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Effect of different environmental variables on the synthesis of Hsp70 in Raphidocelis subcapitata
Comparative Biochemistry and Physiology Part A 120 (1998) 29 – 34
Review
Effect of different environmental variables on the synthesis of Hsp70 in Raphidocelis subcapitata Johan Bierkens *, Wendy Van de Perre, Jef Maes Flemish Institute for Technological Research -VITO, Department of Ecotoxicology, Boeretang 200, 2400 Mol, Belgium Received 24 March 1997; received in revised form 2 August 1997; accepted 6 August 1997
Abstract Heat-shock proteins (Hsp) or stress proteins are strong candidates for biomarkers of environmental pollution since they are activated very early in the cascade of cellular events that follow toxic exposure and at concentrations below the lethal dose. Included in a test battery comprised of different bioassays, Hsp induction could provide a general purpose tier I indicator of pollution. Still, little is known on the induction of Hsp under different environmental conditions. In the present study we have made use of an Enzyme Linked Immunosorbent Assay (ELISA) to detect the synthesis of Hsp70 in Raphidocelis subcapitata in response to changes in pH, temperature, humic acids, nitrates and phosphates. The results show that algae respond to these changes in the environment by a transient increase in Hsp70 levels, the extend of which is dependent on the actual parameter under investigation. Out of these five parameters studied, only temperature and possibly pH were able to induce acquired tolerance, i.e. algae grown at a pH or at a temperature different from control conditions were shown to have acquired resistance to a subsequent challenge with Zn (10 − 5 M). Adjustment of the pH and temperature in two physico-chemically different natural surface waters was demonstrated to be sufficient to obtain similar induction patterns of Hsp70 upon exposure to zinc. These results qualify Hsp70 as a good biomonitor for environmental pollution provided essential environmental parameters such as pH and temperature are kept constant. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Biomonitoring; Ecotoxicology; ELISA; Environmental pollution; Environmental variables; Heat-shock protein 70; Hsp70; Raphidocelis subcapitata
1. Introduction Chemical analyses of complex environmental samples are time consuming and expensive and have the drawback that they cannot predict the ultimate bioavailability of a chemical compound. As with other biomarkers, stress proteins (also referred to as heat-shock proteins or Hsp) share the advantage over analytical methods of measuring the actual effective fraction of pollution that affects an organism, by integrating multiple exposure routes over a given time interval and for any given * Corresponding author. Tel.: + 32 14 335184; fax: + 32 14 320372; e-mail: [email protected] 1095-6433/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S1095-6433(98)10006-5
number of pollutants. Moreover, stress proteins are believed to be activated early on in the cellular events that follow toxic exposure and at concentrations below the lethal dose. Therefore, included in a test battery comprised of different bioassays, Hsp could provide a sensitive general-purpose biomarker of pollution. Four major stress protein families of 90, 70, 60 and 16–24 kDa are the most prominent and are frequently referred to as Hsp90, Hsp70, Hsp60 and low molecular weight (LMW) stress proteins [9,10]. In unstressed cells they are involved in the maintenance of protein homeostasis, i.e. protein folding, aggregation and trafficking (chaperoning) [4–6,8,12]. Under adverse conditions, stress proteins are thought to counter pro-
30
J. Bierkens et al. / Comparati6e Biochemistry and Physiology, Part A 120 (1998) 29–34
teotoxic effects by preventing the denaturation of proteins and holding them in the state of folding or assembly to facilitate repair [6,15]. The importance of stress proteins for the cell is also reflected in the degree of phylogenetic conservation between the isoforms of different organisms from bacteria to plants and man [7]. The broad substrate specificity together with the conserved nature of stress proteins renders them suitable as a subcellular tier I biomarker for ecotoxicological research [16]. As such, an increasing number of studies have been carried out in recent years investigating the induction of stress proteins following exposure to environmentally relevant chemicals [3,6,14,15,17 – 19]. However, little is known on the influence of different environmental conditions on the expression of stress proteins. Still, understanding how the synthesis of stress proteins is affected by various environmental variables, could be of particular interest when ‘crude’ environmental samples have to be analysed or when stress proteins are applied for in situ biomonitoring. In this context we have looked at the effect of pH, temperature, nitrates, phosphates and humic acids on the synthesis of Hsp70, as well as the role of these environmental variables in modifying the Hsp70 response to zinc exposure in Raphidocelis subcapitata.
2. Materials and methods
2.1. Cell culture/organisms Raphidocelis subcapitata was obtained from the Culture Collection of Algae and Protozoa-CCAP (CCAP 278/4). Algae were inoculated at 2×103 cells ml − 1 in a total volume of 50 ml culture medium and kept at a 12/12 light/dark regimen and 24°C throughout the experiments. A culture medium with low ionic strength was chosen (12.5 mM CaCl2.2H2O, 53.3 mM NaCl2, 119.1 mM NaHCO3, 10.1 mM KCl and 5.1 mM MgSO4.7H2O) so as to limit interference of its constituents with the actual environmental parameter under investigation. In order to measure pH, 1 ml 3M KCl/100ml medium sample was added. Nitrate, Phosphate and humic acid concentrations were adjusted by the addition of NaNO3, KH2PO4 and humic acids (Fluka AG, 53680), respectively. In order to study the initial effect of the environmental parameters on Hsp70 levels, the algae were exposed to the environmental parameter under investigation for 4 days. At day 4 a second challenge was mounted consisting in the addition of Zn to a final concentration of 10 − 5 M for 3 more days. While enumeration of the cells was performed daily, protein extractions were done at days 1, 3 and 7. Each treatment group consisted of five replicates. Dunnett testing [2] was performed to assign
significance levels to the different treatment groups as compared to the controls.
2.2. Protein quantitation After treatment cells were collected by centrifugation at 1000 ×g for 5 min. The pellet was dissolved in PBS containing 1 mM ATP and 0.1 mM PMSF and snap frozen in liquid N2. After thawing the cell suspension was sonicated for 20 s (20 Khz, 75 W) and centrifuged at 10000 × g for 5 min at 4°C. Total protein concentration in the supernatant was determined spectrophotometrically (U3000, Hitachi, Tokyo, Japan) using the Bio Rad protein kit (Bio Rad 600–0005).
2.3. Enzyme linked immunosorbent assay for Hsp70 ELISA was performed as described by Anderson et al. [1], with minor modifications. Briefly, samples were diluted to 3 mg per 100 ml using PBS and aliquots containing 0.25–500 ng recombinant human Hsp70 (SPP-755; Stress Gen) were loaded in polycarbonate immunoassay plates (Maxisorp, Nunc). The final volume in each well was 100 ml. The plates were covered and incubated overnight at 4°C. The next day the wells were washed trice with 200 ml PBS prior to addition of 200 ml PBS-3% BSA for 8 h at 4°C (Blocking step). After a washing step (3× PBS) the plate was incubated with 100 ml monoclonal (mouse) anti-Hsp70 antibody (clone 3a3, MA3-006, Affinity Bioreagents) at a dilution 1/500 overnight at 4°C. Subsequently the wells were washed trice with 200 ml PBS containing 0.1% Tween 20, and again incubated with peroxidase conjugated goat anti-mouse IgG (DAKO P0447) (at 1/1000) for 2 h at 37°C. The plate was once again washed three times with PBS containing 0.1% Tween 20, and orthophenylamine (OPD, P6912, Sigma, St. Louis, MO) was added at a concentration of 500 mg/ml. The reaction was stopped using 50 ml 3M H2SO4 and the plate was read in a 96-well multiplate reader (Biorad, Richmond, CA) using a 490 nm filter with reference 595 nm. Background activity was determined in wells containing PBS alone. Non-specific binding was derived from wells only containing secondary antibody.
3. Results Fig. 1 illustrates the effect of different environmental parameters on the synthesis of Hsp70 in Raphidocelis subcapitata expressed as a percentage of control values. The actual Hsp70 levels are given in Table 1. The largest increase was seen in response to a decrease in temperature (panel C). Deviations from control values for pH and nitrates were able to induce significant increases in the basal metabolism of Hsp70. However,
J. Bierkens et al. / Comparati6e Biochemistry and Physiology, Part A 120 (1998) 29–34
31
Fig. 1. Effect of environmental variables on the synthesis of Hsp70 in R. subcapitata expressed as a percentage of the controls and in function of time (days). Control conditions were culture medium at pH 7 and at 24°C.
whereas the effect of pH was transient, the enhanced synthesis of Hsp70 in response to 5.6 mg l − 1 nitrate had not reached a plateau at the end of a 7-day experimental period. The effects of humic acids and phosphates on the total Hsp70 levels were only minute. The last panel shows a comparison of the Hsp70 levels in two physico-chemically very different natural surface waters (Table 2) as compared to the artificial culture medium used in all the other experiments. Whereas algal growth in the canal and boglake water was increased with 25 and 29% as compared with the artificial culture medium, respectively (data not shown), the Hsp70 production was significantly lower in both natural surface waters (Panel F). The response of the Hsp70 synthesis (% of control values) to a second challenge, i.e. 10 − 5 M ZnCl2, is shown in Fig. 2. The actual Hsp70 levels are given in Table 1. The Hsp70 induction was not affected by differences in the concentrations of nitrate (panel E) and humic acid (panel B) in the ambient medium. In contrast, pH and phosphates (panels A and D) did affect the induction of Hsp70 significantly (P B0.1 and
B0.05, respectively). As for pH, algae exposed for 1 day to increasing concentrations of zinc at pH 7 or at pH 5, respectively, displayed a different induction pattern of Hsp70 (Fig. 3). Although the total levels of Hsp70 were significantly higher in algae grown at pH 5 as compared to algae grown at pH 7, the dose-response relationship that existed between the induction of Hsp70 and increasing concentrations of Zn at neutral pH (significance levels between treatment groups: PB 0.1), was entirely lacking at low pH. Finally, a dramatic effect was seen when Zn was added to algae that had been grown at lower temperatures. Algae grown at 13°C that had responded to this decrease in temperature by a steep initial increase in Hsp70 metabolism, completely lacked the ability to respond to the second challenge with Zn. Because pH and temperature were shown to exert the most significant effects on the synthesis of Hsp70, a last experiment was performed in which algae grown in canal and boglake water that had been adjusted for pH and temperature, were challenged with 10 − 5 M ZnCl2. As shown in Fig. 2 (Panel F), mere correction of these
J. Bierkens et al. / Comparati6e Biochemistry and Physiology, Part A 120 (1998) 29–34
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Table 1 Total Hsp70 levels (ng) in Raphidocelis subcapitata for different treatment groups as a function of time Day 1
Day 3 S.D.
x
x
Day 7 S.D.
−Zn x
pH Control pH6 pH5
+Zn S.D.
x
S.D.
8.28 8.90 10.61
0.31 0.83 0.45
2.24 3.86 4.65
0.13 0.31 0.29
2.24 2.29 2.37
0.20 0.10 0.15
2.69 3.33 6.96
0.49 0.40 1.12
Humicacids Control 1.5 mg l−1 5.4 mg l−1
5.35 5.98 6.13
0.54 0.44 0.26
3.07 3.54 3.26
0.39 0.18 0.25
4.04 4.34 4.13
0.61 0.49 0.24
5.23 5.22 6.04
0.47 0.63 1.60
Temperature Control 13°C 19°C
1.63 12.34 5.61
0.14 1.77 0.30
1.43 2.56 3.58
0.09 0.04 0.13
1.22 1.58 4.12
0.09 0.07 0.35
1.46 2.09 4.19
0.11 0.14 0.61
Phosphate Control 0.2 mg l−1 0.9 mg l−1
6.84 5.42 6.28
0.16 0.48 1.25
5.00 5.13 5.37
0.27 0.50 0.84
3.98 4.04 4.63
0.19 0.25 0.36
7.36 6.51 5.57
0.56 0.60 0.72
Nitrate Control 1.5 m gl−1 5.6 m gl−1
10.20 12.75 12.46
1.03 0.55 0.40
5.51 6.38 7.74
0.42 0.45 1.41
4.08 5.95 11.22
0.67 0.67 1.13
6.92 11.75 17.12
0.59 0.75 2.84
1.82 2.01 1.82
0.07 0.10 0.13
1.59 0.94 1.16
0.10 0.11 0.09
1.24 0.42 0.50
0.15 0.06 0.05
2.70 1.01 1.20
0.44 0.19 0.08
Natural surface waters Control Boglake Canal
S.D., standard deviation. Last column represents total Hsp70 levels in algae treated for 3 days with 10−5 M Zinc.
two variables was sufficient to obtain a similar percent induction of Hsp70 in these physico-chemically very distinct surface waters.
4. Discussion Despite the hypothesised advantages of stress proteins as biomarkers there have only been few direct Table 2 Physico-chemical composition of boglake and canal water (mg l−1)
Ba Ca Fe Mg Mn Na Sr Si pH
Canalwater
Boglake water
0.223 60.28 0.006 6.8 0.002 32.9 0.17 0.24 7.8
0.015 4.25 0.145 1.196 0.037 6.7 0.02 0.21 4.1
applications of stress protein induction to environmental toxicology [3,13–15,17–19]. All available data do suggest however that all chemicals that interfere with normal protein metabolism are inducers of the stress response [6,17]. Moreover, the induction of stress proteins by a mild stress factor seems to correlate with the tolerance of the cell or organism to subsequent, more severe stress [11]. Therefore, it was of interest to investigate whether changes of some environmental parameters were perceived by Raphidocelis subcapitata as an initial stress factor, affecting subsequent responses to a supplementary challenge. This would be of particular importance when analysing ‘crude’ environmental samples or when applying stress proteins for in situ biomonitoring. Our results show that only in the case of an important shift in the ambient temperature, the algae had acquired tolerance for a subsequent challenge, as obviated by the lack of response to Zn. Nutrients (nitrates and phosphates) and humic acids most likely did only interfere with the synthesis of Hsp70 by changing the solubility and consequently the bioavailability of Zn for
J. Bierkens et al. / Comparati6e Biochemistry and Physiology, Part A 120 (1998) 29–34
33
Fig. 2. Hsp70 response after 3 days to 10 − 5 M ZnCl2 in Raphidocelis subcapitata under different environmental conditions. Values are expressed as a percentage of non-stimulated control cultures, i.e. pH 7 and 24°C.
the algae. As for changes in pH there remain some doubts. Whereas the increased solubility and bioavailibility of Zn at low pH may be reflected in higher levels
of Hsp70, the absence of a dose-response curve at pH 5 might be indicative of acquired tolerance. Further research need to be performed in order to gain more insight into the effect of pH on Hsp70 metabolism. Finally, we did show that in adjusting the pH and temperature of two physico-chemically entirely different natural surface waters we were able to elicit a similar response to Zn in Raphidocelis subcapitata. This would mark Hsp70 as a good bioindicator for the assessment of environmental samples, provided at least two important environmental variables, pH and temperature, are adjusted.
References
Fig. 3. Hsp70 response (ng Hsp70) after 1 day to increasing concentrations of ZnCl2 in Raphidocelis subcapitata grown at pH 5 and pH 7, respectively.
[1] Anderson RL, Wang CY, Van Kersen I, Lee KJ, Welch WJ, Lavagnini P, Hahn GM. An immunoassay for heat-shock protein 73/72: use of the assay to correlate HSP 73/72 levels in mammalian cells with heat response. Int J Hyperthermia 1993;6:539 – 52. [2] Dunnett CW. A multi comparison procedure for comparing several treatments with a control. J Am Stat Assoc 1955;50:1096 – 121.
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[3] Dyer SD, Dickson KL, Zimmerman EG. A laboratory evaluation of the use of stress proteins in fish to detect changes in water quality. In: Landis WG, Hughes JS, Lewis MA, editors. Environmental Toxicology and Risk Assessment. Philadelphia: American Society for Testing and Materials(ASTMSTP1179), 1993:247 – 61. [4] Hershko A. Ubiquitin-mediated protein degradation. J Biol Chem 1988;263:15237–40. [5] High S, Stirling CJ. Protein translocation across membranes: common themes in divergent organisms. Trends Biochem 1993;3:335 – 9. [6] Hightower LE. Heat-shock, stress proteins, chaperones and proteotoxicity. Cell 1991;66:191–7. [7] Lindquist S, Craig EA. The heat-shock proteins. Annu Rev Genet 1988;22:631 –77. [8] Lis J, Wu C. Protein traffic on the heat-shock promoter: parking, stalling and trucking along. Cell 1993;74:1–4. [9] Morimoto RI, Tissieres A, Georgopoulos C. Stress Proteins in Biology and Medicine. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1990. [10] Nover L., Heat-shock Response, Boca Raton: CRC Press, 1991:509 [11] Roberts PB. Growth in cadmium-containing medium induces resistance to heat in E. coli. Int J Radiat Biol 1984;45:27 – 31. [12] Rutherford SL, Zuker CS. Protein folding and the regulation of signalling pathways. Cell 1994;79:1129–32.
.
[13] Ryan JA, Hightower LEE. Valuation of heavy-metal ion toxicity in fish cells using a combined stress protein and cytotoxicity assay. Environ Toxicol Chem 1994;13:1231 – 40. [14] Sanders BM. Stress proteins: potential as multitiered biomarkers. In: Shugart L, McCarthy J, editors. Environmental Biomarkers. Chelsea: Lewis Publishers, 1990:165 – 91. [15] Sanders BM. Stress proteins in aquatic organisms:an environmental perspective. Crit Rev Toxicol 1993;23:49 – 75. [16] Stegeman JJ, Brouwer M, Di Giulio RT, Fo¨rlin L, Fowler BA, Sanders BM, Van Veld PA Jr. Molecular responses to environmental contamination: enzyme and protein systems as indicators of chemical exposure and effect. In: Huggett RJ, Kimerle RA, Mehrle PM Jr., Bergman HL, editors. Biomarkers. Biochemical, Physiological, and Histological Markers of an Tropogenic Stress. Boca Raton: Lewis, 1992:347. [17] Steinert SA, Pickwell GV. Induction of Hsp70 proteins in mussels by ingestion of tributyltin. Marine Environ Res 1993;35:89 – 93. [18] Theodorakis CW, D’Surney SJ, Bickman JW, Lyne TB, Bradley BP, Hawkins WE, Farkas WL, McCarthy JF, Shugart LR. Sequential expression of biomarkers in bluegill sunfish exposed to contaminated sediment. Ecotoxicology 1992;1:45 – 73. [19] Veldhuizen-Tsoerkan MB, Holwerda DA, Van der Mast CA, Zandee DI. Effects of cadmium exposure and heat-shock on protein synthesis in gill tissue of the sea mussel Mytilusedulis L. Comp Biochem Physiol 1990;96C(2):419– 26.
Review
Effect of different environmental variables on the synthesis of Hsp70 in Raphidocelis subcapitata Johan Bierkens *, Wendy Van de Perre, Jef Maes Flemish Institute for Technological Research -VITO, Department of Ecotoxicology, Boeretang 200, 2400 Mol, Belgium Received 24 March 1997; received in revised form 2 August 1997; accepted 6 August 1997
Abstract Heat-shock proteins (Hsp) or stress proteins are strong candidates for biomarkers of environmental pollution since they are activated very early in the cascade of cellular events that follow toxic exposure and at concentrations below the lethal dose. Included in a test battery comprised of different bioassays, Hsp induction could provide a general purpose tier I indicator of pollution. Still, little is known on the induction of Hsp under different environmental conditions. In the present study we have made use of an Enzyme Linked Immunosorbent Assay (ELISA) to detect the synthesis of Hsp70 in Raphidocelis subcapitata in response to changes in pH, temperature, humic acids, nitrates and phosphates. The results show that algae respond to these changes in the environment by a transient increase in Hsp70 levels, the extend of which is dependent on the actual parameter under investigation. Out of these five parameters studied, only temperature and possibly pH were able to induce acquired tolerance, i.e. algae grown at a pH or at a temperature different from control conditions were shown to have acquired resistance to a subsequent challenge with Zn (10 − 5 M). Adjustment of the pH and temperature in two physico-chemically different natural surface waters was demonstrated to be sufficient to obtain similar induction patterns of Hsp70 upon exposure to zinc. These results qualify Hsp70 as a good biomonitor for environmental pollution provided essential environmental parameters such as pH and temperature are kept constant. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Biomonitoring; Ecotoxicology; ELISA; Environmental pollution; Environmental variables; Heat-shock protein 70; Hsp70; Raphidocelis subcapitata
1. Introduction Chemical analyses of complex environmental samples are time consuming and expensive and have the drawback that they cannot predict the ultimate bioavailability of a chemical compound. As with other biomarkers, stress proteins (also referred to as heat-shock proteins or Hsp) share the advantage over analytical methods of measuring the actual effective fraction of pollution that affects an organism, by integrating multiple exposure routes over a given time interval and for any given * Corresponding author. Tel.: + 32 14 335184; fax: + 32 14 320372; e-mail: [email protected] 1095-6433/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S1095-6433(98)10006-5
number of pollutants. Moreover, stress proteins are believed to be activated early on in the cellular events that follow toxic exposure and at concentrations below the lethal dose. Therefore, included in a test battery comprised of different bioassays, Hsp could provide a sensitive general-purpose biomarker of pollution. Four major stress protein families of 90, 70, 60 and 16–24 kDa are the most prominent and are frequently referred to as Hsp90, Hsp70, Hsp60 and low molecular weight (LMW) stress proteins [9,10]. In unstressed cells they are involved in the maintenance of protein homeostasis, i.e. protein folding, aggregation and trafficking (chaperoning) [4–6,8,12]. Under adverse conditions, stress proteins are thought to counter pro-
30
J. Bierkens et al. / Comparati6e Biochemistry and Physiology, Part A 120 (1998) 29–34
teotoxic effects by preventing the denaturation of proteins and holding them in the state of folding or assembly to facilitate repair [6,15]. The importance of stress proteins for the cell is also reflected in the degree of phylogenetic conservation between the isoforms of different organisms from bacteria to plants and man [7]. The broad substrate specificity together with the conserved nature of stress proteins renders them suitable as a subcellular tier I biomarker for ecotoxicological research [16]. As such, an increasing number of studies have been carried out in recent years investigating the induction of stress proteins following exposure to environmentally relevant chemicals [3,6,14,15,17 – 19]. However, little is known on the influence of different environmental conditions on the expression of stress proteins. Still, understanding how the synthesis of stress proteins is affected by various environmental variables, could be of particular interest when ‘crude’ environmental samples have to be analysed or when stress proteins are applied for in situ biomonitoring. In this context we have looked at the effect of pH, temperature, nitrates, phosphates and humic acids on the synthesis of Hsp70, as well as the role of these environmental variables in modifying the Hsp70 response to zinc exposure in Raphidocelis subcapitata.
2. Materials and methods
2.1. Cell culture/organisms Raphidocelis subcapitata was obtained from the Culture Collection of Algae and Protozoa-CCAP (CCAP 278/4). Algae were inoculated at 2×103 cells ml − 1 in a total volume of 50 ml culture medium and kept at a 12/12 light/dark regimen and 24°C throughout the experiments. A culture medium with low ionic strength was chosen (12.5 mM CaCl2.2H2O, 53.3 mM NaCl2, 119.1 mM NaHCO3, 10.1 mM KCl and 5.1 mM MgSO4.7H2O) so as to limit interference of its constituents with the actual environmental parameter under investigation. In order to measure pH, 1 ml 3M KCl/100ml medium sample was added. Nitrate, Phosphate and humic acid concentrations were adjusted by the addition of NaNO3, KH2PO4 and humic acids (Fluka AG, 53680), respectively. In order to study the initial effect of the environmental parameters on Hsp70 levels, the algae were exposed to the environmental parameter under investigation for 4 days. At day 4 a second challenge was mounted consisting in the addition of Zn to a final concentration of 10 − 5 M for 3 more days. While enumeration of the cells was performed daily, protein extractions were done at days 1, 3 and 7. Each treatment group consisted of five replicates. Dunnett testing [2] was performed to assign
significance levels to the different treatment groups as compared to the controls.
2.2. Protein quantitation After treatment cells were collected by centrifugation at 1000 ×g for 5 min. The pellet was dissolved in PBS containing 1 mM ATP and 0.1 mM PMSF and snap frozen in liquid N2. After thawing the cell suspension was sonicated for 20 s (20 Khz, 75 W) and centrifuged at 10000 × g for 5 min at 4°C. Total protein concentration in the supernatant was determined spectrophotometrically (U3000, Hitachi, Tokyo, Japan) using the Bio Rad protein kit (Bio Rad 600–0005).
2.3. Enzyme linked immunosorbent assay for Hsp70 ELISA was performed as described by Anderson et al. [1], with minor modifications. Briefly, samples were diluted to 3 mg per 100 ml using PBS and aliquots containing 0.25–500 ng recombinant human Hsp70 (SPP-755; Stress Gen) were loaded in polycarbonate immunoassay plates (Maxisorp, Nunc). The final volume in each well was 100 ml. The plates were covered and incubated overnight at 4°C. The next day the wells were washed trice with 200 ml PBS prior to addition of 200 ml PBS-3% BSA for 8 h at 4°C (Blocking step). After a washing step (3× PBS) the plate was incubated with 100 ml monoclonal (mouse) anti-Hsp70 antibody (clone 3a3, MA3-006, Affinity Bioreagents) at a dilution 1/500 overnight at 4°C. Subsequently the wells were washed trice with 200 ml PBS containing 0.1% Tween 20, and again incubated with peroxidase conjugated goat anti-mouse IgG (DAKO P0447) (at 1/1000) for 2 h at 37°C. The plate was once again washed three times with PBS containing 0.1% Tween 20, and orthophenylamine (OPD, P6912, Sigma, St. Louis, MO) was added at a concentration of 500 mg/ml. The reaction was stopped using 50 ml 3M H2SO4 and the plate was read in a 96-well multiplate reader (Biorad, Richmond, CA) using a 490 nm filter with reference 595 nm. Background activity was determined in wells containing PBS alone. Non-specific binding was derived from wells only containing secondary antibody.
3. Results Fig. 1 illustrates the effect of different environmental parameters on the synthesis of Hsp70 in Raphidocelis subcapitata expressed as a percentage of control values. The actual Hsp70 levels are given in Table 1. The largest increase was seen in response to a decrease in temperature (panel C). Deviations from control values for pH and nitrates were able to induce significant increases in the basal metabolism of Hsp70. However,
J. Bierkens et al. / Comparati6e Biochemistry and Physiology, Part A 120 (1998) 29–34
31
Fig. 1. Effect of environmental variables on the synthesis of Hsp70 in R. subcapitata expressed as a percentage of the controls and in function of time (days). Control conditions were culture medium at pH 7 and at 24°C.
whereas the effect of pH was transient, the enhanced synthesis of Hsp70 in response to 5.6 mg l − 1 nitrate had not reached a plateau at the end of a 7-day experimental period. The effects of humic acids and phosphates on the total Hsp70 levels were only minute. The last panel shows a comparison of the Hsp70 levels in two physico-chemically very different natural surface waters (Table 2) as compared to the artificial culture medium used in all the other experiments. Whereas algal growth in the canal and boglake water was increased with 25 and 29% as compared with the artificial culture medium, respectively (data not shown), the Hsp70 production was significantly lower in both natural surface waters (Panel F). The response of the Hsp70 synthesis (% of control values) to a second challenge, i.e. 10 − 5 M ZnCl2, is shown in Fig. 2. The actual Hsp70 levels are given in Table 1. The Hsp70 induction was not affected by differences in the concentrations of nitrate (panel E) and humic acid (panel B) in the ambient medium. In contrast, pH and phosphates (panels A and D) did affect the induction of Hsp70 significantly (P B0.1 and
B0.05, respectively). As for pH, algae exposed for 1 day to increasing concentrations of zinc at pH 7 or at pH 5, respectively, displayed a different induction pattern of Hsp70 (Fig. 3). Although the total levels of Hsp70 were significantly higher in algae grown at pH 5 as compared to algae grown at pH 7, the dose-response relationship that existed between the induction of Hsp70 and increasing concentrations of Zn at neutral pH (significance levels between treatment groups: PB 0.1), was entirely lacking at low pH. Finally, a dramatic effect was seen when Zn was added to algae that had been grown at lower temperatures. Algae grown at 13°C that had responded to this decrease in temperature by a steep initial increase in Hsp70 metabolism, completely lacked the ability to respond to the second challenge with Zn. Because pH and temperature were shown to exert the most significant effects on the synthesis of Hsp70, a last experiment was performed in which algae grown in canal and boglake water that had been adjusted for pH and temperature, were challenged with 10 − 5 M ZnCl2. As shown in Fig. 2 (Panel F), mere correction of these
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Table 1 Total Hsp70 levels (ng) in Raphidocelis subcapitata for different treatment groups as a function of time Day 1
Day 3 S.D.
x
x
Day 7 S.D.
−Zn x
pH Control pH6 pH5
+Zn S.D.
x
S.D.
8.28 8.90 10.61
0.31 0.83 0.45
2.24 3.86 4.65
0.13 0.31 0.29
2.24 2.29 2.37
0.20 0.10 0.15
2.69 3.33 6.96
0.49 0.40 1.12
Humicacids Control 1.5 mg l−1 5.4 mg l−1
5.35 5.98 6.13
0.54 0.44 0.26
3.07 3.54 3.26
0.39 0.18 0.25
4.04 4.34 4.13
0.61 0.49 0.24
5.23 5.22 6.04
0.47 0.63 1.60
Temperature Control 13°C 19°C
1.63 12.34 5.61
0.14 1.77 0.30
1.43 2.56 3.58
0.09 0.04 0.13
1.22 1.58 4.12
0.09 0.07 0.35
1.46 2.09 4.19
0.11 0.14 0.61
Phosphate Control 0.2 mg l−1 0.9 mg l−1
6.84 5.42 6.28
0.16 0.48 1.25
5.00 5.13 5.37
0.27 0.50 0.84
3.98 4.04 4.63
0.19 0.25 0.36
7.36 6.51 5.57
0.56 0.60 0.72
Nitrate Control 1.5 m gl−1 5.6 m gl−1
10.20 12.75 12.46
1.03 0.55 0.40
5.51 6.38 7.74
0.42 0.45 1.41
4.08 5.95 11.22
0.67 0.67 1.13
6.92 11.75 17.12
0.59 0.75 2.84
1.82 2.01 1.82
0.07 0.10 0.13
1.59 0.94 1.16
0.10 0.11 0.09
1.24 0.42 0.50
0.15 0.06 0.05
2.70 1.01 1.20
0.44 0.19 0.08
Natural surface waters Control Boglake Canal
S.D., standard deviation. Last column represents total Hsp70 levels in algae treated for 3 days with 10−5 M Zinc.
two variables was sufficient to obtain a similar percent induction of Hsp70 in these physico-chemically very distinct surface waters.
4. Discussion Despite the hypothesised advantages of stress proteins as biomarkers there have only been few direct Table 2 Physico-chemical composition of boglake and canal water (mg l−1)
Ba Ca Fe Mg Mn Na Sr Si pH
Canalwater
Boglake water
0.223 60.28 0.006 6.8 0.002 32.9 0.17 0.24 7.8
0.015 4.25 0.145 1.196 0.037 6.7 0.02 0.21 4.1
applications of stress protein induction to environmental toxicology [3,13–15,17–19]. All available data do suggest however that all chemicals that interfere with normal protein metabolism are inducers of the stress response [6,17]. Moreover, the induction of stress proteins by a mild stress factor seems to correlate with the tolerance of the cell or organism to subsequent, more severe stress [11]. Therefore, it was of interest to investigate whether changes of some environmental parameters were perceived by Raphidocelis subcapitata as an initial stress factor, affecting subsequent responses to a supplementary challenge. This would be of particular importance when analysing ‘crude’ environmental samples or when applying stress proteins for in situ biomonitoring. Our results show that only in the case of an important shift in the ambient temperature, the algae had acquired tolerance for a subsequent challenge, as obviated by the lack of response to Zn. Nutrients (nitrates and phosphates) and humic acids most likely did only interfere with the synthesis of Hsp70 by changing the solubility and consequently the bioavailability of Zn for
J. Bierkens et al. / Comparati6e Biochemistry and Physiology, Part A 120 (1998) 29–34
33
Fig. 2. Hsp70 response after 3 days to 10 − 5 M ZnCl2 in Raphidocelis subcapitata under different environmental conditions. Values are expressed as a percentage of non-stimulated control cultures, i.e. pH 7 and 24°C.
the algae. As for changes in pH there remain some doubts. Whereas the increased solubility and bioavailibility of Zn at low pH may be reflected in higher levels
of Hsp70, the absence of a dose-response curve at pH 5 might be indicative of acquired tolerance. Further research need to be performed in order to gain more insight into the effect of pH on Hsp70 metabolism. Finally, we did show that in adjusting the pH and temperature of two physico-chemically entirely different natural surface waters we were able to elicit a similar response to Zn in Raphidocelis subcapitata. This would mark Hsp70 as a good bioindicator for the assessment of environmental samples, provided at least two important environmental variables, pH and temperature, are adjusted.
References
Fig. 3. Hsp70 response (ng Hsp70) after 1 day to increasing concentrations of ZnCl2 in Raphidocelis subcapitata grown at pH 5 and pH 7, respectively.
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