In situ measurements of adenylate energy charge and assessment of pollution

Marine Pollution Bulletin Marine Pollution Bulletin, Vol. 18, No. 1, pp. 36-39, 1987. Printed in Great Britain.

0025-326X/87 $3.00+0.0O 0 1987 Pergamon Journals Ltd.

In situ Measurements of Adenylate

Energy Charge and Assessment of Pollution CATHERINE SYLVESTRE and YVES L E G A L Laboratoire de Biologie Marine, Colldge de France, 29110 Concarneau, France

Adenylate energy charge (AEC) measurements were performed on two marine species: Crangon crangon and Nassarius reticulatus sampled in two differently polluted stations, Bay of Seine on the channel and Bay of Concarneau on the Atlantic coast. Significant drops in the AEC and modifications in the nucleotide pool composition were recorded in the Bay of Seine populations. It is proposed that AEC and nucleotide concentration measurements could provide a useful tool in long term in situ monitoring of natural populations.

Management of the marine environment not only requires an understanding of the present state of an ecosystem but also the ability to accurately forecast its future changes. Such a task requires the ability to predict, as precisely as possible, the effects of natural or human-induced perturbations on individuals or on populations of organisms (Bellan & Reich, 1978). The effects of pollutants on living organisms can be evaluated in the laboratory by means of acute toxicity tests. From these, an LDs0 value can be obtained. Such tests can also be used to follow the effects of the concentration and transfer of toxins from one organism to another along a food chain (Aubert, 1972). These tests, however, do not take into consideration the existence of delayed effects induced by concentrations of pollutants which act in a chronic way and are capable of progressively eliminating the less resistant individuals of a population. This necessitates the setting up of more sensitive tests permitting the identification of pollutants and measurement of their most subtle effects. These tests should also be applicable to organisms whilst present in the environment in order to be used as in situ controls and give access to the concept of safe concentrations (Warren, 1972) by affecting an experimentally determined application factor to the values of LDs0 already available. The measurement of adenylate energy charge (AEC) is an example of this type of approach. AEC has been defined by Atkinson (1977) as the ratio of the molar concentrations of adenylic nucleotides according to equation (1): AEC =

36

(ATP) + ½(ADP) (ATP)+ (ADP) + (AMP)

(1)

Under the influence of various types of stress (Barthel, 1984; Ivanovici, 1980) or of experimentally introduced pollutants (Sylvestre et al., 1984) there is a general lowering of the values of AEC which are normally included between 0.75 and 0.90. Values lower than 0.50 testify to the existence of irreversible physiological processes having occurred within an organism. However, examples of the application of AEC measurements to in situ systems are rare, presumably on account of the problems encountered in handling the samples, the conservation of the nucleotides and the variability between individuals. Here we present and discuss the results concerning AEC measurements recorded from natural populations of two invertebrates Nassarius reticulatus and Crangon crangon living in clean or polluted areas. Material and M e t h o d s

The organisms were sampled either in the Bay of Seine which is irrigated by a multi-pollution complex originating from the Seine fiver (Chassard-Bouchaud et al., 1985) or in the Bay of Concarneau which is slightly eutrophic (Fig. 1). The mollusc Nassarius reticulams was obtained by dredging and the crustacean Crangon crangon by trawling. Once collected these organisms were immediately immersed in liquid nitrogen. In the laboratory, the animals were ground up (free of their shell in the case of molluscs) in a Dangoumeau grinder (Prolabo-Paris) at a temperature o f - 1 8 0 " C for 3 min. This procedure, involving the use of ultra-low temperature from the earliest stage of sampling, appeared to be essential for the conservation of the nucleotide pool and particularly of ATP since it reduced the activity of ATPases. The frozen powder was then extracted with 10 ml of 6% perchloric acid and homogenized for 30 s at 4°C. After centrifugation, the extract was neutralized (pH 6.5) by adding 0.5 M K 2 CO 3 and again centrifuged (10 min., 15 000 g). The concentration of the adenylate nucleotides (ATP, ADP and AMP) in the supernatant was measured enzymatically according to Pradet (1967). ATP concentration was determined by measuring photon production after ad lition of luciferine and luciferase to the liquid. Measurement was carried out. exactly 40 s after initiation of the

Volume 18/Number 1/January 1987

CONCARNEAU

klh 4

Fig. 1 Location of the sampling stations, a: Bay of Seine and Seine estuary. Nassarius reticulacus was dredged at stations a, b, and e. Cmngon cmngon was fished at station R. b: Bay of Concarneau. The area of sampling are represented by the dotted circles.

reaction and the temperature was 15°C. In these conditions, the response of the assay is linear from 10-8 M to 2 × 10-6 M of A T E A D P was similarly measured after conversion to ATP by addition of pyruvate kinase in the presence of phosphoenol pyruvate. A M P was measured after intermediate transformation to A D P catalyzed by myokinase and, then treatment as before to produce ATP. Enzymes (myokinase, pyruvate kinase and the preparation of luciferine/luciferase) were obtained from Sigma and the standard nucleotides (ATP, ADP, AMP) from Serva. Proteins were determined on the pellet from the perchloric acid extraction after redissolution in N a O H (Leggett-Bailey, 1980). The values of adenylate energy charge obtained at each sampling station are presented in the form of a cumulated frequency plot. The statistical validity of the differences observed between the samples from different stations was checked by the mean of the T test of Student.

~100__.~,~.w J':://: 50

,.j o<

." i

t

.0.5

1,0

AEC Fig. 2 Distribution of AEC values among individuals of Crangon crangonsampled in R (o, A), Bay of Seineat differentperiods of the year and in the Bay of Concarneau (o) in June. The data were expressedas cumulativepercentageof the total population versus the corresponding AEC. Mean values in May at station R:0.47 (a--0.19); in September at station R: 0.51 (a=0.15). Mean valuein the Bay of Concarnean:0.68 (a ~ 0.085). 100 c @J

A e/t

Cr

Results The results concerning the shrimp Crangon crangon are shown in Fig. 2. The reference curve corresponds to animals collected in the Bay of Concarneau. The samples from the Bay of Seine were collected at station R (Bane du Ratier) at two different times of the year. The values of A E C for the reference animals are significantly higher than those of shrimps collected in the Bay of Seine. One can also notice that the Bay of Seine values are more dispersed than those of the reference system. Similar results were obtained with the mollusc Nassarius reticulatus (Fig. 3). The three sampling

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AEC Fig. 3 Repartition of AEC values among individuals of Nassarius reficulatus sampled in the Bay of Seine. (A): Station a (Mean value 0.51, o--0.15); (e): Station b (Mean value 0.51, o-0.19); (A): Station e (Mean value 0.47, 0 - 0 . 2 9 ) . Bay of Concarneau (o). The data were expressed as cumulative percentage of total populations versus the corresponding AEC values.

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Marine Pollution Bulletin

stations in the Bay of Seine yielded animals with lower values of AEC than in the Bay of Concarneau. Here again, the dispersion of the values is greater for the animals of the Bay of Seine.: These figures do not show any statistical differences (Student's T test) between the stations a, b, and e. However, the differences between the Concarneau sample and those of the Bay of Seine are significant at the 5% level (comparison Concarneau Stn a) at the 2% level (comparison Concarneau Stn b) and at the 1% level (comparison Concarneau Stn e). Similarly, the repartition of the values according to the different classes defined by Atkinson (1977) exhibit a clear gradient from a to e so far as Class I and II are concerned (Table 1). The values of AEC for both series of samples (Figs 2, 3) have been plotted against the actual concentrations of nucleotides used as the basis for the calculation of AEC. For Crangon crangon (Fig. 4a), one can show that some of the high values of AEC observed in the Bay of Seine samples are obtained with lower or, in some instances, higher nucleotides concentrations than in the reference samples (indicated on the graph by the points enclosed in the large drawn circles). The same situation is observed for Nassarius reticulatus (Fig. 4b), where very low nucleotide concentrations account for fairly high AEC values. This is particularly true for the organisms sampled at Station e.

Discussion A large number of physiological or biochemical tests designed for the assessment of sublethal effects of pollutions have been described (Lee et al., 1980; Livingstone, 1982). Some of these tests rely on specific responses of metabolic pathways to the presence of substances undergoing biodegradation (Payne, 1977; Blackstock, 1980; Batrel & Le Gal, 1982). One major drawback of these tests concerns the choice of the enzyme to be measured which is too often determined by technical facilities rather than on the basis of their metabolic or regulatory importance. Moreover, the extreme diversity of biological organization at different evolutionary levels is correlated with large differences in the properties of individual enzymes

TABLE 1 Percentages of N. reticulatus classified according to their AEC values (after Atkinson, 1977) that were collected at stations a, b, and e in the Bay of Seine. Class

Sm a %

Sm b %

Stn e %

I

26.7

14.3

0

II

20.0

28.6

44.5

Ill

53.3

57.1

55.5

Class I: Optimal conditions (AEC = 0.75-0.9), Class II: limiting conditions (AEC = 0.5-0.75, Class III: Severe conditions (AEC < 0.5).

from one species to another. This limits the possibilities of setting up standard methods of estimation applicable to a large series of non-related species. Attention has been drawn to the utilization of broader biochemical indices which are relatively independent of the species considered. For example, the measurement of the synthesis of the nucleic acids (Sutcliffe, 1965; Sellos, 1981; Buckley, 1984; Wright & Hetzel, 1985) or of the lysosomal response to chemically induced stress (Moore et al., 1982; Th4bault & Raffin, 1984). The use of AEC measurements corresponds also to this preoccupation. The above presented results indicate that, in the two populations of the Bay of Seine so far studied, the average values of AEC are significantly lower than those of a reference population located in a less polluted area. The effect of a possible pollution gradient among the different Stations a, b, and e in the Bay of Seine does not appear directly from the average values of AEC. However, the examination of nucleotide concentrations in individual samples shows that high levels of AEC can correspond to different combination of components in the nucleotide pool according to whether or not the organisms are submitted to high environmental stress. Clearly, one set of 'high' values (>0.6) of AEC observed for Crangon crangon indicates a generally poor physiological status of the animals as the nucleotides concentrations range between 1 and 4 pxnol mg -~ protein instead of the average value of 4-5 ~unol mg-L Conversely, other samples exhibit quite high concentrations of nucleotides ( > 6 p.rnol mg-~). Also, for

1.0 1.0

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o

~ 0.5

t../

.<

0,5

o

k

o"

A

oA •

a)

A

•

i

i

SO

I00

Nucteotides (n.moles/m~.prot.)

10

i

i

i

20

30

40

Nudeotides (n.mo(es/mq.prot.)

Fig. 4 Relationship between the AEC values and the intraceUular concentrations of adenine nucleotides. a) Crangon cmngon, b) Nassarius reticulatus. Symbols as in Figs 2 and 3 resp.

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Volume 18/Number 1/January 1987

Nassarius reticulatus, the high values of AEC correspond to low nucleotide concentrations. These data illustrate the existence in these organisms of a strong deregulation of nucleotide metabolism. This deregulation could have its origin in the alteration of the in situ living conditions of the animals. We have previously shown (Sylvestre et al., 1984) that in the course of laboratory toxicity tests, a high energy charge could be maintained at least temporarily by lowering the level of AMP. Our observations on natural populations living in a pollution gradient indicates that a permanent environmental stress induces the same physiological mechanisms. However, our measurements do not allow us to distinguish between the existence of a general stress effect and the cumulative effect of the sampling conditions on damaged and previously physiologically disturbed animals. Recently, Verschraegen et al. (1985), studied the AEC in Nereis diversicolor and in Nephtys sp. collected in polluted areas. These authors did not find any significant effect of the location of the sampling site on the AEC values. On the other hand, artificial stresses (experimental drying of the animals) results in a lowering of the energy charge from 0.85 to 0.55 in less than 2 h. From these data, it can be inferred that such resistant and tolerant organisms as N. diversicolor are able to maintain a high degree of regulation of their AEC and that some kind of selection could have occurred in such heavily polluted areas. The measure of the AEC in these organisms would be therefore of little interest in this context. Verschraegen et al. (1985) also pointed out that the total adenylate concentrations in the 'polluted' population of N. diversicolor are significantly lower than in the 'safe' zone. This should indicate that the lack of responsiveness of the AEC measurements to the pollution actually masks a series of biochemical perturbations alterating the nucleotide metabolism. This result is in agreement with our data (Figs 4a, b) showing that high AEC values could correspond to altered levels of cellular nucleotides. To summarize, it appears that, in sublethal conditions, the AEC response to environmental stress proceeds in several steps. In a first stage the stressed organisms are able to maintain their AEC without altering their nucleotide pool. This can be considered as a normal situation. During the second stage, although the adverse conditions do not allow replenishment of the nucleotide pool, the organisms tend to maintain their AEC above a threshold value. The importance of this essential regulatory mechanism in cell physiology is such that even irreversible degradation processes can be engaged, mediated by enzymes such AMP deaminase (Raffin, 1983). The drop in AEC observed during the last stage reflects, in fact, the inability or the difficulty of the organism to utilize this rescue system. From our results, it can be suggested that AEC measurements, and in a more sensitive way, a complete

survey of the composition of the adenylate and phosphagen pools in representative samples of a population, could be of practical interest in the study of pollution effects since the observation of mean or long term alterations of the AEC or nucleotide patterns represent a convenient alert system to detect and, moreover, anticipate the sublethal effects of low dose pollutants on natural populations.

This work was supported by the CNRS (GRECO-Manchc-Director L. Cabioeh) and the French Ministry of Environment (ASP). A preliminary report concerning this study was presented at the colloqium "Bale de Seine" (Sylvestre etal., 1985).

Atkinson, D. E. (1977). Cellular Energy Metabolism and its Regulation. Academic Press, New York. 293 pp. Aubert, M. (1972). Pollutions chimiques et chaines trophodynamiques marines. R ~ Intern. Oceanogr. Med. 28, 9-25. Barthel, D. (1984). Adenylate energy charge in three marine bivalve species during anoxia. Ophelia 23, 155-164. Barrel, Y. & Le Gal, Y. (1982). La glutamate deshydrogenase dArenicole. Un indicateur du nivcau d'cutrophisation du milieu et de sa pollution. C.R. Soc. Biol. 176,619-623. Bellan, C. & Reich, D. J. (1978). Techniques of studying the modifications of biocenosis. R ~ Intern. Oceanogr. Med. 50, 5-9. Blackstock, J. (1980). The Loch Eil project. J. Exptl. Mar. Biol. Ecol. 46, 197-217. Buckley, L. J. (1984). RNA-DNA ratio: an index of larval fish growth in the sea. Marine Biol. 80,291-298. Chassard-Bouchaud, C., Noel, P., Hubert, M. & Halegot, E (1985). Int6ret de la microscopic analytique pour l'6tude de I'impact de m~taux traces et de terres rares sur le milieu vivant. Application /~ 1'6tude d'une zone pollu6e, la Baie de Seine. Colloquc National CNRS "Baie de Seine" 2,225-230. Ivanovici, A. (1980). Adenylate energy charge. An evaluation of applicability to assessment of pollution. Rapp. P. V r~un. Cons. Int. Explor. Mer. 179, 23-28. Lee, R., Davies, J. M., Freeman, H. C., Ivanovici, A., Moore, M. N., Stegeman, J. & Uthe, J. E (1980). Biochemical techniques for monitoring biological effects of pollution in the sea. Rapp. P. V r~un. Cons. Intern. Explor. Mer 179, 48-55. Leggett-Bailey, J. (1980). Techniques in Protein Chemistry. Elsevier, Amsterdam. 310 pp. Livingstone, D. R. (1982). General biochemical indices of sublethal stress. Mar. Pollut. B,dl. 13,261-263. Payne, J. K (1977). Mixed function oxydascs in marine organisms in relation to petroleum hydrocarbon metabolism and detection. Mar. Pollut. Bull. 8, 112-116. Pradet, A. (1967). Etude des AMP, ADP, ATP dans les tissus v6g&aux 1. Dosage enzymatique. Physiol. Veget. 5,209-221. Raffin, J. P. (1983). AMP deaminase from dogfish erythrocytes. Comp. Biochem. Physiol. 75 B, 461-464. Sellos, D. (1981). Acides nucl~iques et protEines en rant qu'indices de pollutions subl&ales. Colloque GABIM Brest, Abstract Vol. p. 41. Sutcliffe, W. H. (1965). Growth estimate from ribonueleic acid content in some small organisms. Limnol. Ocenogr. 10 (suppl.), 253-258. Sylvestre, C., Beaupoil, C., Batrel, Y. & Le Gal, Y. (1984). Evolution de la charge energEtique adfnylique sous l'effet d'une pollution expdrimentale. CR. Soc. Biol. 178, 512-51Y. Sylvestre, C., Barrel, Y. & Le Gal, Y. (1984). La d6teetion des effets sublEtaux des pollutions. Utilisation in situ d'un indice biochimique, la charge 6nerg6tique. Colloque National CNRS "Baie de Seine" 2, 237-241. Verschraegen, K., Herman, P. M. S., Van Gansbeke, D. & Braeckman, A. (1985). Measurement of the adenylate energy charge in Nereis diversicolor and Nephtys sp. Evaluation of the usefulness of AEC in pollution monitoring. Marine Biol. 86,233-240. Warren, C. E. (1972). Biologyand Water Pollution Control. W. B. Saunders, Philadelphia. 434 pp. Wright, D. A. & Hetzel, E. W. (1985). Use of RNA-DNA ratio as an indicator of nutritional stress in the american oyster Crassostrea virginica. Marine Ecology Progress series 25, 199-206.

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