ADENYLATE ENERGY CHARGE AS a TOOL for EVALUATING SUB-LETHAL TOXICITY By

QatarUniv. Sci J. (1999), 19:

ADENYLATE ENERGY CHARGE AS A TOOL FOR EVALUATING SUB-LETHAL TOXICITY By.. Picado A. and LeGal Y. Yves LeGal: Laboratoire de Biologie Marine Museum National d'Histoire Naturelle et College de France, 29900 Concarneau, France; Tel. 33 (0)2 98 97 06 59, Fax 33 (0)2 98 97 81 24, E-mail: [email protected])

Running Title : Adenylate energy charge and sub-lethal toxicity

SUMMARY Adenylate energy charge (AEC) is directly related to the cellular concentrations of the three adenylates nucleotides ATP, ADP and AMP. This biochemical index reaches high values (0.9) under optimal conditions but drops rapidly in the presence of stressing agents. In vertebrates, AEC is strongly regulated and maintained within narrow limits. In contrast, in invertebrates, AEC displays a wide range of values according to the importance of the internal stress or to the variations in the external environment of the organisms (natural or anthropogenic). This work concerns the use of adenine nucleotides concentrations, ATP/ ADP ratios and AEC as physiological markers in different species. Our results show that AEC is modified in animals submitted to experimental pollution or sampled in polluted areas. Results of zinc contamination in adults of clam Ruditapes decussatus show that after one month exposure, AEC values change significantly.

The use of AEC in the bivalve Ruditapes decussatus for in situ evaluation allowed a classification among different sampling sites in what concerns environmental quality. In this way, AEC proved to be an accurate bi

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INTRODUCTION:. diagnostics are needed [2]. Chemical surveys that monitor the quality of coastal and estuarine waters allow the In these conditions, methods such as scope inventory of overall pollutant levels but for growth [6] appear very interesting as are inadequate per se for assessing the they have a global character and respond actual impact of pollutants on living not only to specific contaminants but to a systems [ 1]. large series of natural or man-induced stressing agents generally present in the Various approaches and procedures have environment at low concentrations. Such been proposed for environmental hazard total or global indexes do have their assessment [2,3]. Within this framework, natural place in any analytical approach of the various techniques of biological long-term effects of low level monitoring proposed so far are based on contaminants present m manne different biological principles but have environment [7]. also different meanings. Mixed function oxydases enzymes [4], metallothioneins An index based on the measurement of the [5] mainly account, in a first approach, for metabolic energy pool offers several the detoxification processes in response to advantages, in particular, the university of the presence of environmental the biological systems and therefore the contaminants. Biochemical methods based adenylate energy charge was proposed as on both systems are able to detect zones an index of environmental stress [8]. contaminated by hydrocarbons or by metals with a precision similar to or higher ACE is the ratio of the concentrations in than that of chemical methods. ATP, ADP, and AMP and var1es theoretically form 0 to 1. the value of AEC Another aspect of these indexes 1s that reflects the energy balance at a given time they are reflecting only the presence of for an organism or a part of it [9]. molecules or substances of a particular type : P AH, metals, etc. Due to more loose regulations and particularly to the low efficiency of the In addition to the classical tests and enzyme AMP deaminase invertebrates are ecological surveys, it is thus necessary to able to survive to lower energy charge establish techniques which measure the (0.3-0.4) than vertebrates (0.5-0.6) [10]. «well-being» of apparently normal AEC Values Physiological conditions organisms and which allow the assessment 0.75 < AEC < 0.90 Optimum: growth, of possible future damages (or recovery) reproduction of biological system. 0.50 < AEC < 0.75 Perturbation: slow growth, no reproduction reversible effects. Because man-induced modifications of the AEC < 0.5 No growth, environment are likely to stress organisms, no reproduction, irreversible effects. Death. ecophysiological or ecobiochemical-based -37- Picado A. and LeGal Y.

AEC measurements have been performed phosphate and can be used in some cases either on orgamsms submitted to on small living organisms. experimental contaminations in the laboratory or directly sampled in the The interpretation of the results has also to natural environment [11-14]. Generally take in account the overall physiology of these measurements lead to the conclusion the regulation of AEC, in which two key that AEC used either in the laboratory or enzymes are involved : Adenylate kinase in situ constitutes a sensitive mean of and Al\t1P deaminase. appreciating sub lethal effects undergone / by organisms living in polluted areas. adenylate kinase 2ADP ATP+AMP A large number of factors can affect ACE IMP of organisms. Handling of organisms can, AMP desaminase in some instances, modify drastically AEC. Bad conditions of conservation of The result of the activity of these two the samples can also be at the origin of enzyme systems is a protection of the cell erroneous values of AEC. A rapid freezing against a drop of AEC at the expense of or freezeclamping of the living samples in the global stock of adenylates. liquid nitrogen is an absolute condition of Invertebrates, AMP deaminase is success for the subsequent treatments. The particularly efficient and thus !imitates any choice of the method for estimating variation of AEC in stress conditions. adenylate nucleotides species is relatively However, in invertebrate species the low unimportant and depends mainly of the catalytic activity of AMP deaminases availability of equipments. B iolumines- authorises large variations of AEC that . cence is based on the enzymatic can be used for testing the biological production of light in presence of ATP. It response of organisms to natural of requires a prior enzymatic conversion of man-induced stress modifications of their AMP and ADP into ATP. However it environment. allows a simultaneous treatment of a large series of samples. METHODS Laboratory experiments The second type of method is based on the Adults of Ruditapes decussatus with an separation of the adenylates nucleotides by average length of 3.5 - 4.0 em were HPLC and gives directly the measure of sampled at Ria Formosa (Fig. 1- site B). ATP, ADP and AMP. After laboratory acclimation (35.6% pH 8.0, 15°C, aeration, medium renewal every A third method is based on the measure of other day and feed with Phaeodactylum 31 P by NMR techniques. This methods tricornutum ) organisms were exposed to offers the advantage of giving also access sub lethal concentration of 1 mg/1 Zn Cl2 to the phosphagens and to inorganic for 15 and 30 days. After these period the

• 38. Adenylate energy charge as a tool for Evaluating Sub-Lethal toxicity edible part was frozen under liquid After a 30 days exposure a drop on AEC nitrogen for adenylates analysis. mean values is observed. Mean value of AEC for in situ individuals is 0.8 (s.e.m. Field experiments : Individuals of Rudi 0.01 n=10). tapes decussatus. were sampled at sites A and B (Fig. 1) and frozen under liquid Figure 2 present in the form of cumulated nitrogen till analysis. frequency plots individual AEC values for different exposure times. Figure 3 presents Analysis of the nucleotides. For all tested AEC variation versus ATP/ADP ratios. organisms tissues of each individual were Contaminated organisms present very pulverised under liquid nitrogen and ex dispersed values either for AEC or for tracted with perchloric acid [8]. ATP/ADP ratio. Analysis of adenylates was performed by high-performance liquid chromatography Field experiments. Individuals of Rudi according to Leray [15] with LKB tapes decussatus sampled in different Bromma equipment. The work column (5 sites of the portuguese coast were utilised j..tm) and pre-column (7 j..tm) were Lichro for site monitoring study. In table II re sorb RP 18 and the elution was carried out sults on mean AEC values and total adeny with 0.35 M phosphate buffer, pH 5.5. All lates are presented. samples and buffer were filtered (0.45 j..tm) prior to analysis. The adenylate concentra Results presented in the form of tions were referred to the protein content cumulated frequency plots for individual of the extracts [16] with bovine serum al AEC values in different sites are presented bumin as standard. Values of the adeny late in figure 4. Results obtained shows energy charge obtained at each concentra different site conditions. tion (laboratory experiments) or at each sampling station (field experiments) and In decreasing order : Faro, A veiro and defined [9] as the ratio of the molar Culatra. Organismes form Culatra present concentrations of the adenylate nucleo some high values concerning total tides according to adenylates and great dispersion. Figure 5 AEC= present AEC variation with ATP/ADP (ATP + 1/2ADP) (ATP + ADP + AMP) ratio. The lowest ATP/ADP values were are presented in the form of cumulated obtained for orgamsms sampled at frequency plots. Culatra.

RESULTS Discussion Laboratory experiments. AEC values and One of the maJor Issues arising from total adenylates for Ruditapes decussatus ecotoxicological studies concerns the exposed to a sub lethal zinc concentration relationship between the effects noted for different times are presented in table 1. during short-term laboratory experiments

-39- Picado A. and LeGal Y. and the future of the organisms in their index of reaction or of counteraction to environment. Adenylate energy charge adverse situation. Appearance of the analyses is seen to resolve this issue. reaction sigl)al is only possible within a given interval of intensity of the stressor. At the laboratory level and for zinc The study of this signal can thus indicate exposure AEC ts able to · detect the limits of active response of an contamination effect on the bivalve. organism (counteractive capacity).

Similar results have been described for REFERENCES: different organisms placed under stressing 1. Chapman, A.G. & Atkinson, D.E. conditions [8, 17] experiments with a paper 1973. Stabilization of adenylate mill effluent not causing mortality within energy charge by the adenylate 96 hr, lead to significant variations m deaminase reaction. J.Biol. Chern. AEC values after 24 hr in the case of 248:8309-8312. Cerastoderma edule [4]. 2. Livingtone, D.R. 1982. General biochemical indexes of sublethal The use of AEC for field studies allowed a stress. Mar. Pollut. Bull., classification of different sites according 13:261-263. environmental conditions. Culatra, 3. Gray, J.S., Calamari, D., Duce, R., considered a priori as a reference showed Portmam, J.E., Wells, P.G. & Win the lowest quality. Also, tranfert dom, H. L. 1991. Scientifically based experiments from non polluted areas to strategies for marine environmental contaminated sites allow the use of this protection and management. Mar. Pol biomarker to detect effects on C. edule lut. Bull., 22:432-440. due to effluent discharges [ 14]. 4. Stegeman J. J. 1995. Diversity and regulation of cyt P 450 in aquatic Cellular AEC seems to be strictly species, in Arinc A., Schenkamn J. controlled. It can be stabilized by a And Hogson E., eds. NATO A SI Se reduction in the total adeny late nucleotide ries H. Molecular mechanisms of tox pool through a degradation of AMP icity pp 135-158 catallyzed by AMP deaminase [1] 5. Viarengo, A., Mancinelli, G., Marti considered a crisis enzyme by Raffin [18]. no, G, Pertica, M., Canesi, L. & Results demonstrate that under stress Mazzucotelli, A. 1988. Integrated cel conditions and after 24 hr a series of lular stress indices in trace metal con regulations processes operates to ensure a tamination : critical evaluation in a stabilized AEC level. Thus, we can field study. lVIar. Ecol. prog. Ser. assume that area the evolution of the 46: 65-70. nucleotide pool is more important than the 6. Bayne, B. L., Brown, D. A., Burns, AEC itself [12, 19]. K., Dixon, D. R., Ivanovici, A., Liv AEC is not a signal of discomfort but an ingstone, D. R., Lowe, D. M.,

• 40. Adenylate energy charge as a tool for Evaluating Sub-Lethal toxicity

Moore, M. N., Stebbing, A. R. D. & 13. Savari, A., Sylvestre, C., Sheader, Widdows, J. 1985. The Effects of M., Le Gal, Y. & Lockwood, A. P. stress and pollution on marine ani M. 1989. Stress studies on the com mals. Praeger Publishers, New Youk. mon cockle (Cerastoderma edule L.) 384 pp. in Southampton water. Scient. Mar. 7. Howells, G., Calamari, D., Gary, J. & 53:729-735. Wells, P.G. 1990. An analytical ap 14. Picado, A.M. & Le Gal, Y. 1990. As proach to assessment of long term ef sessment of industrial sewage impacts fects of low level of contaminants in by adenylate energy charge measure the marine environment. Mar. Pollut. ments in the bivalve Cerastoderma Bull. 21: 371-375. edule. Ecotoxicology and Environ 8. Ivanovici, A.M. 1980. Adenyalte ener mental Safety 19, 1-7. gy charge : an evaluation of applica 15. Leray, C. 1979. patterns of purine nu bility to assessment of pollution ef cleotides in fish erythrocytes. Comp. fects and directions for future Biochem. Physiol. 64B: 77-82. research. Rapp. p.v. Reun. Cons. int. 16. Gornall, A. G., Bardawill, C. J. & Explor. Mer 179:23-28. David, M. M. 1949. Determination of 9. Atkinson, D. E. & Walton, G. M. serum proteins by means of the biuret 1967. Adenosine triphosphate conser reaction. J. Bioi. Chern. 177: 751-766. vation in metabolic regulation. Rat liv 17. Sylvestre, C. 1988. La charge energe er citrate cleavage enzyme. J Bioi. tique adenylique: possibilites d'utilisa Chern. 242 : 3239-3241. tion dans la gestion de 1' environnment 10. Raffin, J. P. And Thebault M.T. marin. These de Doctorat Univ. Bre 1996. Modelization of coordinated tagne Occidentale. 125 pp. changes of adenylate energy charge 18. Raffin, J. P. 1983. Metabolisme ener and ATP/ADP ratio : application to getique branchial chez les poissons te energy metabolism in invertebrate and leosteens: Etude des proprietes de vertebrate skeletal muscle. C.R. I' AMP deaminase en relation avec Acad.Sci. Sciences de la Vie quelques facteurs du milieu. These e 319,9-15. Doctorat es Sciences. Univ. Stras 11. Din, Z.B. & Brooks, J. M. 1986. Use bourg. of adenylate energy charge as a physi 19. Skjodal, H. R. & Bakke, T. 1978. Re ological indicator in toxicity experi lationship between A TP and energy ments. Bull. of Env. Contam. and charge during lethal metabolic stress Toxicol. 36 (1):1-8. of the marine isopod Cirolona boreal 12. Sylvestre, C. & Le Gal, Y. 1987. In is. J. Bioi. Chetn. 253,3355-3356. situ measurements of adenylate energy charge and assessment of pollution. Mar. Pollut. Bull. 18 (1): 36-42.

• 41- Picado A. and LeGal Y.

Table I - Mean AEC values and total aden Table II - Mean AEC values and total ylates for Ruditapes decussatus exposed adenylates for Ruditapes decussatus sam to a concentration of lmg/lduring 0, 15 pled in different sites. and 30 days AEC (ATP)+(ADP)+(AMP) Site AEC (ATP)+(ADP)+(AMP) s.e.m (nmole/m3 prot.) s.e.m Day s.e.m (nmole/mg prot.) s.e.m Aveiro (A) 0.75 (n=7) 40.43 0.02 6.20 0 0.79 (n=5) 33.30 0.01 2.05 Culatra (C) 0.67 (n=8) 53.63 0.04 9.86 15 0.70 (n=9) 89.10 0.02 9.77 Faro (F) 0.81 (n=7) 27.86 0.02 2.10 30 0.67 (n=13) 67.56 0.02 4.4

Table I - Mean AEC values and total adenylates for Ruditapes decussatus exposed to a

concentration of lmg/1 during 0, 15 and 30 days.

Day AEC (ATP)+(ADP)+(AMP)

s.e.m (nmole/mg prot.) s.e.m

0 0,79 (n=5) 33,30

0,01 2,05

15 0,70 (n=9) 89,10

0,02 9,77

30 0,67 (n=13) 67,56

0,02 4,4

• 42. Adenylate energy charge as a tool for Evaluating Sub-Lethal toxicity

Table II - Mean AEC values and total adenylates for Ruditapes decussatus sampled in

different sites

Site AEC (ATP)+(ADP)+(AMP)

s.e.m. (nmole/mg prot.), s.e.m

Aveiro (A) 0,75 (n=7) 40,43

0,02 6,20

Culatra (C) 0,67 (n=8) 53,63

0,04 9,86

Faro (F) 0,81 (n=7) 27,86

0,02 2,10

A - Ria A veiro

B- Ria Formosa- 2 sites, Culatra, Faro

Fig. 1 : Sampling sites for Ruditapes decussatus .

. 43. Picado A. and LeGal Y.

dO 100 cf(%) • d 15 80 • d30 60 • 40 20 0 0,4 0,6 0,8 1 AEC

1 AEC 0,8 ,..• ~\ .. 0,6 ••• • •• 0,4 I I 0 50 100 150 (ATP)+(ADP)+(AMP) (nmole/mg prot.)

Fig. 2 : Cumulated frequencies versus individual AEC values and total adenylates evolution for Ruditapes decussatus exposed to a concentration of lmg/lduring 0,15 and 30 days. 1

o,s AE 0,6

0,4 1,4 1,9 2,4 AlP/AD Fig. 3 : AEC evolution versus ATP/ADP ratio for Ruditapes decussatus exposed to a concentration of lmg/lduring 0,15 and 30 days.

-44- Adenylate energy charge as a tool for Evaluating Sub-Lethal toxicity

100 • Faro • Culatra 80 A Aveiro 60 cf(%) 40 20 0 0,4 0,6 0,8 1 AEC

Fig. 4 : Cumulated frequencies versus individual AEC values and total adenylates evolution for Ruditapes decussatus in different sampling sites.

0,8 • AEC .. ~ • • 0,6 .~ • • • 0,4 • 0 50 100 150 (ATP+ADP+AMP) nmole/mg prot.)

Fig. 5 : AEC evolution versus ATP/ADP ratio for Ruditapes decussatus sampled m different sites.

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