Histophysiology of polysaccharide and lipid reserves in various tissues of Littorina littorea exposed to sublethal concentrations of cadmium

Vol. 94C,

Camp. Biochem. Physiol.

No. 2, pp. 641-648,

1989

Printed in Great Britain

0

0306~4492/89 $3.00 + 0.00 1990 Pergamon Press plc

HISTOPHYSIOLOGY OF POLYSACCHARIDE AND LIPID RESERVES IN VARIOUS TISSUES OF LITTORINA LITTOREA EXPOSED TO SUBLETHAL CONCENTRATIONS OF CADMIUM J. M. GIL, J. A. MARIGOMEZ* and E. ANGULO Laboratorio de Citologia e Histologia. Departamento de Biologia Celular y Ciencias Morfologicas, Facultad de Ciencias, Universidad de1 Pais Vasco, Apdo. 644, 48080-Bilbao, Spain Abstract-l. Sublethal exposure to cadmium causes glycogen depletion in connective tissues of the mantle, kidney folds, and digestive gland-gonad complex. Glycogen levels are lower at higher environmental concentrations of metal and at longer exposure times. 2. Simultaneously with glycogen level reduction in reserve tissues, higher levels of glycogen than in control specimens have been detected in the digestive gland of cadmium exposed winkles. Phosphoglucomutase activity has been detected in kidney, connective tissues, and intestine, but not in digestive tubules. This suggests glycogen mobilisation through digestive tubule epithelia. 3. Phosphoglucomutase activity in gills is associated with glycogen level increases in blood vessels and in distal portion of gill lamellae after proximal epithelium disruption. 4. Lipid contents of the studied organs are only decreased when glycogen levels are largely reduced. Lipase activity has been demonstrated in digestive tubule, kidney and gill epithelia, but not in connective tissues. It is concluded that lipidic store is intracellular while the polysaccharidic one is organismic, 5. Sublethal concentrations of cadmium do not cause impairment of phosphoglucomutase and lipase activities: enzymatic activity is well correlated with reserve consumption, demonstrable activity being lost only after substrate (glycogen or lipid) depletion.

INTRODUCTION

deal with the development of pollution/stress indices; however, it must be underlined that basic research concerning toxicity and detoxification mechanisms is required in order to use properly such environmental quality bioindicators. At the cellular level, basic metabolic processes are quite similar in most organisms, and hence the response of different organisms to a variety of pollutants is qualitatively identical (Blackstok, 1984). Subacute toxicity should not be attributed to the pollutant nature but to a prolonged stress situation leading to organismic exhaustion (Marigomez et al., in press). It is thought that one of the most general sublethal effects caused by environmental irritants is the decline of organismic reserves. Stress sources such as temperature can inhibit the synthesis of glycogen in mussels (Cracium, 1980). Richard and Ireland (1978) observed an inverse relationship between glycogen absorption in the earthworm Dendrobaena rubida and the environmental concentrations of lead. LoweJinde and Niimi (1984) described the loss of glycogen in cadmium exposed Salmo gardnieri. Recio et al. (1988) obtain similar results in zinc exposed slugs (Arion ater). Polysaccharidic metabolism is basic in the energy budget of marine gastropods, and its biochemical alterations might be indicative of regulatory metabolic responses against environmental stress (Blackstock, 1984). Concretely, glycogen is the major polysaccharide involved in the storage of metabolic energy in gastropods (Livingstone and De Zwaan, Many

investigations

*To whom correspondence

1983). On the other hand, some species of the genus Littorina, as L. planaxis, can use the lipidic reserves

after long-term starvation (Emerson, 1967). Also in other gastropods, as Helix aspersa (Sumner, 1965) and Arion r&v (Janssen, 1985) the lipid consumption in association with linase activity has been reported after starvation. In some instances (Marigomez et a[., 1986), sublethal exposure to metalic pollutants has been related to a starvation-like situation. Cadmium toxicity includes inhibition of mitochondrial enzyme activity (Sastry and Subhadra, 1985), as well as activation of other enzymes as carboxipeptidase (Vallee and Ulmer, 1972). Toxic mechanisms consist of competition or collaboration with metallic cofactors. This cation also affects glucose-6-phosphatase and pyruvate carboxilase (Viarengo, 1985), phosphoglucomutase (Whelar, 1975), and esterases (Vallee and Ulmer, 1972). Since phosphoglucomutase (PGM), target enzyme for glycogen catabolism in marine gastropods (Bolognani et al., 1987), and non-specific esterases, including lipases (Drury and Wallington, 1967), are impaired by toxic concentrations of cadmium and other metals, and also because both enzymes (or enzyme groups) play a role in reserve consumption, they might offer a key to assess whether sublethal effects of cadmium on reserve materials are due to direct toxicity or, on the contrary, to non-specific sublethal damage. The present work attempts to clarify this question on the basis of semiquantitative histochemistry of glycogen and lipids and of microespectrophotometry of PGM and lipase activities in several tissues of experimentally Cd-exposed Lit torina littorea.

should be addressed. 641

J. M. GIL et al.

642

The other purposes of the present study are to investigate whether glycogen or lipids are used by winkles in subacute toxicity conditions, and whether changes in their tissular distribution are related to sublethal cadmium exposure. MATERIALS AND METHODS

Experimental procedure L. littorea (collected from Clew Bay, Eire) were purchased from a commercial dealer and transferred to the laboratory. The selected individuals (20-23 mm collumelar height) were maintained in 15 1 (16 individuals per litre) glass tanks with filtered natural seawater (Zierbena, Biscay) for 1 week period, in absence of food, in order to get acclimation to laboratory conditions and homogenization of the physiological condition. Tanks were continuously aerated and water temperature ranged between 16 and 18°C. Afterwards, animals were exposed to two sublethal concentrations of cadmium (0.5 and 1.25 mg/l, Marigomez et al., 1987) in replicate series for 28 days. Cadmium was supplied as cadmium chloride. One replicate control set was also carried out. During the experimental period, animals were fed on Fucus vesiculosus continuously. Water and food were changed every second day. Specimens for histochemical analysis of lipids and glycogen were removed after 4, 6, 10, 13, 17, 20, 24, and 27 experimental days. Those for histoenzymological studies were removed after 3, 6, 10, 14, 18, 22, and 28 experimental days. Gills and adjoining mantle, digestive gland-gonad complex, and kidney were dissected out and processed for analyses.

Glycogen and lipid histochemistry Two individuals per group of treatment were studied. Tissues were fixed in Bouin’s liquid (32 hr, YC) for glycogen histochemistry; and in Ciaccio’s fixative (18 hr, 5°C) with posterior chromation (5 days, room temperature) for lipid histochemistry (Martoja and Martoja-Pierson, 1970). Paraffin-sections (8-10pm thick) were stained with Best’s carmine and diastase control for glycogen demonstration, with PAS plus diastase as confirmative test for glycogen, and with Sudan B to demonstrate lipids (Martoja and Martoja-Pierson, 1970). Control stains for lipids were made by using material fixed in Bouin’s liquid because lipids are easily cleaned during paraffin embedding unless chromation is realized. After accurate observation of the stained slides a semiquantitative valuation (i.e.: -, +, + +, + + -, . .) of glycogen and sudanophilic substances was considered for different zones in each organ (i.e., brush border, eosinophilic granules, dark granules, cytoplasm, yellow granules and basal lamina-histological sense-of digestive cells). The global result for one organ was the sum of all its elements. Afterwards, maximum and minimum values were established for each zone, and for the whole organ. Then, the subjective traditional histochemical values (- = 0, + = 1, + + - = 1.5, . . .) were transformed into percentage values after considering the whole range of variation between the maximum and the minimum for each zone and organ. Mean values of these percentages (arbitrary units in graphics) were calculated from the replicate series of experimental exposure. Histoenzymology and microspectrophotometry

Small pieces of freshly excised tissues were freezcd with Bright Cryo-Spray (dichlorodifluoromethane, -SO/ - 55°C) and stored at -26°C. Samples were then placed on aluminium cryostat specimen holders and embedded in Bright Cryo-M-Bed. Sections (1Opm) were cut in a Bright’s cryostat (5030 microtome) at a cabinet temperature of - 28/- 30°C. Sections were then collected on glass slides at

the cabinet temperature and slightly heated with the finger and stored at -70°C until staining. The histochemical reaction for Phosphoglucomutase (PGM) activity was demonstrated in cryostat sections, after fixation in cold acetone at 5”C, by the Meijer’s method described by Martinez (1979). Incubating medium in absence of glucose-l-phosphate was used as a control. Lipase activity was not directly demonstrable in digestive gland because of the procedural interferences with the high amounts of tissular calcium (Lillie, 1965). The esterases are a major group of enzymes in which lipases are included. Esterase activity was demonstrated in unfixed cryostat sections according to the 1-naphthylacetate procedure described bv Loida et al. (1976). After inhibition nrocedures for a, b, and cksterases ihcludmg pre-treament with metallic salts (silver and copper nitrate, Oxford, 1973) no significant changes in the enzymatic activity were recorded. The pretreatment with a phenol solution for a few minutes abolishes the positive reaction (Oxford, 1973). Therefore, it was concluded that lipase activity is properly shown in the studied organs of Littorina littorea by the general procedure for esterases (Gil et al., in prep.). Enzyme activity quantification was carried out with the aid of a Carl Zeiss V MSP-05 microspectrophotometer, with fast scanning slide-guidance and MPC 64 control units, attached to a M-20 Olivetti microcomputer. Energy source was one halogen lamp (12 volt), and the light beam pierces a monochromatizer with manual adjustment of the trackwidth. Objective used for measuring was 25 x Image is projected onto a screen with a measurement diaphragm, and piercing light arrived at a photomultiplier. Then, the signal is digitized by the MPC-64 control unit and recorded by the microcomputer by means of a IEEE-488 interface. The position of the slide-guidance unit is precisely controlled by hand, by a joistick, or by the computer. The system also offers an auxiliary source of visible light. The procedure used to realize the measurements is based on the method of Casperson (1951) and consists of two steps: selection of measurement points by the operator, and automatic recording by the system (Esteban, 1986). Once the enzymatic reaction was detected in tissue sections, the chromophore precipitate was measured by the one-wavelength microspectrophotometer. Absorption spectra of both PGM and lipase activities were recorded and maximum peak values were shown to be 530 and 580 nm, respectively. Therefore, measurements were realized at these two wavelengths. The sample size was estimated after random measuring from 25 to 85 record points in each section. Results indicated that mean extinction values kept constant from 50 records on, and hence this value, which represents the minimum sample size, was selected as sample size for subsequent recordings of both enzymatic activities. One sole section provided enough for measurements, because variability between sections was smaller than intravariability. RESULTS

Glycogen and PGM activity Glycogen has been demonstrated in the cytoplasm of basophilic cells, in eosinophilic and dark granules of digestive cells, and also in the basal third of all epithelial cells in the digestive gland of control winkles. The strongest positive reaction is extensively evidenced in the cytoplasm of the interstitial connective cells which surround gonadal and digestive tubules. A exposure-time and Cd-concentration dependent decrease in glycogen content from this connective tissue has been observed (Fig. 1A). Thus, lower levels of histochemically demonstrable glycogen were detected in those specimens exposed to

Cd reduces Littorina glycogen

4

6

lo

13

II

20

24

27

4

6

643

l0

13

17

20

24

27

50

50

4

0

10

14

17 20

24

L

i

27

a

TIME I days) ITCONTROL El O.SmgCd/ I

I 1.25mgCd/ 1

Fig. 1. Semiquantitative estimation of the glycogen levels (arbitrary units) against the exposure time (days) in the interstitial connective tissue (A), in the digestive gland epithelium (B), in the renal folds (C), and in the connective tissue of the mantle (D) after staining with Best’s carmine. higher doses at longer times. A cyclic variation in glycogen occurrence has been observed in the digestive epithelium of control winkles: maximum

levels have been recorded on the 4th and 17th experimental days (Fig. 1B). A similar dynamics occurs in exposed animals; however, maximum peak values are higher as a result of experimental Cd-concentration. Exceptionally, the second maximum peak value for 1.25 mg Cd/l exposure (17th to 24th days) is lower than expected after the observation made above. This might be related to the glycogen exhaustion observed in adjoining reserve connective tissue at this exposure condition (Fig. 1A). PGM activity was not evidenced in digestive tubule epithelium in any instance. Positive reaction was only demonstrated in the interstitial connective cells and in the basal third of intestine epithelium (Table 1). The presence of PGM activity in connective tissue is related to negative slopes in glycogen level curves in control and 0.5 mg Cd/l exposed L. littorea. On the contrary, glycogen depression from the connective tissue is not associated to demonstration of PGM activity in winkles exposed to 1.25 mg Cd/l seawater. PGM activity in the intestine epithelium corresponds to positive slopes or maximum peak values of glycogen level in the digestive gland cells (Table 1, Fig. 1B). Glycogen has been located in granules and basal cytoplasm of nephrocytes, as well as in the underlying blood sinuses (Fig. 3C). False positive reaction, not inhibited by diastase pre-treatment, was evidenced in apical cytoplasm, brush border, and lumen. Glycogen levels have also shown an exposure-time and dose dependent depression in kidney folds (Fig. 1C) and in connective cells from their adjoining mantle (Figs lD, 3A, and 3B). A cyclic variation in glycogen

histochemical levels has kidney folds. Minimum the 17th experimental values take place around

been also found in control values have been recorded day, while maximm peak the 6th and the 20th days

Table 1. Values of PGM activity (arbitrary units) in the interstitial connective tissue (A), in the intestine (B), in the renal folds (C), and in the proximal portion of the gill lamellae (D). Exposure time in days, and external cadmium concentration in mg/l Concentration Tissue

Time

0

0.5

I .25

A

3 6 10 14 18 22 28

0.401 + 0. I43

n.d. n.d. 0.251 + 0.063 n.d. n.d. n.d. 0.303f0.111

n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.190*0.052 n.d. 0.316 + 0.079 0.231 * 0.049 -

n.d.

B

3 6 10 14 I8 22 28

n.d. 0.382 f 0.142 n.d. n.d. n.d. n.d. 0.160 + 0.025 n.d. n.d. 0.120 * 0.030

C

3 6 10 14 18 22 28

0.190 f 0.071 n.d. n.d. 0.124 f 0.039 n.d. 0.296 + 0.135

0.205 k 0.075 n.d. 0.298 * 0.101 n.d. n.d.

n.d. 0.201 + 0.066 0.179+0.076 n.d. n.d.

D

3 6 10 14 18 22 28

n.d.

0.200 * 0.107 n.d. 0.469 -1:0.163 n.d. 0.549 5 0.168 0.301 f 0.172

n.d. 0.384 f 0.113 n.d. nd. 0.526 + 0.139 0.480 k 0.084 0.173 f0.104

n.d. n.d. 0.395 + 0.104 nd.

0.699+ 0.291

0.300 + 0.072

(n.d.) non-demonstrated

n.d. 0.175 & 0.076 0.225 2 0.066 n.d. n.d.

activity; (-)

no sample.

n.d.

J. M. GIL et al.

644

(Fig. 1C). Glycogen levels are mainly depressed after 6 day exposure to 0.5 mg Cd/l, and even before the first analysis day (4th) in animals exposed to 1.25 mg Cd/l (Fig. 3D). Therefore, the oscillatory dynamics observed in controls is not evidenced in renal glycogen levels of treated winkles. PGM activity has been located in nephrocytes and mantle connective cells. This enzymatic activity is associated, similarly to the instance of connective pore cells from the digestive gland-gonad complex, with negative slopes in glycogen level curves for kidney (Table 1, Fig. 1C). Three different zones have been distinguished in gill lamellae according to previous workers on histochemistry and morphology of the gill epithelium of Littorina littorea: proximal with cubic ciliated cells, mid-zone with cubic cells, and distal epithelium with high cells but mainly mucocytes (unpublished data). Glycogen is mainly located at the apex and basal third of the cells comprising the proximal epithelium of the gills. After 17 days under Cd-exposure conditions, Best’s carmine positive material is demonstrated inside the intralamellar blood vessels. From the 1417th Cd-exposure day onwards, this material is also present in the epithelial basal third of cells from the distal zone of the gill lamellae. This event occurs in association with disruption of proximal epithelium cells and also concomitantly with larger depressions in the glycogen levels of the adjacent connective tissue of the mantle (Fig. 1D). In agreement with these observations, data from Table 1 indicate that PGM activity is demonstrated only from the 14th and 17th day on. Sudanophilic materials and lipases

Lipidic materials are located at the basal half of digestive cells, in association with the zones where Table 2. Values of lipase activity (arbitrary units) in the digestive cells (A), in the blood sinuses and connective tissue of the kidney folds (B), in the excretory cells (C) and in the proximal portion of the gill lamellae (D). Exposure time in days, and external cadmium concentration in mg/l Concentration Tissue

Time

A

3 6 10 14 18 22 28

B

3 6 IO 14 22 28

0 I.088 1.473 1.303 0.842 0.149 0.914

0.5

k 0.299 +_0.330 + 0.363 f 0.289 & 0.277 f 0.269 -

0.704 + 0.187 0.697 _+0.193 0.595 + 0.325 -

I.163 + 0.290 I.419 + 0.258 I.193 + 0.307 0.913+0.311 0.866 + 0.222 I.193 + 0.256

-

0.645 + 0.199

I .629 _+0.293 -

-

D

3 6 IO I4 I8 22 28

0.632 ltO.171 0.444 * 0.201 0.595 + 0.325 0.578 * 0.371 I.180 f 0.443 0.733 k 0.192

1.228 + 0.264

activity;

-

-

0.622 IO.257 -

IO 22 28

(n.d.) non-demonstrated

0.828 f 0.314 0.698 k 0.318 1.393_+0.313 0.783 + 0.246 0.919 + 0.284

0.570 k 0.299 0.633 + 0.257

C

-

1.25 0.461 +O.llB -

0.623 0.995 I.101 1.783

+ 0.253 + 0.268 f0.318 _t 0.342 0.445 f 0.243 0.576 f 0.189 (-)

no sample.

0.423 f 0.21 I 0.399 _+0.175 n.d. 0.993 * 0.307 0.801 f 0.299 0.841 + 0.347 0.527 0.555 0.356 1.433 0.677 0.731

k 0.260 + 0.327 + 0.209 _+0.350 + 0.191 f 0.271

4

6

10

13

17

20

24

17

20

24’

1 27

TIME (days) OCONTROI.

Ei

O.bmgCd/l

I1.25,,w,CdlI

Fig. 2. Semiquantitive estimation of the sudanophilic substance levels (arbitrary units) against the exposure time (days) in the dark granules of the digestive cells (A), and in the renal folds (B) after staining with Sudan B.

dark and yellow granules are present. Total lipid content shows a cyclic pattern in histochemical detection in control and 0.5 mg Cd/l exposed winkles (Fig. 2A). A similar trend is observed in 1.25 mg Cd/l exposed specimens, but this is broken by a final strong decrease. Sudan B positive reaction has been also located in intestinal epithelium. This seems not to be affected by stabulation or cadmium exposure. Lipase activity has been mainly detected in granules and cytoplasm from the basal third of digestive cells (Table 2). However, it must be outlined that a precise cellular distribution is unfeasible because required incubation causes damage to cell integrity (Janssen, 1985). Lipase activity is slightly decreased through experimental time in control digestive glands. It is kept quite constant in the 0.5 mg/l exposure series, while evidently increases with exposure time in animals exposed to 1.25 mg Cd/l. Moreover, at this exposure series, lipase activity showed to be more sparse by the cytoplasm of the digestive cells than in control series. Lipase activity is shown to be extremely high in intestinal epithelium, where no changes have been recorded. Lipid content increases in nephrocyte granules and in subjacent blood sinus connective cells of control L. Zittorea, maximum levels being detected after 20-27 stabulation days (Fig. 2B). Nephrocytes of animals exposed to 0.5 mg Cd/l show a slightly higher lipid content during the first days, and a subsequent strong decrease from the 20th experimental day on (Fig. 2B). A very poor level of histochemically demonstrable lipids has been evidenced in all the instances of winkles exposed to 1.25 mg Cd/l. Lipase activity has been observed in the blood sinuses underlying the renal epithelium. As a result of procedural damage leading to poor tissular integrity

Cd reduces Littarinaglycogen

645

Fig. 3. (A) Histochemical demonstration

of glycogen in the mantle connective tissue of control winkles of glycogen in the connective tissue of the mantle in animals exposed to 1.25 mg Cd/l for 24 days. S.B.: SOpm. (C) Glycogen in the renal folds of a control winkle after 24 days of stab~lat~o~. S.B.: 100 ,um. (D) Glycogen demonstration in the renal folds of a winkle exposed to 1.25 mgCd/l for 24 days. S.B.: 50pm.

af&r a siabuiat~o~ of 20 days. LB.: 50 pm. (6).DeGonstration

observations on nephrocytes have been realized only occassionally, enzymatic activity being located in basal granules. Positive intensity in these granules of control nephrocytes is higher than that recorded in adjoining blood sinuses (Table 2), but it decreases due to cadmium exposure. On the other hand, the maximum values for lipase activity in blood sinuses have been recorded during the first days in the 1.25mg Cd/l exposure series (0.993 + 0.307 arbitrary u.). Further, activity decreases significantly from the 14th Cd-exposure day on, and no lipase activity has been demonstrated the 28th day because the blood sinuses are completely reduced (Fig. 3D). Lipids have been located only at the apical and basal thirds of the cells from the proximal epithelium in gills. As a result of stabulation some positive material has been evidenced in the basal portion of the cells from the mid-epithelium sometimes. No changes have been observed as a result of cadmium exposure. Lipase activity has been also found in gill lamellae+ Although tissue disruption makes it difficult to ensure its location, it seems to be associated with lipid sites, Reaction intensity is quite variable throughout the time, and the observations indicate that there is not a specific trend due to stabulation or to cadmium exposure (Table 2). DISCUSSION

In most of molluscan species, glycogen is shown to be an organismic reserve pool whose location is

essentially deserved to mantle and interstitial connective tissues (Cabbott, 1976; Bayne et al., 1978, 1982), as well as to the kidney (Andrews, 1981; Livingstone and De Zwaan, 1983). In some concrete instances, glycogen storage has been also attributed to the digestive and basophilic cells of gastropod digestive gland (Walker, 1970), but the general thought is that major intracellular reserves are lipids (Sumner, 1965; Walker, 1970; Livingstone and De Zwaan, 1983; Janssen, 1985). As a result, lipid reserve consumption would be restricted to only some extreme instances as, for example, starvation or hibernation (Sumner, 1965; Walker, 1970; Oxford and Fish, 1979; Janssen, 1985), and environmental stress (Bayne et al., 1978). The present observations indicate that both glycogen and lipids are used in Littu~~~ffiit~or~#. According to some authors (Sumner, 1969; Janssen, 1985), fat metabolism is fundamental in gastropods molluscs; however, Livingstone and De Zwaan (1983) assert that glycogen is the major reserve substance involved in energy storage for the general metabolism in gastropods. An alternative energy source (protein or glycogen) dependent on metabolic requirements has been proposed by Abdullah and Ireland (1986) in the prosobranch N~e~~~aIa~~~~~s. The uItrastr~ctura1 evidence of a close spatial relation between lipid droplets and glycogen fields in kidney and digestive gland of gastropods (Moya, 1973; Marigomez et al., in press), and the demonstration of an integration of glycolisis and fatty acid biosynthesis in molluscs (Livingstone and De Zwaan, 1983) are indicative of

646

J. M. GIL et al.

an interdependence of both reserve resources. Moreover, the similar alternation in levels of histochemitally demonstrable glycogen in the kidney and digestive gland are parallel to those found in lipid levels whose sole difference is a 3 day displacement in the time. Thus, it is feasible to hypothetize that alternative lipid or glycogen consumption would depend on the particularities of the inhabitat of a population, or even, in the same species, of the environmental conditions which impose metabolic requirements. In this context, the present observations indicate that after a pre-experimental starvation period, glycogen levels, which are rapidly recovered (4 days), show very slight decreases in reserve connective tissue, and kidney, whilst lipid storage increases in kidney epithelium. Simultaneously, mobilisation of glycogen across the digestive epithelium occurs at alternate periods, indicating a normal glycogen utilization, maybe in gametogenesis (Bayne et al., 1982), or even the effect of experimental stabulation. According to Bayne et al. (1978), lysosomal destabilization in both the digestive cells and interstitial connective cells is associated with the demand of energy. The mechanism consists of a controlled autolysis coupled to the mobilization of glycogen within membrane bound vesicles (Bayne et al., 1982). In fact, presently, vesicular traffic of glycogen has been observed in both the kidney and the digestive tubule epithelia. Although such mobilization is associated with PGM activity in nephrocytes, PGM activity is not relevant in digestive cells, larger mobilizations across the digestive gland being related to maximum peak values of PGM activity in the intestinal epithelium. This suggests that glycogen released from digestive epithelium might be reabsorbed in the intestine. Consequently, glucose could be transported to the systemic circulation across the intestinal epithelium for its distribution throughout the organism (Barber et al., 1975). A similar mechanism for glucose distribution directly from renal epithelium (Taylor and Andrews, 1987) also might occur. According to Whelan (1975) PGM activity is inhibited by several metals which compete with enzymatic magnesium, and hence, it could be supposed that cadmium also interacts with the active core of the enzyme. On the other hand, cadmium enhances nonspecific esterase (included lipases) activity (Vallee and Ulmer, 1972). PGM and lipase activities do not seem to be directly affected by cadmium in the present experimental conditions because both enzymatic activities were demonstrated similarly in all the studied tissues, The sole exception is the interstitial connective tissue of the digestive gland of winkles exposed to 1.25 mg Cd/l where PGM activity has not been found in any case. This could indicate a different mechanism for glycolysis in this tissue under extreme environmental conditions. Lysosomal hydrolases could be involved in rapid glycolytic processes (Bayne et al., 1982) but the present results are not sufficient to clarify this aspect which will concern further investigation. Thus, the toxic impairment of enzymatic activities should occur only over threshold metal concentrations which probably would interfere with enzymatic working, resulting in an acute toxicity. The presently tested concentrations of cadmium

do not cause significant mortality at any time in these experimental conditions, which cover the whole range of sublethal response (Marigomez et al., 1987). The observations are in agreement with a general stress response mediated by an extra demand of energy which leads to an increased reserve consumption. Simultaneously, routine observations during the experiment indicated lower locomotor and feeding activities in highly exposed animals. This suggests the concomitant induction of a defficient nutritional stage. This event has been previously reported by Leakey and Dutton (1975) and Marigomez et al. (1986) in terrestrial gastropods. As a result, the decline in the glycogen content of reserve tissues should be interpreted as a non-specific response. In fact, quite different environmental stresses have been demonstrated to cause it. Lead on earthworms (Ireland and Richards, 1977), cadmium on the prosobranch N. lapillus (Abdullah and Ireland, 1986), and abnormal temperature and starvation on mussels (Cracium, 1980; Bayne et al., 1978, 1982) cause inhibition of glycogen synthesis and enhancement of its degradation. Similar observations have also been made in vertebrates (Reddy et al., 1982; Lowe-Jinde and Niimi, 1984). In conclusion, although the quantitative response can be quite dissimilar from one species to another and from one stressor to another, the qualitative response is shown to be general (Blackstock, 1984). In this context, some of the present findings resemble the observations of other authors about the histochemical changes associated with long-term starvation in several gastropods. Janssen (1985) indicates that although the body fat content is rather constant during starvation in the slug Arion rujii, the fat contents in the digestive gland is reduced after 3 weeks. This author generalizes this conclusion to pulmonate and prosobranch gastropods, including the marine species Patellu uulgata (Sumner, 1969). In agreement with the above results, the loss of lipid from the digestive gland becomes apparent after 20 days of treatment in the present work. Sumner (1965) and Walker (1970) report similar lipid level decline in the digestive gland of terrestrial pulmonates, in association with a previous migration of Sudan black positive granules to the apex of digestive cells. In these instances, lipid depletion is related to a nonspecific esterase activity located in digestive cell granules (Sumner, 1969; Oxford, 1979; Janssen, 1985). Esterase activity has been shown, in agreement with the present results, to be sparsed by the cytoplasm after longer starvation periods. Gills are not believed to be reserve tissues in gastropods, however, glycogen, lipids, and PGM and lipase activities have been found in the present investigation. Normal low levels of glycogen and lipid are basically restricted to the proximal epithelium which is suppossed to play an osmotic regulatory role (unpublished data). Then, these materials could participate in the energy supply for gill working, or even in the direct regulation of osmosis (Nevenzel et al., 1985). The sole significant change observed during the present cadmium exposure conditions is the displacement of glycogen towards the distal epithelium and the large increase in glycogen contents in the underlying blood vessel, indicating a recovery

Cd reduces Lirtorina glycogen

of materials. Thurberg et al. (1977) conclude that low concentrations of cadmium do not cause alterations in crustacean gill physiology while high ones do. Instead of damage, enhanced gill tissue respiration takes place under low Cd-exposure conditions. These results reflect again the conceptual differences between acute and sublethal damage. Similarly, sublethal concentrations of metals also causes increased working in other organs as kidney (Marig6mez et al., in press) and digestive gland (Recio et al., 1988). The overall effect is a tissular premature ageing which results in partial disruption of the tissues after exhaustion. In these conditions of high energetic demand and reserve decline, materials from disrupted tissues might be recovered. A mechanism similar to the resorption of gametes during normal life-cycles or in stress conditions in association with glycogen mobilization (Bayne et al., 1982) might be envisaged from the present observations. Summarizing, sublethal concentrations of cadmium cause deficient nutritional conditions and increased energetic requirements in winkles. The response does not include enzymatic impairment but increased utilization of glycogen, and subsequent utilization of lipids. This resembles the mechanisms described during starvation and hibernation in several molluscan species. Acknowledgemenu-We gratefully appreciate the technical assistance of Mr A. Mateo, Mr J. L. Esteban, and Mrs A. A. Alvarez. This investigation was in part financed by the Basque Government (Research Project X-86041) and by the Spanish Ministry of Education (FPI, A.18, 1985). REFERENCES

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