Effects of cadmium and zinc on the composition of phosphate granules in the marine snail Littorina littorea

Aquatic Toxicology, 25 (1993) 43 54 © 1993 Elsevier SciencePublishers B.V. All rights reserved 0166-445X/93/$06.00

43

AQTOX 00557

Effects of cadmium and zinc on the composition of phosphate granules in the marine snail Littorina littorea J.A. Nott and W.J. Langston Plymouth Marine Laboratory, Citadel Hill, Ply,mouth, UK (Received 1 July 1992;accepted 10 November 1992)

In the hepatopancreas of the marine snail Littorina littorea there are intracellular granules of magnesium/ calcium phosphate which act as sites of metal accumulation, detoxification and excretion. Previous X-ray microanalysis (XRMA) has shown that cadmium is localized at the limiting membranes of the granules and does not enter the mineral component. The present XRMA work on the effect of cadmium on zinc uptake from seawater suggests that this 'membrane' cadmium does not affect the mass fraction of zinc in the granules but does reduce the mass fraction of magnesium. However, treatment with cadmium reduces tissue concentrations of potassium and zinc, and from this it is concluded that the metal may cause a reduction in the total number of granules in the hepatopancreas. Treatment with zinc causes an increase in the mass fraction of calcium in the granules and a decrease in magnesium. Zinc does not affect the elemental concentrations of the whole hepatopancreas tissue. The high variability of the concentrations of magnesium in the granules for each treatment contrasts with the low variability of phosphorus, calcium and, particularly, potassium.

Key words: Cadmium; Zinc; Littor&a; Phosphate granules; X-ray microanalysis

INTRODUCTION The periwinkle Littorina littorea a n d other m a r i n e snails (gastropods) have a large digestive gland which takes the form of a d i v e r t i c u l u m of the gut. It has n u m e r o u s f u n c t i o n s which include extracellular a n d intracellular digestion, a n d storage of nutrients. It is c o n s t r u c t e d of n u m e r o u s tubules with a n epithelial lining o f digestive a n d basophil cells; digestive cells are characterised by lysosomes a n d lipid, a n d basophil cells by r o u g h e n d o p l a s m i c reticulum a n d spherical, concentrically-structured, m i n e r alized granules. These granules are only 1 - 2 / l m in diameter b u t they occur in very large n u m b e r s a n d c o n s t i t u t e a d o m i n a n t feature o f the gland. T h e y consist s u b s t a n -

Correspondence to: J.A. Nott, Plymouth Marine Laboratory, Citadel Hill, Plymouth PLI 2PB, UK.

44 tially of hard acid metals, namely, magnesium, potassium and calcium which occur as phosphates (Mason and Nott, 1981; Mason and Simkiss, 1982). These granules also provide electrostatic binding sites for intermediate metals, including manganese, zinc, iron, cobalt and nickel (Simkiss and Mason, 1983; Mason et al., 1984; Nott and Nicolaidou, 1989b; Taylor et al., 1990) and thus, act as sites of localization and accumulation. They are eliminated via the gut in faecal pellets (Mason et al., 1984; Nott and Nicolaidou, 1989b) when basophil cells disintegrate into the lumen of the digestive gland during normal turnover of the epithelium. Soft acid metals, including mercury and cadmium, do not occur in tissues as phosphate deposits. Intracellularly, they are usually bound covalently to sulphur rich proteins (Mason et al., 1981; Langston and Zhou, 1986; Bebianno and Langston, 1991; and reviews - Roesijadi, 1992; Viarengo and Nott, 1992) either in the cytosol with metallothionein or within residual lysosomes which are confined to digestive cells rather than basophil cells. However, in the digestive gland of L i t t o r i n a l i t t o r e a , cadmium, in association with sulphur, occurs in the region of the bounding membrane of phosphate granules (Nott and Langston, 1989). It has been proposed that if cadmium is bound to active groups in the membrane it might affect the efficiency of transport of metals into the granules. This could form the cytological basis for some synergistic and antagonistic interactions which occur between cadmium and other metals during uptake from seawater (Eisler and Gardner, 1973; Wright, 1977; Bryan et al., 1985; Viarengo et al., 1985; Roesijadi and Fellingham, 1987; Engel and Brouwer, 1989; Luk'yanova and Evtushenko, 1989).

2000

,A

P

Mg

Zn .J Bremsstrahltmg r X-ray energy ->

--~f

/ j , , , _ , i l ....

~

--

....

5keV

Fig. 1. L i t t o r i n a littorea. X-raymicroanalyticalspectrumfroman intracellulargranuleshowingthe elemental peakson the non-specificbackground(Bremsstrahlung).The net integralfor each peak is calculatedby subtractingthe underlyingbackground.An integralfor an extendedregionof background(shadedarea) is recorded as a relativemeasureof total mass.

45 The present work investigates the effects of pretreating Littorina littorea with cadm i u m on the accumulation of zinc and other elements in phosphate granules of the digestive gland. X-ray microanalysis is used to compute the elemental composition of individual granules within basophil cells and techniques of cryo-fixation are used to retain labile elements at in vivo locations within the tissues. MATERIALS AND METHODS Adult Littorina littorea were kept in aerated seawater at a temperature of 12°C. They were divided into four groups of 20 which were kept in separate aquaria. The seawater was changed every three days. The first group was kept for 30 days in seawater which contained 0.4 mg Cd 1~. The second group was kept similarly in cadmium followed by 16 days in 1 mg Zn 1~. The third group was kept in 1 mg Zn 1l for 16 days without pretreatment with cadmium. The fourth group was kept in clean seawater as controls. After the treatments, groups of six animals were removed from the shell and divided into digestive gland and remaining soft tissues. Individual tissue samples were digested in HNO3 and finally made up in 1 N HC1 before being analysed by atomic absorption spectrophotometry for magnesium, calcium, zinc and cadmium. Potassium was analysed by flame emission. Small pieces of fresh digestive gland from separate individuals were prepared for an X-ray microanalytical study of the magnesium/calcium phosphate granules. A cryopreparation method prevented extraction and redistribution of elements; the tissue was quench frozen in liquid ethane at a temperature of -180°C (Ryan and Purse, 1984; Ryan et al., 1987, 1990), freeze-dried at - 8 0 ° C and embedded in epoxy resin at r o o m temperature. Sections were cut dry at a thickness of 1.5/.tm and pressed onto

TABLE 1 Summary of materials and methods. Specimen preparation quenched in ethane at -180°C freeze-dried at - 80° C embedded in resin Sections thickness: 1.5/lm cut dry grids: copper or titanium grid holder: graphite Electron microscope voltage: 200 keV mode: STEM stationary spot tilt angle: 30° X-ray microanalyser analysis time = 50 s dead time = 23-30% count rate -- 2000 counts s-t

46 f o r m v a r - c o a t e d , single-holed t i t a n i u m grids. This p r o c e d u r e has been d e s c r i b e d in detail in N o t t a n d L a n g s t o n (1989). S p e c i m e n grids were e x a m i n e d at 200 k V in a Jeol 2 0 0 C X t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e with the electron b e a m focussed as a p r o b e on the p h o s p h a t e granules. T h e resulting X - r a y fluorescence was d e t e c t e d a n d p r o c e s s e d in a m i c r o a n a l y s e r . X - r a y s can be d i v i d e d into two categories (Fig. 1): (1) B r e m s s t r a h l u n g , c o n t i n u u m or non-specific b a c k g r o u n d r a d i a t i o n with an overall intensity t h a t is related to the a t o m i c m a s s o f the p r o b e d v o l u m e o f the specimen; (2) c h a r a c t e r i s t i c X - r a y p e a k s with energies t h a t are specific for the fluorescing elements. A n a l y t i c a l c o n d i t i o n s for the present w o r k are given in T a b l e 1. F r o m X - r a y spectra, integrals are r e c o r d e d for p e a k s a n d intervening regions o f B r e m s s t r a h l u n g o r non-specific b a c k g r o u n d (Fig. 1). B a c k g r o u n d is s u b t r a c t e d f r o m each p e a k to give a net integral. A n integral for an e x t e n d e d region o f b a c k g r o u n d is r e c o r d e d for each s p e c t r u m as a relative m e a s u r e o f mass. E l e m e n t a l p e a k s are r a t i o e d to the e x t e n d e d b a c k g r o u n d integral a n d the values used for detecting c h a n g e s in m a s s fractions in different granules ( N o t t , 1991). RESULTS AND DISCUSSION

Analysis of whole tissue In o r d e r to m o n i t o r a n y effects o f t r e a t m e n t s on levels o f elements in the b o d y o f the a n i m a l , digestive g l a n d a n d r e m a i n i n g soft tissues were s e p a r a t e d a n d a n a l y s e d indiv i d u a l l y by a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y (Tables 2 a n d 3).

TABLE 2

Littorina littorea. Analysis of the digestive gland and remaining soft tissues by atomic absorption spectrometry. Results are given in/lg/g dry weight together with the standard deviation of the mean value (n = 6). The results for calcium show extreme variation and are not included. These results are analysed further in Table 3. Treatments

Cd

Zn

Digestive gland Control Zn Cd/Zn Cd

110 ± 17 95 ± 21

98 ± 994 ± 831 ± 73 ±

Remaining soft tissues Control Zn Cd/Zn Cd

69 ± 5 63 ± 25

ll 178 120 11

76 ± 6 106 ± 22 114 ± 23 61 ± 5

Mg

K

20805 ± 4295 20540 ± 5539 19130 ± 4797 15945 ± 2979

10748 ± 1803 9530 ± 427 9858 ± 996 8254 ± 1053

10278 ± 1616 10162 ± 1158 12893 ± 3040 13210 ± 2623

12287 ± 746 12328 ± 807 12059 ± 411 9382 ± 426

47 In the digestive gland, cadmium treatment causes a loss of potassium and zinc when compared with the controls, and a loss of potassium when compared with zinc treatment. After cadmium/zinc treatment the tissue has regained the quantity of potassium which occurs in controls. In the remaining soft tissue, cadmium treatment is associated with a loss of potassium with respect to controls but this is restored in the cadmium/zinc treatment. Results for calcium were too variable for statistical analysis.

Microanalysis of individual granules All the results are derived from counts of X-rays arriving at the detector from the specimen which is ' p r o b e d ' in the microscope by the electron beam. Peaks (Fig. 1) are related to elemental concentrations in the specimen but it is obvious that beam intensity, counting time and section thickness will have direct effects on the absolute values of peak integrals for X-ray counts. Generally in situations of controlled analysis, these parameters have similar effects on all elemental peaks so that peak to peak and peak to background ratios (mass fractions) remain constant. Also, the efficiency of X-ray collection for different elements varies to the extent that for isoatomic mixtures of magnesium and calcium, the peak integral for magnesium is 25% of the integral for calcium. This means that the ratios of peak integrals shown in Table 4 are not a direct measure of concentration ratios. Nevertheless, changes in the ratios do reflect changes in the relative concentrations of elements and changes in the ratios of elements with

TABLE 3

Littorina Bttorea. Student 't' treatments of the AAS data in Table 2. The matrix shows the occurrence of significant differences in the mean values between treatments, dg = digestive gland; st -- remaining soft tissues. "ql Treatments Zn

Cd

Cd/Zn

dg K* dg Zn*

Control

st K***

dg K*

Zn

dg K* st K*** *Significantly different (P = 0.01 - 0.05). * * *Significantly different (P < 0.001). Differences between all other values in Table 2 are not significant (P > 0.05).

Cd

48 TABLE 4 Littorina littorea. X-ray microanalysis of phosphate granules in the digestive gland after four treatments. The results are analysed further in Table 5. A Mass fractions of the elements measured as the ratios of the net peak integrals to an extended region of background (see Fig. I). Mass fractions

Control Mean SD Variability (%) Zn treatment Mean SD Variability (%) Cd/Zn treatment Mean SD Variability (%) Cd treatment Mean SD Variability (%)

Mg/b

P/b

S/b

K/b

Ca/b

Zn/b

1.26 0.94 75

6.2 2.3 38

0.3 0.2 76

1.5 0.4 29

3.5 0.9 26

0.50 0.53 107

4.8 2.2 46

0.3 0.1 43

1.9 0.4 23

5.6 1.9 34

0.59 0.29 49

1.05 0.67 64

5.7 1.5 26

0.5 0.2 52

1.9 0.3 17

4.8 1.3 28

0.48 0.28 58

0.45 0.55 124

4.7 1.8 39

0.3 0.1 51

1.4 0.4 26

3.7 0.8 21

B Elemental ratios measured as ratios of the net integrals for the peaks (see Fig. 1). Elemental ratios

Control Mean SD Variability (%) Zn treatment Mean SD Variability Cd/Zn treatment Mean SD Variability (%) Cd treatment Mean SD Variability (%) Variability =

P/Mg

P/K

P/Ca

4.2 3.2 76

4.4 1.9 44

7.3 2.7 37

7.7 6.9 90

2.6 1.2 44

3.7 1.7 45

53 44 83

6.8 3.4 51

17 13 79

7.0 3.0 44

3.1 0.8 25

4.8 1.1 24

101 90 90

6.1 5.5 90

20 16 78

6.0 8.6 141

3.5 1.6 47

4.9 2.1 42

SD × 100 Mean

P/Zn

K/S

Ca/Zn

9.0 10.8 120

6.8 3.7 54

49 respect to the background (e.g. K/b) do show changes in the mass fractions of the elements in the granules. In Table 4 it is apparent that mass fractions derived from the X-ray spectra show marked differences in variability when the standard deviation is calculated as a percentage of the mean. Thus, in controls, variability ranges from 26% and 29% for Ca/b and K/b, respectively, to 75% and 76% for Mg/b and S/b, respectively. This pattern is not disturbed by the three experimental treatments. All zinc ratios show high variability which could reflect a lack of equilibrium resulting from a short-term (16 day) dosage experiment. These results show that calcium and potassium are stable components of granules. It has been established that calcium occurs in the form of amorphous calcium phosphate but potassium could be linked to phosphate and/or protein ligands. However, when sulphur content is taken as an indicator of protein content, the 120% variability of the K/S ratio (Table 4) suggests that potassium is not linked to total protein content. Indeed, the lower variability of P/K ratios shows that potassium is more closely linked with total phosphate content. Stabilities of the mass fractions for calcium (Ca/b) and potassium (K/b) contrast with the extreme variability of the mass fraction for magnesium. Net integrals for magnesium can range from values in excess of calcium to zero. Efficiency of detection for magnesium is 25% of that for calcium which indicates that its molar concentration in some granules can greatly exceed that of calcium. All ratios which include magnesium show extreme variation, even in control animals. Data in Table 4 have been analysed by the Student's t-test to determine the significance of differences between mean values generated by the treatments (Table 5). It is apparent that treatment with zinc causes more elemental readjustments within granules than treatment with cadmium when compared with controls. Furthermore, zinc continues to have an effect after pretreatment with cadmium. Zinc treatment vs. control (Table 5)

Compared with controls zinc treatment granules show an increased mass fraction for calcium (Ca/b). There are reductions in the ratios of potassium and calcium to phosphorus (P/K and P/Ca) which suggest that potassium and calcium increase relative to phosphorus or phosphate. The mass fraction of phosphorus (phosphate), (P/b) is not affected significantly by any treatment. On the other hand, the mass fraction of magnesium (Mg/b) is decreased by treatment with zinc, which corresponds with an increase in the phosphorus to magnesium ratio (P/Mg), suggesting that magnesium decreases relative to phosphorus. Since the mass fraction of phosphorus remains constant, it is possible that magnesium is substituted on the phosphate ligand by calcium and zinc.

50 Cadmium treatment vs. control (Table 5)

Treatment with cadmium has less effect on the mass fractions of other elements in the granules than zinc although there is an indication that magnesium (Mg/b) is reduced. Cadmium-zinc treatment vs. control (Table 5)

This treatment produces increases in the mass fractions of potassium (K/b) and calcium (Ca/b) in granules but there are no significant changes in magnesium ratios. A reduction in the P/Ca ratio suggests that the treatment causes calcium to increase relative to phosphorus. Zinc treatment vs. cadmium treatment (Table 5)

Mass fractions of potassium (K/b) and calcium (Ca/b) in the granules have increased in zinc treated specimens by comparison with cadmium specimens. There is no significant difference in the mass fraction of magnesium (Mg/b) between the two treatments. Zinc treatment vs. cadmium-zinc treatment (Table 5)

There are no significant differences between the two treatments for mass fractions and elemental ratios. Cadmium-zinc treatment vs. cadmium treatment (Table 5)

Compared with cadmium treatment, further treatment with zinc (cadmium-zinc) causes an increase in the mass fraction of potassium (K/b), and calcium (Ca/b). Phosphorus and sulphur

Mass fractions of these elements are not affected, significantly, by the treatments. In granules, it is assumed that phosphorus is present as mineral phosphate and that sulphur is a constituent of the amino acids cysteine, cystine and methionine. Generally, the mass fraction of phosphorus (P/b) shows less variability within treatments than that of sulphur (S/b) (Table 4). Magnesium

Within treatments, variability of the magnesium mass fraction (Mg/b) exceeds that for potassium (K/b) and calcium (Ca/b). After treatments with cadmium and zinc, the

51 standard deviation for the Mg/b ratio exceeds 100% of the mean. Despite this variability, however, separate cadmium and zinc treatments cause a reduction in the Mg/b ratio, with respect to controls, which is significant at the 5% level. This contrasts with the increased Ca/b ratio after treatment with zinc, which is significant at the 1% level. Potassium

Potassium is a dominant element in intracellular phosphate granules although its retention in tissue for X-ray microanalysis can only be guaranteed by cryofixation. It is noteworthy that the mass fraction (K/b) recorded for each treatment shows the least variation of all the ratios (Table 4) and that after treatment with cadmium/zinc the standard deviation is only 17% of the mean. Potassium is, therefore, a soluble and quantitatively constant biochemical component of granules and it is probably linked with low molecular weight ligands. Functionally, it is likely to be a component of a buffering or ionic balancing system, since the granule is within a membrane-bound c o m p a r t m e n t of the cell. The mass fraction of potassium (K/b) in granules is increased significantly by treatment with cadmium/zinc with respect to controls and by zinc with respect to cadmium.

TABLE 5 Littorina littorea. Student 't' treatments of the X-ray microanalysesof phosphate granules in Table 4. The matrix shows the occurrence of significant differences in the mean values between treatments for mass fractions, e.g. Mg/b, and elemental ratios, e.g. P/Mg. b -- background, see Fig. 1.

• Treatments Zn

Cd

Cd/Zn

Mg/b* Ca/b** P/Mg*

Mg/b*

K/b* Ca/b* P/Ca*

Control

P/K*

P/Ca** Zn

K/b*

Ca/b* K/b** Ca/b* *Significantly different (P = 0.01-0.05). **Significantly different (P = 0.001-0.01). Differences between all other values in Table 4 are not significant (P > 0.05).

Cd

52

Calcium The mass fraction, Ca/b, remains constant within controls and within treatments. However, relative to controls, both zinc and cadmium-zinc treatments cause a significant increase in the mass fraction of calcium. Reduction in the P/Ca ratio after treatment with zinc indicates that the proportion of calcium increases relative to phosphate and that it possibly replaces magnesium. It can be concluded that although cadmium associates with the membrane which encloses phosphate granules in Littorina littorea, it does not affect the uptake of zinc by the mineral component. Also, cadmium does not affect the mass fractions of other elements in granules except for a reduction in magnesium. However, cadmium does react antagonistically with other elements in digestive gland tissue. Cadmium treatment reduces the concentrations of potassium and zinc with respect to controls as measured by AAS. The missing potassium and zinc may be associated with the cytosolic fraction but if, however, these elements are associated with the mineral fraction, it can be suggested that cadmium treatment reduces the frequency of granules in the tissue. Zinc treatment, on the other hand, does affect the elemental composition of the granules. It increases the mass fraction of calcium (Ca/b) and decreases the mass fraction of magnesium (Mg/b) with respect to controls. Zinc treatment after pretreatment with cadmium (cadmium/zinc) increases the mass fractions of potassium (K/b) and calcium (Ca/b) and increases the calcium content relative to phosphorus (P/Ca). Extreme variability of the mass fraction of magnesium (Mg/b) both within treatments and between treatments, indicates that it is readily incorporated into and lost from granules. It could, therefore, be replaced by calcium, zinc and other metals and form an active component of a very responsive buffering system for toxic cations. This effect has been reported previously in Mediterranean gastropods where zinc apparently replaced magnesium in granules (Nott and Nicolaidou, 1989a). High variability of magnesium within treatments contrasts with the low variability of phosphorus, calcium and potassium. It is presumed that calcium and phosphorus form the basic phosphate mineral of granules. This may or may not be the case for potassium which shows the least variability of all elements after treatment with zinc and cadmium/zinc. This stability for potassium contrasts with its high solubility. It is removed completely from granules which are treated with chemical fixatives before X-ray microanalysis. It is only retained in specimens which are prepared by freezequenching and freeze-drying. From these characteristics it is assumed that potassium is a readily exchangeable component of a system concerned with buffering and ionic balance of the compartment within the granule with respect to the rest of the cell.

53 ACKNOWLEDGEMENT

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