Biochemical and histochemical observations on effects of low-level heavy metal load (lead, cadmium) in different organ systems of the freshwater crayfish, Astacus astacus L. (crustacea: Decapoda)

ECOTOXICOLOGY

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ENVIRONMENTAL

SAFETY

21, l37- 156 (199 1)

Biochemical and Histochemical Observations on Effects of LowLevel Heavy Metal Load (Lead, Cadmium) in Different Organ Systems of the Freshwater Crayfish, Astacus astacus L. (Crustacea: Decapoda) W. MEYER,* *Ins&it

M. KRETSCHMER,~

A. HOFFMANN,*

AND G. HARISCH~

jiir Zoologie and tlnstitut fiir Physiologische Chemie, Tieriirztliche Hochschule Hannover, Biinteweg I7, D-3000 Hannover 71. Federal Republic of Germany Received May 14. 1990

The effects of low-level lead (20 &liter) and/or cadmium (2 &liter) exposure on the structure and function of different organ systemsof the freshwater crayfish, Astacus astacus L. (Crustacea: Decapoda) were estimated by several biochemical and histochemical methods. The animals were incubated during 10 weeks (max.) at a temperature of 10°C and a normal diurnal rhythm. Lead accumulated in high amounts especially in the digestive gland, carapax, and gills. whereas the hindgut and musculature exhibited very low lead levels. Cadmium accumulated particularly in the digestive gland and gills. Lead and cadmium levels were definitely lower in the digestive gland, gills, and carapax of animals incubated in water containing a double, i.e., lead and cadmium load, than in animals kept in water containing only one of these heavy metals. Histochemically both metals could be visualized in a typical distribution within the tissues, such as the carapax, digestive gland, or gills. After several weeks of poisoning, all organs, but especially the digestive gland, showed severe structural impairment. The activities of oxidative enzymes in the digestive gland and gills were significantly lowered after 2 weeks of incubation. Enzyme histochemical evaluation demonstrated changes of reaction intensities within the organs as compared to the controls. GSH Stransferase activities and GSH contents were also distinctly decreased following lead and/or cadmium intoxification. The histochemical demonstration of SH and S-S groups exhibited a stronger staining reaction after 10 weeks of exposure, especially in digestive gland and gills. The results obtained are discussed in view of the specific impairment of function of the organ systemsstudied, as related to the typical biology ofthe animal species tested. Q 1991 Academic Press. Inc.

INTRODUCTION

The biological impact of heavy metals in aquatic systems has become a major concern in recent years. One of the most interesting aspects, in particular in view of long-term effects, is the influence of low-level heavy metal concentrations on different ecological systems. In this connection, invertebrates may serve as helpful indicator organisms, as has already been shown for different artificial or natural microcosms (see, e.g., Hendrix et al., 1982) or for macroinvertebrate community structures, exhibiting a predictable, graded response to heavy metal pollution (Schulz-Baldes, 1973; Winberg, 1978; Winner et al., 1980; Wachs, 1983). It has been substantiated that invertebrates tend to concentrate lead and cadmium more than other living aquatic organisms. In addition, with low concentrations of both metals in the water, enrichment of these metals in organisms was clearly higher than it was with relatively higher metal concentrations in the medium (see, e.g., Enk and Mathis, 1977: Anderson, 1978: Taylor, 1983; Meyer et al., 1986). 137

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Until now, our knowledge on specific ecological effects of low-level heavy metal concentrations on aquatic arthropods has been rather limited (Sprague, 1984). Moreover, examination of recent literature reveals that many studies performed on heavy metal accumulation and influence in this animal group reveal a more or less nonbiological approach to the problem; i.e., most of the experiments done used relatively high concentrations of the metals in question. This implies, for example, values ranging from 100 pg/liter to 10 mg/liter for cadmium, or 1 to 100 mg/liter for lead (see, e.g., Amiard-Triquet et al., 1986; Green et al., 1986; Weis and Kim, 1986; Hong and Reish, 1987; Tulasi et al., 1987; Del Ramo et al., 1989; Ramirez et al., 1989; Torreblanca et al., 1989; Reddy et al., 1989; Wong and Wong, 1990). Considering, however, the problems referred to previously and the manifold and various ways in which heavy metals may be uptaken and mediated in the organism, it seems worthwhile to get a more detailed insight into the different organ systems of invertebrates, as influenced by environmentally present low-level heavy metals. This may be especially true of freshwater organisms, which are sensitive to cadmium levels an order of magnitude higher than seawater organisms (Stebbing, 1976; Engel and Fowler, 1979). This phenomenon is related to the fact that cadmium complexes readily with chloride ions, and that increasing salt concentrations are inversely correlated with the concentration of free metal ions (Hahne and Kroontje, 1973; Engel and Fowler, 1979). With these problems in mind, and in view of the few relevant data on freshwater crustaceans (Fiirstner and Wittmann, 1983), the present study was designed to get a broader basis for a more differentiated interpretation of the reactions of different organisms under low-level heavy metal impact, such as the freshwater crayfish (Astacus astacus L.). The approach combines several biochemical as well as histological and histochemical methods, focusing on the demonstration of important oxidative processes. The results obtained are discussed in view of better estimating the direct consequences of a lowlevel heavy metal load on the environment. MATERIALS

AND METHODS

Animal Material The animals used in this study were 101 specimens (82 $8, 19 OP; length 8-10 cm) of the freshwater crayfish, AstacusastacusL., which had been obtained from a breeding company in southern Germany (Teichwirtschaft Bernbachtal, Dr. Max Keller, Augsburg). In this connection it was possible to get a rather uniform animal material.

Holding Conditions The animals were kept separately in plastic containers (length 45 cm, width 28 cm, height 20 cm) with a plastic tube as a hide-out. The plastic material was chosen to minimize any adsorption of heavy metals at the surface of the holding tanks. The normal holding conditions included a water temperature of 10°C and a 16-hr day/& hr night diurnal rhythm. The animals were fed once a week with small pieces of pork heart and carrots. The containers were continuously ventilated and cleaned. All crayhsh were allowed to acclimatize for 3 weeks before the experiments started. For the different tests, four groups of 20-24 animals each were formed. Under experimental conditions, the first group (I) lived in water containing 20 &liter lead, and the second group (II) in water with 2 pg/liter cadmium; in the third group (III)

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the water contained both, 20 pg/liter lead and 2 pg/liter cadmium. The fourth group (IV) served as the control group with fresh water under the same general conditions but without the addition of heavy metals. Primary stock solutions of the metals were prepared by diluting reagent grade AAS standard solutions ( 1 mg/ml in dilute HN03, Sigma) in distilled water. The desired heavy metal concentrations were obtained by adding aliquots of tertiary stock solutions to measured volumes of fresh water. The lead- and/or cadmium-poisoned water was changed every 5 days in order to maintain the heavy metal concentrations at approximately the same indicated value. After 2, 5, 7, and 10 weeks, 5-6 crayfish of each group were taken for the different methods outlined below. Tissue Preparation Upon removal from the water, the animals were rinsed with aqua dest. and treated as follows. For the biochemical analyses and specific histochemical purposes (heavy metals, enzymes, GSH), the specimens were killed at once by short immersion in liquid nitrogen; afterward, the gills, digestive (midgut) gland (empty after starving), abdominal musculature, hindgut (empty), and carapax were dissected out or prepared ice cooled, and stored in liquid nitrogen until further processing. For the histochemical assays, the relevant parts of the animals were directly frozen and stored as tissue blocks in liquid nitrogen before sectioning in a SLEE cryostat (Type “HS”). Several tissue blocks ofeach test animal were also fixed in 4% buffered formalin (pH 7.4) and Bouin’s solution and embedded without ethanolic media in the plastic resin Technovit 7 100 (Kulzer; see Gerrits and Smid, 1983), or after dehydration via ethanol and Histosol (Shandon) in paraffin wax (Histoplast, Serva). The animals were always killed at 10: 00 AM in order to avoid diurnal variations. Only stainless-steel dissecting equipment, glass, and plastic labware, washed with 10% nitric acid and distilled water, were used in preparations of the tissues for metal analysis (see also Kruse, 1980). Biochemical

Methods

Heavy metal analysis. The amount of accumulated lead in the different tissue samples was determined after dry ashing and dissolution of the resulting ash in nitric acid. The aqueous samples were analyzed in a flameless atomic absorption spectrophotometer (Perkin-Elmer Zeeman 5000 with HGA 400) at a wavelength of 217 nm. The cadmium amounts were determined by the direct injection method with a GriinZeeman SM 20 atomic absorption device at a wavelength of 228.8 or 326.1 nm. Other methodical aspects are described in detail by Kruse ( 1980) and Stoeppler and Niimberg (1979). If necessary, the standard addition method or matrix matched calibration graphs were applied to minimize as far as possible matrix influences (especially for lead). Enzyme activity determinations. For enzyme activity analysis, the tissue samples were homogenized in ice-cold saccharose-Tris buffer (0.25 M saccharose, 0.005 M Tris, pH 7.6). The mitochondria of the homogenate were sonicated and used as the enzyme sample, while the postmitochondrial supematant served as the enzyme sample for the microsomal NADPH-cytochrome P450 reductase. Succinic dehydrogenase was measured using the method of Kramar (197 1). The primary electron acceptor was menadione and the secondary acceptor was 2-(p-iodophenyl)-3-(pnitrophenyl)-5-phenyltetrade (INT) (Sigma). The enzyme

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activity is given in nmol INT (reduced) X mg protein-’ X min-‘. The NADPHcytochrome P450 reductase activity was determined with the method of Phillips and Langdon (1962) with cytochrome c as electron acceptor. The results are given in nmol cytochrome c (reduced) X mg protein-’ (from postmitochondrial supematant) X min-‘. Glutathione Stransferase was measured according to Habig et al. ( 1974). After elimination of mitochondria and cytosol the 105,OOOg pellet served as the source of the microsomal enzyme. 1-Chloro-2,4-dinitrobenzene served as the glutathione-conjugatable substrate at a final concentration of 0.5 n&L The final concentration of GSH was 1.0 m&L Formation of the glutathione conjugate was recorded at a wavelength of 340 nm (25°C). The results are expressed as nmol of the conjugate X mg protein-’ X min-‘. A Zeiss PM Q3 was used for the enzymatic optical measurement at a cuvette temperature of 25°C. Protein was determined using the biuret method, which was checked at regular intervals by addition of certain amounts of bovine serum albumin (fraction V, COHN). GSH measurements. GSH content was determined fluorometrically as described by Hissin and Hilf (1976) and modified by Harisch et al. (1979). GSH content is expressed as pmol/g of organ tissue. Histological

and Histochemical

Methods

The Technovit-embedded tissue was cut at a thickness of 3 pm and the paraffin material at a thickness of 8 pm. For normal histology, the sections were stained with hematoxylin (after Delafield) and eosin or with methylene blue-azure II (Morgenstem, 1969). Heavy metal distribution. The distribution of lead and cadmium within the tissues was visualized with the help of the autometallographic procedure of Danscher (1984) as modified by the silver enhancement technique (Danscher et al., 1987; chemical kit from Janssen). Additionally, cadmium distribution was demonstrated according to Sumi et al. (1984a,b), using benzothiazolylazo-/3-naphthol as the staining reagent. SH and S-S groups. The demonstration of sulfhydryl and disulfide groups for the indirect detection of metallothioneins was done in paraffin-embedded tissue according to the method of Sippel as modified by Morselt et al. (1984), using tri-n-butyl-phosphine (Sigma) as the reducing reagent, in combination with the dichloroquinone-chloroimide method of Hallit and Damas (198 1). Enzyme histochemistry. For the succinic dehydrogenase the method of Nachlas et al. (1957) was used, with tetranitroblue tetrazolium salt (TNBT) (Merck) as the electron acceptor (substrate concn. 60 mM, TNBT concn. 1.5 mM, incubation time 30 min at 37°C). The NADH and NADPH diaphorases (NADH- and NADPH-tetrazolium reductases) were demonstrated according to Scarpelli et al. (1958) (NADH and NADPH (Boehringer) concn. 3 mg/ml, TNBT concn. 0.5 mM, incubation time 20 min at 37°C). The cytochrome c oxidase activity was visualized with the amine-amine method of Burstone (1960) (amine (Sigma) concn. 20 mM, incubation time 45 min at 37°C). The different control reactions were done according to Hentschel and Meyer (1982). All enzymes were demonstrated in IO-pm fresh frozen sections. As in the demonstration of heavy metals within the different tissues, the sections of control animals were always incubated in the same medium, i.e., together with the sections of the test

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animals. Note that intact, usable frozen sections of the digestive gland could not be obtained regularly, probably because the high lipid content of the cells sometimes prevented total freezing of the organ. Statistical Evaluation After control for normal distribution, Student’s t test was applied for evaluation of significant differences (P < 0.01) between test and control animals. RESULTS During the incubation in water containing heavy metals, the test animals showed no general behavioral disorders. After 3-4 weeks, the locomotory activity seemed somewhat lowered, but the crayfish still fed relatively constantly. The first signs of disturbance of normal biology followed 1 to 2 weeks later, when several animals molted. Now it became obvious that the new cuticula did not harden as usual, but remained more or less smooth. For these animals food uptake as well as rapid movements was clearly troublesome. From a histological point of view, a distinct impairment of tissue structure could be discerned best in the digestive gland (midgut gland). Here, the epithelium of the glandular tubules normally contains absorptive cells (R-cells) and secretory cells (Bcells) (see, e.g., Dall and Moriarty, 1983). After several weeks of heavy metal poisoning, the epithelium of several tubules or parts of tubules showed an overall presence of numerous pathologically altered, extremely enlarged cells with voluminous cytoplasmic vacuoles. These cells seemed to be degenerated B-cells. They dominated the glandular tubule in that their epithelial organization was lost, because the large vacuoles of several neighboring cells obviously had fused, including a disintegration of the cell walls (Figs. 1b-ld). However, this phenomenon was confined to individual tubules and did not affect the entire digestive gland, so that some intact glandular epithelium was always present. Heavy Metal Accumulation The results of the organ lead and cadmium analyses are shown in Figs. 2-4. Tissue metal content is represented as mean concentration with the range indicated by bars. The values obtained varied in relation to the different organs studied, although the heavy metal content generally increased in all organs compared to that of the controls, with the exception of cadmium values in the musculature. It was obvious that lead and cadmium accumulated rather rapidly in the organs within the first 2 weeks of incubation, after which the metal content remained rather constant, with the exception of the digestive gland, where both heavy metals concentrated continuously, and the carapax, where lead contents developed in the same way. In general, lead accumulated in very high amounts especially in the digestive gland, the carapax, and the gills, while the hindgut and the musculature exhibited very low lead levels. Cadmium accumulated particularly in the digestive gland and the gills. It was interesting to note that the lead level was definitely lower in the digestive gland of animals incubated in water with a lead and cadmium load than in animals that had been kept in water containing lead ions alone. Accordingly, cadmium contents were also lower in the digestive gland, gills, and carapax of animals that had been incubated with both heavy metals than in

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FIG. 1. Heavy metal induced structural changes in the epithelium of tubules of the digestive gland. (a) Control, (b) 2 weeks cadmium, (c) 10 weeks cadmium, (d) 10 weeks lead; all Technovit sections, H&E staining; bar represents 50 pm (L, lumen of tubule).

animals living in cadmium water. The differences between the different test groups were statistically significant (P < 0.01). In analyzing the lead and cadmium concentrations found, as related to the heavy metal concentrations of the water under test conditions, it appeared that accumulation rates in the organs were different from those of absolute heavy metal tissue concentrations; i.e., the enrichment factor of the heavy metal was different in relation to the respective organ studied. In this connection, both heavy metals also interacted with each other when applied together (Fig. 3). The histochemical results on lead and cadmium distribution generally agree with the biochemical findings. Compared to the controls, exposed animals showed heavy

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IN CRAYFISH Pb water Pb+Cdwatw

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FIG. 2. Heavy metal accumulation in different organs after incubation in lead and/or cadmium water: controls in black (DG, digestive gland; Cl. gills; MU, musculature: HG. hindgut: CA, carapax).

metal precipitations in the tissues studied. This was best visualized with the autometallographic procedure of Danscher ( 1984). In the digestive gland, heavy metal deposits were found in the apical cytoplasm of the epithelial cells (mostly R-cells), but also below and around the large vacuoles, near the basal membrane (Fig. 4~). In the hindgut and the gills, the heavy metals were more or less concentrated at the surface of the epithelia and near the basal membrane (Fig. 4b). In the carapax, the heavy metal load was confined to the mesocuticular layers and absent in the exocuticula (Fig. 4a). The staining procedure of Sumi et al. (1984a,b), using benzothiazolylazo-@-naphthol for the demonstration of cadmium, produced positive results only in the digestive gland and the gills. In the latter, strongly positive reactions could be observed in the epithelial cells as well as in the hemolymph of the afferent and the efferent hemolymph channels (Fig. 4e). In the digestive gland, positive staining was visible in the cytoplasm of the epithelial tubule cells and, in particular, at the surface-probably the microvilli and the surface enteric coat-of the cells, above all of the extremely voluminous, pathologically altered secretory cells (Fig. 4d).

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Pb

DG Pb+Cd

Pb+Cd II Cd

Cd

b

Cd P

FIG. 3. Enrichment of lead and cadmium in different organs as related to heavy metal concentrations in the water (for abbreviations see the legend to Fig. 2).

Oxidative Activities The development of the activities of important oxidative enzymes (succinic dehydrogenase, NADPH-cytochrome P450 reductase) in the different organs after lead and cadmium poisoning is shown in Fig. 5. Significant differences (P < O.Ol), i.e., distinct decreases in enzyme activities, were generally visible after 2 weeks of incubation, independent of the heavy metals applied or their combination, respectively. Lead, in particular, caused the most severe effects, while cadmium or the combined lead and cadmium incubation resulted in somewhat lower activity losses. The rather low levels of enzyme activities, typically reached after 2-4 weeks of incubation, remained more or less stable during the following time. The histochemical procedures applied for the demonstration of different oxidative enzymes (succinic dehydrogenase, cytochrome c oxidase, NADH and NADPH diaphorases) revealed results comparable to those of the biochemical measurements. The

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reactions of all these enzymes clearly decreased in intensity in tissues or organs of animals incubated in water containing heavy metals. This was obvious in the respiratory cells of the gills (Figs. 6a-6d), and also in the epithelium of the digestive gland and the hindgut (Figs. 6e, 6f). GSH Contents and GSH S- Transferase Activities As already demonstrated for the oxidative enzymes studied, the GSH contents as well as the GSH S-transferase activities exhibited distinct decreases following lead and/ or cadmium intoxification. This could be seen best in the gills, whereas the digestive gland reacted somewhat less sensitively (GSH contents). Here the combination of both heavy metals caused a less severe impairment of enzyme activity, and, vice versa, a reduction in GSH amounts after several weeks of incubation (Fig. 7). SH and S-S Groups The histochemical demonstration of SH and S-S groups exhibited a stronger staining reaction after 10 weeks of incubation in the digestive gland, with special reference to the disulfide bonds and a somewhat lower reaction of the sulfhydryl groups. The staining of SH and S-S groups was restricted to the apical portion of a few reactive epithelial cells that formed small groups within the tubular epithelium (Figs. Sa, 8d, 8e). In comparison with the controls, the hemolymph in the efferent hemolymph channels of the gills reacted also more intensely for S-S groups (Fig. 8~). while reactions in the carapax were negative. DISCUSSION The findings of the present study confirm the view that low-level heavy metal poisoning affects organs of aquatic arthropods in a varying manner. The grade of impairment of normal organ or tissue structure and function depends greatly on the specific constructive principles developed for each organ in relation to biotope and mode of life of the species or animal group concerned. It is obvious that highly active metabolic organ systems, such as the midgut, offer the most effective possibilities to encounter such dangers during long-term incubation. Other organs, such as the brain or the gills, react more readily with a slow but continuous decrease in optimal functioning, an aspect which indicates behavioral changes as connected with problems in dealing with normal life (see also Meyer et al., 1986). A more detailed view of the different organ systems concerned emphasizes numerous problems of structure and function impairment. Evaluating the results obtained in the course of this study, concentration development of lead and cadmium seems to be similar in most aquatic arthropods and other invertebrates: lead accumulates in high levels in the exoskeleton, but also in the gills and the digestive gland; cadmium is found highly concentrated especially in the digestive gland, but also in the gills; while the hindgut, and particularly the musculature, contain only very small amounts of these metals (see, e.g., Thurberg et al., 1977; Anderson, 1978; Anderson and Brower, 1978; Dickson et al., 1979; Stoeppler and Ntirnberg, 1979; Stinson and Eaton, 1983; Amiard-Triquet et al., 1986; Meyer et al., 1986; Engel and Brouwer, 1986; Tulasi et al., 1987; Uthe et al., 1987).

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FIG. 4. Histochemistry of heavy metals in different organs after 10 weeks of exposure. (a-c) Autometallographic procedure, plastic sections, dark-field microscopy: (a) carapax; (b) gills with strong reaction in mucous layer, (c) apical part of epithelial cells in digestive gland tubule. (d, e) benzothiazolyl-azo-~-naphtholmethod for cadmium; (d) epithelium of digestive gland tubule; (e) gill filament; bar represents 50 pm (EX, exocuticula; ML, mucous layer; BE, branchial epithehum; BM, basal membrane; L, lumen of tubule; AF and EF, afferent and efferent hemolymph channels).

The digestive gland, particularly in crustaceans, has a well-defined role in the storage, metabolism, and detoxification of a number of metals. Its role as an excretory organ, however, is unclear, but it may be used for storage of excretion (Dal1 and Moriarty, 1983). In this respect, different authors have found that cadmium, for example, induces metallothionein formation in Decapoda (Jennings et al., 1979; Olafson et al., 1979; Overnell and Trewhella, 1979; Wiedow et al., 1982; Overnell, 1986; Del Ramo et al., 1989). Other potentially toxic heavy metals are presumably detoxified in the same

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0

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I 4

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1 7

I lOweek<

-

FIG. 5. Activity of oxidative enzymes (NADPH-cytochrome P450 reductase. succinate dehydrogenase) in digestive gland (DG) and gills (GI) during heavy metal exposure.

way. Nevertheless, although comparisons between animals from polluted and nonpolluted areas have shown that in polluted biotopes the digestive gland is able to concentrate more than lOO-fold higher amounts of heavy metal than in nonpolluted regions (Overnell and Trewhella, 1979; Uthe et al., 1987), the protective capacities of the animal against heavy metal intoxification cannot be assessed critically from the amounts of metallothioneins found. Other parameters, e.g., alterations in oxidative metabolism, seem to be more appropriate indicators (see below). As previously discussed (Hughes, 1976; Ramirez et al., 1989) the effects of heavy metals on metabolic rates depend to a considerable degree on the organism’s reaction mechanisms, the entrance speed of substances into the organism, and the competition which may occur for an active place in several enzymes. Our biochemical as well as histological and histochemical findings demonstrated distinct impairment of metabolism in the digestive gland during exposure to relatively low heavy metal levels. Histologically this could be seen from the increasing appearance of degenerated epithelial cells with voluminous vacuoles and a tendency of the epithelial arrangement to disintegrate. Comparable effects have been reported for other crustaceans only after

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FIG. 6. Histochemical demonstration of oxidative enzymes in gills (a, c) and hindgut (e) after heavy metal exposure of 10 weeks as compared to controls (b, d. f). (a, b) NADH diaphorase (NADH-tetrazolium reductase); (c, d) succinate dehydrogenase: (e, f) cytochrome c oxidasc: bar represents 100 pm for (a-f).

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A GSH-S-T

200-

PbCd -

DO

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as

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00 0

2

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7

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FIG. 7. Development of GSH Stransferase activity and GSH contents in digestive gland (DC?) and gills (GI) during heavy metal exposure.

very high levels of lead intoxification (Amiard-Triquet et al., 1986), and it seems reasonable that all these effects reflect a general response to different types of pollution. This view may also apply to mollusks, in which the digestive gland appeared to be the organ most damaged after heavy metal poisoning (Benedetti et al., 1982). The decrease of oxidative enzyme activities in the digestive gland as well as in the gills or other metabolically active organs of the freshwater crayfish could be due to an increase in degenerated mitochondria, which generally was the major cadmium-induced alteration observed in other invertebrates as well (Thurberg et al., 1977; Seidman et al., 1986). Results comparable to our findings have been published by Reddy et al. ( 1989) for the freshwater field crab (Barytelphusa guerini), although incubation took place at much higher cadmium concentrations. In addition, mitochondrial respiration is shown to be even more sensitive to lead (Bull, 1980). For freshwater invertebrates this was demonstrable in the brain of odonate larvae (Meyer et al., 1986). With regard to succinic dehydrogenase, for example, it has been shown that this oxidative enzyme

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FIG. 8. Histochemistry of SH and S-S groups in digestive gland (a, h, d, e) and gills (c) after heavy metal exposure. (a, b) SH groups: (a) after 7 weeks lead, (b) control. (c) S-S groups in gills after 10 weeks lead. (d, e) S-S groups in digestive gland epithelium after 10 weeks cadmium-typical reaction in single cell groups (arrow); bar represents 100 grn (EF, efferent hemolymph channel; L, lumen).

possesses sulfhydryl groups that are very sensitive to heavy metals and, as it is buried deeply in the inner mitochondrial membrane, the cadmium and lead ions may become stably associated with the enzyme as it passes through the membrane (Jacobson and Turner, 1980). The problems discussed above may also be related to GSH metabolism. In mammals, GSH Stransferase reacts very rapidly with reduced activity after cadmium administration (Grose et al., 1987). These results, generally similar to our own findings, point to a noncompetetive inhibition of this enzyme by the heavy metal (Reddy et al., 198 1). An induction of GSH S-transferase activity by lead nitrate (Dock, 1989) may be a short-term aspect, because we also found distinctly decreasing activities for this enzyme after lead incubation. As far as reduced glutathione is concerned, it is the most abundant intracellular thiol, and it may be involved in the elimination of metals by complexation with these toxic elements (Klaasen et al., 1985). In mammals, reduction of hepatic GSH concentrations, for example, results in a marked increase in the toxicity of cadmium and liver damage (Wong and Klaasen, 198 1). Basically, GSH in invertebrates

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may, likewise, be involved in heavy metal detoxification, apart from the fact that sulfhydryl in the form of metallothionein effectively inactivates the reactive cadmium or lead ion. The strong histochemical reaction for S-S groups might be related to the accumulation of this binding protein (see also Suzuki and Yamamura, 1980; Morselt et al., 1984). For long-term low-level heavy metal impact, both mechanisms may be necessary and probably complement each other, considering that in such circumstances only one portion of the crustacean digestive gland tubules is more or less intact and metabolically active and that the regeneration cycle of epithelial digestive cell types may be disturbed and/or only partially effective. The first areas to suffer from functional alterations by any intoxicant, including heavy metals, are the gills. The large surface area for adsorption, the large volume of water passing over the gill surfaces, the adsorption on mucous sheaths secreted as a protective response, and the relatively small biomass of the gills compared to their surface area all result in high heavy metal concentrations, especially of cadmium, in this respiratory organ (see also Anderson and Brower, 1978). Similar problems are known to exist in different molluscan species (see, e.g., Hemelraad et al., 1986; Robinson and Ryan, 1986), as well as aquatic vertebrates, such as the trout, where waterborne cadmium accumulates in the branchial epithelial cells (Verbost et al., 1989). In crustaceans such cadmium is rapidly transported into the hemolymph (Wright, 1977; Janssen and Scholz, 1979), as bound to hemocyanin, and then deposited in the digestive gland (Brouwer et al., 1984). In addition, other studies have shown that lead causes equally severe damage to the gill filaments of crayfish under the same inescapable conditions described before (Anderson, 1978; Torreblanca et al., 1989). All these observations correspond to our histochemical findings on heavy metal distribution, and our biochemical and hi&chemical results on the decrease of oxidative enzyme activities reflect the problems arising for normal oxygen uptake and handling in the gills of decapods. Impairment of epithelial cell structure and function can be compensated for by increasing the amount of oxygenated water passing over the gill surface for shorter periods. Such short-term (Anderson, 1978; Ramirez et al., 1989) as well as contradictory effects on the stimulation of oxygen uptake have been reported (Vemberg et al., 1974; Thurberg et al., 1977; Uma Devi and Rao, 1989). Longer and chronically low-level heavy metal poisoning, however, reduces the respiratory rates for the animal, leading to severe problems for general body metabolism (see also Jacobson and Turner, 1980). This implies that heavy metal ions are specifically stored in the mucous film covering the gill surfaces (Anderson, 1978; Anderson and Brower, 1978). and so are continuously supplied to the epithelial cells. A problem specifically connected with gill function under cadmium burden needs to be discussed separately in view of the way the crustacean exoskeleton is formed. The primary method by which the cuticula is hardened in this arthropod group is calcification, a process in which calcium salts are deposited in the organic matrix of the cuticula, filling the spaces between the chitin fibrils (Stevenson, 1985). Micromolecular concentrations of cadmium in water inhibit branchial Ca*+ uptake and induce, therefore, hypocalcemia in teleost fishes (Verbost et al., 1987, 1989). Our observations of incomplete cuticula hardening in test animals incubated in cadmium water could, thus, be explained by hypocalcemial influences in the crayfish. In this way our findings confirm earlier proposals of cadmium metabolism in the common shrimp (Crungon crangon) by Dethlefsen (1978). Moreover, the crustacean digestive gland also serves as an organ of accumulation and metabolism of calcium, which is stored as small

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granules. These granules appear to have the additional role of immobilizing lead (Dal1 and Moriarty, 1983). Because there is evidence that cadmium inhibits intestinal calcium absorption in mammals (Foulkes, 1986), the cadmium burden in the crayfish may also disturb normal calcium metabolism in the digestive gland, in competing not only for calcium but also for lead, as can be seen from our biochemical measurements of heavy metal accumulation (see also below). Another effect of heavy metal intoxification on normal body metabolism in the crayfish becomes obvious when the results from animals incubated in water containing both lead and cadmium ions are considered. In contrast to mollusks (see, e.g., Phillips, 1976; Elliott et al., 1985; Marcus and Thompson, 1986), certain interactions of these heavy metals could be supposed. For example, not only lead uptake but also cadmium uptake was clearly reduced in the digestive gland and in the gills, when both metals were present. This is probably due to mutual competition, as can be seen from heavy metal enrichment in the carapax of decapod crustaceans, where lead compensates for cadmium in competing for calcium (Wright, 1977). In the metabolically highly active digestive gland, the relatively low lead burden in the presence of cadmium may, to some extent, be influenced via enzyme metabolic aspects; i.e., cadmium can obviously reverse negative lead effects on enzymes when present in higher concentrations (Jacobson and Turner, 1980). This latter aspect may be connected with long-term incubation as well as with the concentration of cadmium in the hemolymph and the digestive gland cells themselves. Furthermore, as can be deduced from experiments with mammals, lead ions are able to inhibit cadmium uptake through absorptive epithelia (Foulkes, 1986). This aspect is confirmed by our results, demonstrating lower contents of cadmium in the hindgut and the gills after incubation in water containing both heavy metals. Ultrastructural observations corroborate some of the views discussed above. The formation of intracellular lead inclusion bodies, for example, is highly susceptible to the presence of other metals such as cadmium, presumably due to competition for the initial binding proteins (Fowler, 1987). So all the data again stress the importance of metal-metal interaction with regard to the understanding of the bioavailability and bioeffects of heavy metals. CONCLUSION A continuous, low-level heavy metal stress in aquatic systems may have a gross biological impact comparable to that of intermittent insults of much greater intensity (Winner et al., 1980). In addition, the available experimental evidence also suggests that the uptake of pollutants from the water may be the major source of residues in the different animal species concerned (Moriarty, 1988). Thus, it appears to be important to meticulously verify the influences of low-level effects on aquatic invertebrates and their organ systems, by means of which it could be possible to broaden our perception of the dangers arising, dangers that may stem from the fatal handling of rather “low,” but still permissible, heavy metal limiting concentrations for aquatic biological systems in industrial countries (see, e.g., Aurand, 1986). Nevertheless, heavy metals, whatever the concentration, are quite unbiological, and, therefore, merely limiting them is a basically unsuccessful approach to this ecological problem. ACKNOWLEDGMENTS The skillful technical assistance of Mrs. K. Bosch, Mrs. B. Hassfurther, and Mrs. K. Loeppen is gratefully acknowledged. We are greatly indebted to Dr. R. Kruse and Mrs. A. Schmidt (Staatl. Veteriniruntersuch-

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ungsamt Cuxhaven) as well as Dr. M. Stoeppler and Mrs. C. Mohl (Institut fur Physiologische Chemie, Kemforschungsanlage Jtilich) for their expert technical advice and support. Finally, we thank Dr. Y. Sumi (St. Marianna University, School of Medicine, Kawasaki, Japan), who kindly supplied a sample of the benzothiazolylazo-/3-naphthol reagent.

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