Evaluation of heat shock protein 70 as a biomarker of environmental stress in Fucus serratus and Lemna minor

Heat shock proteins (Hsps) are known to be induced in response to short-term stress. The present study aimed to evaluate the potential of Hsp70 as a biomarker of stress produced by increased temperature, osmotic pressure, and exposure to cadmium and sodium chloride in marine macroalgae and fresh water plant species. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed with a working range of 0.025–10 μg?ml^?1 using a monoclonal antibody raised against purified Hsp70 of Phaseolus aureus (mung bean). Fucus serratus (toothed wrack), Chondrus crispus (Stackhouse or Carrageen moss), Ulva lactuca (sea lettuce) and Lemna minor (common duckweed) sample extracts were stressed for up to 24 h and then tested in the IC-ELISA. The presence of Hsp70 and cross-reactivity of the monoclonal antibody was confirmed by Western blot. The heat shock response was confirmed in each species using a 2-h 42°C treatment. Following heat shock, Hsp70 concentrations increased to a peak at 2 h (F. serratus) or 4 h (L. minor), after which concentrations decreased. Osmotic and cadmium stresses also resulted in elevated Hsp70 concentrations in samples of F. serratus and L. minor when compared with unstressed controls. In both, osmotic and metal stress, the production of Hsp70 increased to a maximum and subsequently decreased as the stressor levels increased. Results suggest that Hsp70 IC-ELISA could potentially be applied to the detection of stress in these aquatic species, although it would probably be most effective when used in conjunction with other measurements to provide a stressor-specific biomarker profile or fingerprint.     

Hsp70 instability and induction by a pesticide in Folsomia candida

The heat shock protein Hsp70 has been shown to be a promising biomarker in aquatic and terrestrial organisms. However, its analysis in the soil insect Folsomia candida (Collembola) poses many problems as the protein is particularly unstable in this species. Western blotting has shown that the principal degradation fragment has a size of 48 kDa. We have developed a Western blot method that avoids the degradation of Hsp70 and was successful in detecting the protein in the springtail F. candida after a heat shock (12, 18 and 24 h at 32°C). In the second part of the study the organisms were exposed to artificial compressed soil contaminated with the dinitrophenol dinoseb (10, 15 and 20 µg g^-1 dry weight [DW]). Hsp70 was analysed in pooled samples (40 to 150 collembola according to age) after 1, 2, 3, 4, 5, 6, 7, 11 and 14 days. The only significant induction was observed after 5 days at 20 µg g^-1 DW of dinoseb. The induction patterns over time were dissimilar for the different concentrations and a relatively high variability between the replicates was observed. Our results show that we must be cautious when interpreting biomarker results, especially those for Hsp70.     

Hsp70 response in pigs is affected by their Halothane genotypes after heat stress

 Heat shock protein ([Hsp70]) and ionized calcium ([Ca²⁺]) concentrations were examined in pigs’ blood at different times after heat stress (HS). Pigs were from different halothane genotypes (HAL) (NN, n=7; Nn, n=5; nn, n=6). [Hsp70] increased significantly from 1–2 h after HS and [Ca²⁺] decreased significantly from HS-end in NN and Nn. No significant differences in [Hsp70] and [Ca²⁺] were detected among NN and Nn. [Hsp70] and [Ca²⁺] in nn did not vary significantly from baseline values. [Hsp70] was significantly higher in NN and Nn than in nn. Briefly, HAL affected [Hsp70] after HS in pigs probably via a disordered [Ca²⁺] regulation.     

Toxicity of cypermethrin: hsp70 as a biomarker of response in transgenic Drosophila

Heat shock protein induction is often associated with a cellular response to a harmful environment or to adverse life conditions. The main aims of our study were (1) to evaluate the cytotoxic potential of cypermethrin; and (2) to investigate the suitability of stress-induced heat shock protein Hsp70 as a biomarker for environmental pollutants in transgenic Drosophila melanogaster (Hsp70-lacZ)Bg⁹. Different concentrations of cypermethrin (0.002, 0.2, 0.5 and 50.0 p.p.m.) were mixed with food. Third instar larvae of transgenic Drosophila melanogaster were allowed to feed on these mixtures for different time intervals (2, 4, 6, 12, 24 and 48h). Following feeding, hsp70 induction and tissue damage were evaluated. In the highest concentration treatment group (50 p.p.m.), 100% larval mortality was recorded after 12 h exposure. Hsp70 was found to be induced even at the lowest concentration (0.002 p.p.m.) of the insecticide, while tissue damage was observed in the larvae exposed for 48 h. While an insignificant decline in hsp70 expression was observed in the larvae exposed to cypermethrin at a dietary concentration of 0.002 p.p.m. after 48 h compared with those exposed for 24 h, in the next two higher concentrations of the toxicant, a similar but significant decline in hsp70 expression was evident in the exposed larvae after 48 h. The present study reveals the cytotoxic potential of cypermethrin and further proposes that hsp70 induction in transgenic Drosophila melanogaster could be used as a sensitive biomarker in risk assessment.     

Western blotting versus ELISA detection of stress proteins induced in the blue mussel Mytilus edulis exposed to cadmium and tributyltin

Stress proteins of the Hsp70 family induced in the blue mussel Mytilus edulis exposed in the laboratory to increased concentrations of cadmium (Cd) or tributhyltin (TBT) were analysed using Western blotting and/or ELISA tests. Statistical evaluation of results indicated that increased concentrations of Hsp70 were detected by means of the ELISA tests as compared with control organisms in extracts from the gills of mussels exposed to both Cd or TBT (p = 0.022). Results of analysis by means of Western blotting showed no differences in the levels of Hsp70 in the extracts (p = 0.151). It was concluded that the ELISA test allowed a more sensitive detection of Hsp70 than did Western blotting.     

丛枝菌根真菌对薰衣草耐热性的影响

于不同温度（25℃/20℃、35℃/30℃和40℃/35℃）下测定接种丛枝菌根真菌（arbuscular mycorrhizal fungi,AMF）摩西斗管囊霉Funneliformis mosseae、变形球囊霉Glomus versiforme和F. mosseae+G. versiforme 混合菌种处理对狭叶薰衣草Lavandula angustifolia耐热性的影响。结果表明,供试AMF能与狭叶薰衣草根系形成菌根共生体,以混合菌种处理的侵染率最高,达到68%。40℃/35℃下,与不接种AMF对照相比,混合菌种处理的狭叶薰衣草叶片可溶性糖、可溶性蛋白、脯氨酸和叶绿素等含量以及根系活力分别提高了46%、68%、65%、29%和70%;与不接种AMF对照相比,3种温度处理下接种AMF显著增加了狭叶薰衣草植株超氧化物歧化酶、过氧化物酶、过氧化氢酶、抗坏血酸过氧化物酶和硝酸还原酶活性,而降低相对电导率及丙二醛含量。表明接种AMF能增强狭叶薰衣草抗氧化酶活性,减轻高温造成的伤害,增强耐热性,与单一接种相比以混合接种摩西斗管囊霉和变形球囊霉提高狭叶薰衣草耐热性的效应最大。     

硒对镉胁迫下大球盖菇菌丝生长的影响

选用大球盖菇Stropharia rugosoannulata为试验材料,分析在镉胁迫处理下（2mg/L）添加不同浓度硒（0、6、15、20mg/L）对大球盖菇菌丝生长的影响,初步研究了施加硒对镉胁迫下大球盖菇菌丝生长抑制的缓解效应和作用机制。结果表明：镉对大球盖菇生长速度和生物量具有明显抑制作用,镉造成了大球盖菇渗透调节物质、过氧化氢和丙二醛含量显著增加;添加硒后,镉对大球盖菇菌丝的生长速度、生物量抑制作用均有所改善,且增加了渗透调节物质,促进镉胁迫下大球盖菇渗透调节能力;硒诱导抗氧化酶的活性上调,提高活性氧清除能力,降低了菌丝中丙二醛和过氧化氢的含量,缓解细胞膜过氧化程度,增强了大球盖菇对镉的耐受性。本研究重点探讨外源添加硒对镉胁迫下大球盖菇菌丝生长、生理特性及抗氧化胁迫能力的影响,为进一步研究硒在食用菌抗镉栽培中的应用奠定了一定的理论基础。     

A novel immunocytochemistry technique to measure hsp70 induction in L929 cells exposed to cadmium chloride

The heat shock response has been suggested as a potential biomarker in toxicology. A vast amount of stimuli have been shown to induce heat shock proteins and new techniques to measure the response are constantly being assessed. In this study we use a novel immunocytochemistry technique to measure heat shock protein 70 (hsp70) induction in L929 cells exposed to cadmium chloride. Hsp70 induction was quantifiably measured using a soluble coloured substrate and qualitatively measured using a coloured substrate that precipitated at the location of hsp70. Using the insoluble coloured substrate hsp70 was identified predominantly within the cytoplasm of control cells. At intermediate cadmium concentrations hsp70 was observed to translocate to the nucleus. At these intermediate concentrations a heterogeneous heat shock response was observed. At lethal concentrations a strong heat shock response was observed with a widespread cellular response. This study demonstrates the potential of this immunocytochemistry technique to measure toxicological effects in cells by identifying the response quantitatively and qualitatively.     

A novel immunocytochemistry technique to measure hsp70 induction in L929 cells exposed to cadmium chloride.

The heat shock response has been suggested as a potential biomarker in toxicology. A vast amount of stimuli have been shown to induce heat shock proteins and new techniques to measure the response are constantly being assessed. In this study we use a novel immunocytochemistry technique to measure heat shock protein 70 (hsp70) induction in L929 cells exposed to cadmium chloride. Hsp70 induction was quantifiably measured using a soluble coloured substrate and qualitatively measured using a coloured substrate that precipitated at the location of hsp70. Using the insoluble coloured substrate hsp70 was identified predominantly within the cytoplasm of control cells. At intermediate cadmium concentrations hsp70 was observed to translocate to the nucleus. At these intermediate concentrations a heterogeneous heat shock response was observed. At lethal concentrations a strong heat shock response was observed with a widespread cellular response. This study demonstrates the potential of this immunocytochemistry technique to measure toxicological effects in cells by identifying the response quantitatively and qualitatively.     

Relationship between tomato fruit growth and fruit osmotic potential under salinity

To investigate the relationship between fruit growth and fruit osmotic potential (Ψ_s) in salty conditions, a sensitive tomato cultivar (Lycopersicon esculentum Mill.) and a tolerant accession of the wild species Lycopersicon pimpinellifolium Mill. were grown in a greenhouse with 0 and 70 mM NaCl, and the growth of the fruit studied from 15 to 70 days after anthesis (DAA). L. pimpinellifolium did not reduce significantly fruit weight in salty conditions throughout the growth period, whereas L. esculentum fruit weights decreased significantly with salinity from 45 DAA. L. esculentum fruit fresh weight reductions resulted from both less dry matter and water accumulation, although the fruit water content was affected by salinity before the fruit weight. In both species, fruit osmotic potential (Ψ_s) decreased significantly with salinity during the rapid fruit growth phase, although the changes were different. Thus, fruits from L. pimpinellifolium salt treated plants showed a Ψ_s reduction at the beginning (15 DAA) twice as high as that found in L. esculentum. As the advanced growth stage (from 15 to 55 DAA), the Ψ_s reduction percentages induced by salinity were quite similar in L. pimpinellifolium fruits, while increased in L. esculentum. Under saline conditions, the solutes contributing to reduce the fruit Ψ_s during the first 55 DAA were the inorganic solutes in both species, while in the ripe fruits they were hexoses. L. esculentum fruits accumulated K⁺ as the main osmoticum in salty conditions, while L. pimpinellifolium fruits were able to use not only K⁺ but also the Na⁺ provided by the salt.     

镉胁迫对绢毛委陵菜-丛枝菌根真菌共生体根系结构和生理的影响

为从生理水平上揭示绢毛委陵菜（Potentilla sericea L.）-丛枝菌根真菌共生体对镉胁迫的应答机制,以绢毛委陵菜多年生植株为试验材料,分别接种根内根生囊霉（Rhizophagus intraradices）和摩西球囊霉（Funneliformis mosseae）,80 d后以不同浓度的镉胁迫处理并测定菌根侵染率,研究侵染率较高菌种的共生体植株的生理指标变化和结构变化。结果表明：摩西球囊霉较根内根生囊霉对绢毛委陵菜菌根侵染率高,达100%,而根内根生囊霉侵染率为89.33%;接种摩西球囊霉真菌的植物,当镉质量浓度达10 mg?L^-1时,细胞壁小范围破裂、液泡出现变小的趋势,高浓度镉（20 mg?L^-1）处理时,根系细胞壁破裂,细胞无法维持原有形态;镉胁迫下,Cd（5 mg?L^-1）~ Cd（10 mg?L^-1）MDA含量显著上升（P<0.05）,Cd（0 mg?L^-1）~Cd（5 mg?L^-1）SOD活性和脯氨酸含量变化显著（P<0.05）,可溶性蛋白含量和叶绿素含量显著减少（P<0.05）,Cd（10 mg?L^-1）~Cd（15 mg?L^-1）可溶性蛋白含量和叶绿素含量变化不显著（P>0.05）,植物可以抵抗一定浓度范围的镉胁迫。综合来看,接种摩西球囊菌可以增强绢毛委陵菜耐镉胁迫能力,为利用绢毛委陵菜修复镉污染土壤提供了理论依据。     

Cross-tolerance in the tidepool sculpin: the role of heat shock proteins

Cross-tolerance, or the ability of one stressor to transiently increase tolerance to a second heterologous stressor, is thought to involve the induction of heat shock proteins (Hsp). We thus investigated the boundaries of cross-tolerance in tidepool sculpins (Oligocottus maculosus) and their relationship to Hsp70 levels. Survival of sculpins exposed to severe osmotic (90 ppt, 2 h) and hypoxic (0.33 mg O(2)/L, 2 h) stressors increased from 68% to 96%, and from 47% to 76%, respectively, following a +12 degrees C heat shock. The magnitude of this heat shock was critical for protection. A +10 degrees C heat shock did not confer cross-tolerance, while a +15 degrees C heat shock was deleterious. Sculpins required between 8 and 48 h of recovery following the +12 degrees C heat shock to develop cross-tolerance. There was no association between Hsp70 levels before the onset of the secondary stressor and cross-tolerance. However, branchial Hsp70 levels following osmotic shock were highly correlated with the time frame of cross-tolerance. Thus, Hsp70 induction by the priming stressor may be less important than the ability of the cell to mount an Hsp response to subsequent stressors. The time frame of cross-tolerance is similar to the interval between low tides, suggesting the possible relevance of this response in nature.     

Field validation of hsp70 stress proteins as biomarkers in Asian clam (Potamocorbula amurensis): is downregulation an indicator of stress?

The focus of this paper is to consider the applicability of the hsp70 stress protein response as a biomarker in field studies. Stress proteins (or heat shock proteins, hsp) of the hsp70 family are induced by sublethal concentrations of a variety of environmental pollutants. However, few studies have applied these proteins as biomarkers of environmental stress under field conditions. Our laboratory is investigating hsp70 proteins and other responses of Asian clam (Potamocorbula amurensis) as potential biomarkers in laboratory and field studies. Our efforts include two studies presently being conducted in northern San Francisco Bay: (1) monthly collection of clams from four sites along a cadmium contamination gradient; (2) 7 day in situ exposure of clams at two selected sites at Mare Island Naval Shipyard. Here we present results on hsp70 proteins in P. amurensis in field-collected and outplanted clams. Both field projects are ongoing, therefore the results presented here do not represent completed studies; rather, they illustrate a portion of our experience. For this workshop, we illustrate weaknesses and strengths of these proteins as biomarkers, and we underscore where additional work is needed. In field-collected clams (study no. 1), site-specific differences in levels of two hsp70 proteins, hsp70 and hsp76, were measured in May and June 1997. Although an inverse correlation exists between cadmium tissue concentrations and hsp70 protein levels, differences detected may be reflective of a salinity gradient. Results from recent laboratory exposures to cadmium and a range of salinities are discussed. After in situ exposure for 7 days (study no. 2), both hsp70 and hsp76 levels were significantly reduced in clams from site R. However, given a brief heat-shock in the laboratory, hsp70 protein levels were significantly higher in clams from this site than in controls. Results indicate that downregulation as well as upregulation of hsp70 proteins may be indicators of stress in P. amurensis.     

Species richness in macroalgae and macrofauna assemblages on Fucus serratus L. (Phaeophyceae) and Zostera marina L. (Angiospermae) in Skagerrak, Norway

The macrophytes Fucus serratus and Zostera marina form similar substrates for associated flora and fauna in shallow waters in Norway. While F. serratus forms a more or less continuous belt on rocky substrate along the coast, Z. marina forms disjunct populations on sandy or muddy bottoms. This study focused on the organisms associated with these two macrophytes in two localities in the Skagerrak region. In total, 130 taxa of epiphytic organisms were identified: 22 green algae, 41 red algae, 32 brown algae, eight diatoms and 27 sessile animals. One hundred and twenty-seven taxa of mobile macrofauna were registered. The dominant group was crustaceans, with amphipods as the order containing most species. Many species of both plants and animals preferred one or the other habitat. It is concluded that coastal macrophyte systems have high species diversity.     

The Biology and Host Range of Falconia intermedia (Hemiptera: Miridae), a Potential Biological Control Agent for Lantana camara (Verbenaceae) in Australia

The life history and host specificity of Falconia intermedia in Australia were investigated. Adults and nymphs feed on the intercellular tissue on the underside of Lantana camara leaves. Eggs are deposited singly or in small clusters alongside veins and, on average, hatch in 12 days. Development to adult takes about 15 days and there are 5 instars. Females live for approximately 30 days and lay an average of 1.5 eggs/day. Oviposition occurred on all five L. camara phenotypes tested but subsequent development was significantly poorer on the pink-flowering phenotype. Forty-six plant species were tested to determine host specificity. The only species upon which adults fed and oviposited were L. camara and another introduced weed, Lippia alba. Both plant species supported populations of F. intermedia over several generations. F. intermedia did not display any predatory behaviour towards eggs, nymphs or larvae of either Aconophora compressa or Ectaga garcia, two other introduced biocontrol agents of L. camara. F. intermedia was approved for release in Australia in 2000.     

Hsp70 and larval thermotolerance in Drosophila melanogaster: how much is enough and when is more too much?

Heat shock proteins (Hsps) and other molecular chaperones perform diverse cellular roles (e.g., inducible thermotolerance) whose functional consequences are concentration dependent. We manipulated Hsp70 concentration quantitatively in intact larvae of Drosophila melanogaster to examine its effect on survival, developmental time and tissue damage after heat shock. Larvae of an extra-copy strain, which has 22 hsp70 copies, produced Hsp70 more rapidly and to higher concentrations than larvae of a control strain, which has the wild-type 10 copies of the gene. Increasing the magnitude and duration of pretreatment increased Hsp70 concentrations, improved tolerance of more severe stress, and reduced delays in development. Pretreatment, however, did not protect against acute tissue damage. For larvae provided a brief or mild intensity pretreatment, faster expression of Hsp70 in the extra-copy strain improved survival to adult and reduced tissue damage 21h after heat shock. Negative effects on survival ensued in extra-copy larvae pretreated most intensely, but their overexpression of Hsp70 did not increase tissue damage. Because rapid expression to yield a low Hsp70 concentration benefits larvae but overexpression harms them, natural selection may balance benefits and costs of high and low expression levels in natural populations.     

Zinc pretreatment prevents hepatic stellate cells from cadmium-produced oxidative damage

Pretreatment with zinc produces tolerance to several cadmium toxic effects. This study was performed to further elucidate the mechanism of zinc-induced tolerance to cadmium cytotoxicity in a rat hepatic stellate cell line (CFSC-2G). Twenty four hours after seeding, cells were treated with 60 micromol/L ZnCl2 for 24 h. Following zinc pretreatment, cells were exposed to 3 micromol/L and 5 micromol/L CdCl2 for an additional 24 h. The toxicity of cadmium was significantly reduced in the zinc-pretreated cells. Zinc pretreatment produced a decrease in lipid peroxidation damage of cadmium-treated cells. Glutathione cell content diminished 33% and 43% as a result of 3 micromol/L and 5 micromol/ L CdCl2 treatment, respectively. Cell pretreatment with zinc recovered glutathione content at control cells level. Catalase and glutathione peroxidase activities were also recovered to control values with zinc pretreatment. Cadmium (5 micromol/L) was able to induce 39% the expression of alpha1 collagen (I) gene after 1 h treatment, while zinc pretreatment prevented this cadmium profibrogenic effect. We also examined the production of heat shock protein 70 (Hsp70) as a cellular response to oxidative stress produced by cadmium. By Western blot analysis, a 1.3 and 3 times increment in Hsp70, with 3 micromol/L and 5 micromol/L CdCl2 treatment, respectively, was observed. Zinc pretreatment prevented the production of Hsp70. Metallothionein-II (MT-II) gene expression was induced by cadmium, but the induction was unaffected with zinc pretreatment. These data suggest that zinc-induced protection against the cytotoxicity of cadmium in stellate cells may be related to the maintenance of normal redox balance inside the cell.     

Over-expression of highly conserved mitochondrial 70-kDa heat-shock protein in the sea anemone Anemonia viridis

Organisms react to cellular stress by inducing the synthesis of heat-shock proteins (Hsp). One such protein is the mitochondrial 70-kDa Hsp (mHsp70). The expression of mHsp70 in organisms that undergo stress in their natural habitat is unknown. We used a biochemical approach enabling to identify an mHsp70 from the sea anemone Anemonia viridis, which is abundant in highly fluctuating marine habitats. Antibodies raised specifically against yeast mHsp70 recognized a 70-kDa protein from A. viridis. We found that A. viridis mHsp70 is constitutively expressed at 22–23 °C, but over-expressed upon exposure to heat shock (31 °C) or to temperature fluctuations, suggesting that mHsp70 plays a significant role in adaptation of sessile marine invertebrates to highly fluctuating environmental conditions. Using an affinity column we were able to obtain a partially purified fraction of this protein. Partial amino acid sequences proved that the purified Hsp70 functions in the mitochondria.     

Toxicity of cypermethrin: hsp70 as a biomarker of response in transgenic Drosophila

Heat shock protein induction is often associated with a cellular response to a harmful environment or to adverse life conditions. The main aims of our study were (1) to evaluate the cytotoxic potential of cypermethrin; and (2) to investigate the suitability of stress-induced heat shock protein Hsp70 as a biomarker for environmental pollutants in transgenic Drosophila melanogaster (Hsp70-lacZ)Bg⁹. Different concentrations of cypermethrin (0.002, 0.2, 0.5 and 50.0 p.p.m.) were mixed with food. Third instar larvae of transgenic Drosophila melanogaster were allowed to feed on these mixtures for different time intervals (2, 4, 6, 12, 24 and 48h). Following feeding, hsp70 induction and tissue damage were evaluated. In the highest concentration treatment group (50 p.p.m.), 100% larval mortality was recorded after 12 h exposure. Hsp70 was found to be induced even at the lowest concentration (0.002 p.p.m.) of the insecticide, while tissue damage was observed in the larvae exposed for 48 h. While an insignificant decline in hsp70 expression was observed in the larvae exposed to cypermethrin at a dietary concentration of 0.002 p.p.m. after 48 h compared with those exposed for 24 h, in the next two higher concentrations of the toxicant, a similar but significant decline in hsp70 expression was evident in the exposed larvae after 48 h. The present study reveals the cytotoxic potential of cypermethrin and further proposes that hsp70 induction in transgenic Drosophila melanogaster could be used as a sensitive biomarker in risk assessment.