish. We assessed the expressions of cellular stress markers, including cytochrome P4501A (Cyp1A), heat shock protein 70 (hsp70), and glucocorticoid receptor (GR) in feral charr from the two lakes. The average polychlorinated biphenyl (PCB) load in the charr liver from Ellasj?en was approximately 25-fold higher than in individuals from ?yangen. Liver Cyp1A protein expression was significantly higher in individuals from Ellasj?en compared with ?yangen, confirming differential PCB exposure. There was no significant difference in hsp70 protein expression in charr liver between the two lakes. However, brain hsp70 protein expression was significantly elevated in charr from Ellasj?en compared with ?yangen. Also, liver GR protein expression was significantly higher in the Ellasj?en charr compared with ?yangen charr. Taken together, our results suggest changes to cellular stress-related protein expression as a possible adaptation to chronic-contaminant exposure in feral charr in the Norwegian high-Arctic.     

Effect of glutathione depletion, hyperthermia, and a 100-mT static magnetic field on an hsp70/luc reporter system

Heat shock proteins, in particular hsp70, are induced under conditions of cellular stress. It has been reported that environmental stimuli such as hyperthermia, oxidative stress, and exposure to magnetic fields increase levels of hsp70. It has also been reported that hyperthermia in combination with magnetic field exposure results in a synergistic increase in hsp70 production. We tested the hypothesis that oxidative stress induced by glutathione (GSH) depletion in combination with static magnetic field (SMF) exposure will produce a similar synergistic increase in hsp70 production. We exposed cells to heat, SMF, and diethylmaleate (DEM), which depletes GSH levels alone and in combination with each other, and measured hsp70 production using an hsp70/luciferase reporter and mRNA levels using PCR. We found that treatment with DEM significantly reduced the rate of luciferase bioluminescence production, particularly in the presence of heat. There was no significant effect of a 100-mT SMF exposure either alone or in combination with heat, DEM, or both on bioluminescence, however there was a significant interaction between SMF and DEM on hsp70 mRNA levels. Therefore, under our exposure conditions, GSH depletion reduced hsp70 levels but a synergistic effect of combining this stress with other external stimuli was only observed at the level of mRNA.