Chem.-Biol. Interactions, 27 (1979) 111--123 © Elsevier/North-Holland Scientific Publishers Ltd.
111
ACCUMULATION OF CADMIUM AND LEAD IN THE GILLS OF M Y T I L US EDULIS: X-RAY MICROANALYSIS AND CHEMICAL ANALYSIS
A.T. MARSHALL and V. TALBOTa'b
Department of Zoology and aDepartment of Inorganic Chemistry, La Trobe University, Bundoora, (Melbourne) Victoria 3083 (Australia) (Received December 10th, 1978) (Revision received March 19th, 1979) (Accepted March 21st, 1979)
SUMMARY
The accumulation of Cd and Pb in the gills of the lamellibranch mollusc Mytilus edulis has been studied by electron microscopy, X-ray microanalysis, atomic absorption spectroscopy and radionuclide monitoring. The patterns of accumulation of the two elements differ markedly as do the sites of deposition within the gills. Lead is found extracellularly as crystalline deposits in the basal lamina which forms the capillary walls of the gill lamellae. The Pb is found associated with Ca in equiatomic ratios and occurs either as a mixed or complex carbonate. Cadmium is always associated with S and frequently with P in membrane bound vesicles within the cells of the gill epithelium and in the amoebocytes. The S is probably attributable to the presence of cysteine residues in a metal binding protein which can be extracted from the gills. Analysis of the metal binding protein shows t h a t it binds Ag, Cd, Cu, Fe, Hg, Sn and Zn. Its amino acid composition is similar to that reported for eels and limpets but has a lower cysteine c o n t e n t than mammalian metal binding protein.
INTRODUCTION
Anthropogenic discharges of heavy metals into the marine system adjacent to Melbourne, Australia, have resulted in the pollution of the recent fluvial [1,2] and marine (3--4) sediments and the food chain [5--9]. The mussel Mytilus edulis has been reported to contain levels of Cd in excess of 200 ppm dry wt [10]. Lead levels in the same mussel are consistently above health standard regulations along the same coastline. bpresent address: Department of Environmental Science, Rusden State College, Clayton, Victoria 3168, Australia.
112 The uptake of Pb at the cellular level has been investigated in Mytilus kidney, which is reported as a major storage organ for Pb [11]. Lead has been shown to be present with P and Ca in membrane bound granules in the kidney cells. Cadmium uptake, however, does not appear to have been examined at the cellular level. It has been shown [12] that the gills are a major route of uptake of Fe in Mytilus. Uptake is attributed to pinocytosis in the epithelial cells and the Fe is subsequently transferred to amoebocytes and thence to the byssal gland and kidney. It was considered to be of some interest to examine the gills as sites of uptake of Cd and Pb and to investigate their accumulation pathways. The latter might be expected to differ from each other since it has been shown t h a t Cd is bound to a metal binding protein (MBP) in Mytilus whereas Pb is not so bound [13]. Toxicological experiments have shown that Pb is considerably less toxic to Mytilus than is Cd. The toxicological difference may also reflect different accumulation pathways; certainly accumulation rates of both Cd and Pb in Mytilus suggest that Cd has a much shorter biological half-time (tl/2) than Pb (Talbot, unpublished results). MATERIALS AND METHODS
Accumulation of cations Mussels (Mytilus edulis) 5.5 cm long were collected in Port Phillip Bay, adjacent to Melbourne. They were allowed to normalise in acid-washed glass tanks each containing 8 1 filtered (0.45 gm) seawater at pH 8.1, O2 concentration 6--7.5 ppm, salinity 35.5% and at a temperature between 16--18°C. The seawater was changed daily. Cadmium chloride (1 ppm) was administered to one set of tanks and PbC12 {10 ppm) to another. The mortality rates of mussels in these concentrations of toxin are low [13], consequently mussels could be sampled after 60 days exposure. A further series of tanks were used to monitor the metal uptake of M. edulis. The radionuclide Cd 115 m {approx. 1 t~Ci/1) in the chloride form was added to one set of tanks while stable Pb {10 ppm) was used in the other. Mussels were sampled regularly to construct accumulation graphs. The assimilation of this nuclide by the visceral mass, gills, mantle and foot of Mytilus was monitored using a Packard Tri-card Liquid Scintillation Spectrometer. The accumulation levels of Pb in these tissues were monitored using a Varian Atomic Absorption Spectrometer A.A.5. in conjunction with a HNO3/HC104 acid digestion of the tissues [7].
Sample preparation for electron microscopy (EM) studies After 50 days accumulation of stable Cd and Pb, the gills of the healthiest mussels were excised for electron probe X-ray microanalyses and morphological electron microscopy. At the same time samples were taken and stored in uncontaminated seawater at 2°C for chemical analysis. Gill samples were fixed for morphological electron microscopy in 3% glutaraldehyde {w/v) in
113 seawater for 2 h at 4°C, followed by 1% (w/v) OsO4 in seawater for 2 h at 4°C. Dehydration was carried out via a graded series of acetone solutions. The specimens were embedded in Spurr's resin. Ultrathin sections were stained in uranyl acetate and lead citrate. Thick sections (1 ~m) were m o u n t e d on glass slides and stained with methylene blue for light microscopy. Gills from Pb loaded specimens of M. edulis were fixed in 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) for 2 h at 4°C. Other specimens were fixed in 1% (w/v) OsO~ in seawater for 2 h at 4°C. Dehydration and embedding were carried o u t as before. Ultrathin sections were stained in uranyl acetate and lead citrate for morphological purposes. Thick sections (0.5 pm and 1.0 pm) were placed on titanium or carbon coated nylon grids for analysis. Gills from Cd loaded specimens of M. edulis were fixed in 2.5% (w/v) glutaraldehyde in seawater for 2 h at 4°C, or in a mixture of equal parts of 1% (w/v) OsO4 and 2.5% glutaraldehyde in seawater for 2 h at 4°C. Dehydration and embedding were carried out as before. Ultrathin sections were stained with uranyl acetate and lead citrate for morphological examination and thick (0.5 ~m to 1.0 ~m) sections were placed on carbon coated n y l o n grids for analysis. Analyses were carried out in a JEOL JEM 100B STEM fitted with an energy dispersive X-ray spectrometer (Edax 707A). The spectrometer resolution was 165 eV at Mn Ks. Spectra were processed by means of the Edit 7 EM computer programme (Edax Laboratories) running in a Nova 2 minicomputer. Data are expressed in terms of peak counts (1.2 × FWHM) and atomic ratios. Grids were m o u n t e d in a carbon specimen holder tilted 25 ° from the horizontal. Analyses were made in the TEM and STEM modes with beam currents (measured by Faraday cup) of I × 10 -IU A (STEM mode) and 5 × 10 -9 A (TEM mode). Selection of the TEM or STEM mode was made on the basis of granule size and the degree of contrast obtainable in the section. Thus, Cd-containing granules of relatively low contrast and small size were analysed by STEM, whereas Pb containing crystalline deposits of relatively large size and high contrast were analysed by TEM.
Metal binding protein The MBP fraction which contained Cd were isolated by centrifuging the homogenised gill tissue at 80 × 103 g for 1 h in a Beckman LS-65 ultracentrifuge. The water soluble MBP fractions were separated on a Sephadex G-75 column equilibrated with 0.05 M NH4HCO3 [13]. The MBP was then eluted through a G-50 column. The MBP eluate from this column was freezedried for X-ray microanalysis and amino acid analysis. This yielded a discrete fraction [ 13] which was considered to be sufficiently pure for a preliminary amino acid analysis. Amino acid analyses were made on a Beckman 120 Amino Acid Analyzer with the ion exchange column at a temperature of 55°C.
114
115
Papain digestion Gill samples f r o m Pb-loaded Mytilus were digested r e p e a t e d l y with an a q u e o u s solution o f papain and centrifuged. T h e final inorganic s e d i m e n t was r e t a i n e d . RESULTS T h e n o r m a l gill e p i t h e l i u m is s h o w n in Fig. 1 and consists o f several t y p e s o f ciliated and non-ciliated cells. At t h e level o f the e l e c t r o n m i c r o s c o p e , it can be seen t h a t the m a j o r i t y o f the epithelial cells contain e l e c t r o n dense bodies which are m e m b r a n e b o u n d {Fig. 2). These a p p e a r to be lysosomal in n a t u r e (Fig. 3~4,5) and are f o u n d at every level in the cell b u t are particularly p r o m i n e n t at the apical (Figs. 3 and 6) and t h e basal regions o f the cell. T h e y o c c u r in b o t h ciliated and non-ciliated cells. T h e basal lamina o f the epithelial cells is relatively thick' (2--4 p m ) and c o n t a i n s granular and fibrillar c o m p o n e n t s (Fig. 7). It f o r m s the wall o f the gill capillary. Within t h e capillary l u m e n are a m o e b o c y t e s which c o n t a i n electron-dense bodies (Fig. 8). T h e cells in t h e gills o f Cd l o a d e d M. edulis a p p e a r to be severly disrupted. V a c u o l i s a t i o n is extensive and t h e r e is a m a r k e d r e d u c t i o n in the hyaloplasm. M i t o c h o n d r i a and cilia a p p e a r to be relatively u n d a m a g e d . T h e r e are n u m e r o u s m e m b r a n e b o u n d vesicles o f varying size which c o n t a i n e l e c t r o n dense material {Fig. 9). In gills f r o m Pb l o a d e d M. edulis, the cells are highly-vacuolated b u t the vacuoles t e n d t o be larger t h a n in Cd l o a d e d gills. T h e m i t o c h o n d r i a are very swollen and t h e r e are n u m e r o u s m e m b r a n e b o u n d vesicles with e l e c t r o n dense c o n t e n t s (Fig. 10). In addition, crystalline deposits are f o u n d in the t h i c k e n e d basal lamina which f o r m s the capillary walls (Figs. 10 and 11). In o r d e r to d e t e r m i n e w h e t h e r Pb or Cd was leached f r o m tissues during f i x a t i o n and d e h y d r a t i o n , A.A.S. analyses were p e r f o r m e d o n all fluids used f o r the f i x a t i o n and d e h y d r a t i o n o f Pb and Cd l o a d e d gills. T h e levels o f Pb were e x t r e m e l y low b u t s o m e w h a t higher for Cd {Table I). T h e concent r a t i o n s o f Pb and Cd in u n t r e a t e d samples o f loaded gill tissues were 78 604 and 6 8 2 0 p p m d r y wt respectively. Since the u l t r a s t r u c t u r a l preservation with g l u t a r a l d e h y d e in p h o s p h a t e Fig. 1. Light micrograph of gill filaments seen in horizontal section (C: capillary lumen, L: lateral cilia, PL: post lateral cells). Fig. 2. Normal gill epithelium consisting of several types of ciliated and non-ciliated cells (F: frontal, LF: laterofrontal, I: intermediate, L: lateral cells). Membrane-bound electron dense bodies (arrows) are present in the epithelial cells and in the amoebocytes (A) within the capillary vessel (C). Fig. 3. Membrane-bound electron dense bodies (arrows) in the frontal cells. They are particularly prominent in the apical region of the cells (AM: apical plasma membrane). Fig. 4. Multivesicular body in a post lateral cell.
Fig. 5. M e m b r a n e - b o u n d e l e c t r o n d e n s e b o d i e s ( a r r o w s ) f o u n d at every level o f t h e lateral cells. Fig. 6. E l e c t r o n d e n s e b o d i e s ( a r r o w s ) in t h e l a t e r o f r o n t a l cells. Fig. 7. G r a n u l a r ( G ) a n d fibrillar c o m p o n e n t s ( F ) o f t h e basal l a m i n a o f t h e p o s t lateral cells w h i c h f o r m s t h e capillary wall. E l e c t r o n d e n s e b o d i e s (D) are p r e s e n t in t h e cells. Fig. 8. E l e c t r o n d e n s e b o d i e s (D) in t h e a m o e b o c y t e s (A) in t h e capillary l u m e n .
117 TABLE I QUANTITIES OF Pb and Cd LOST TO PROCESSING FLUIDS AS A PERCENTAGE OF A V E R A G E T O T A L GILL CONTENT Percentage of total gill content Processing Fluids
Pb loaded
Cd loaded
Glutaraldehyde in phosphate buffer 3 0 % Acetone 50% Acetone 7 0 % Acetone 8 0 % Acetone 1 0 0 % Acetone
0.40
7.05
0.00 0.01 0.00 0.06 0.06
I.I0 0.65 0.35 0.20 0.05
Total loss
0.53
9.40
buffer was n o t adequate, glutaraldehyde and OsO4 were subsequently used in seawater. Lead and Cd losses were not measured in these fixatives but it seems probable that there would not be a marked increase. Analyses of electron dense bodies in the Cd-loaded specimens were carried out. These dense bodies are si~ed in the cells of the gill epithelium and in amoebocytes which correspond to those seen in normal cells {Fig. 2). They reveal that Cd is always associated with S and frequently with P. A typical spectrum is shown in Fig. 12 and a series of analyses from laterofrontal cells are given in Table II. It can be seen that the atomic ratio of Cd:S is highly variable as also is P content. It is interesting to note that S also exists in the absence of Cd in similar dense bodies. T A B L E II X-RAY MICROANALYSIS
OF GILL EPITHELIA FROM
Site
Element
Apical granule
Cd S P
I~ Ks Ks
Basal granule
Cd S P
Apical granule
Peak intensity
Cd-LOADED MUSSELS
Atomic ratio
Mode
67 416 86
1.0 17.7 4.5
STEM
L~ Ks Ks
185 285 196
1.0 4.4 3.7
STEM
Cd S P
I~ Ks Ks
326 570 59
1.0 5.0 0.6
STEM
Apical granule
Cd S P
La K{x Ka
27 521 14
1.0 55.1 1.8
STEM
Multi-vesicular body
Cd S
I_~ Ks
1122 2389
1.0 6.1
TEM
118
119 | ¥S:
|||S|C 251 HS:
IC/S ~ 2|E¥/CH
0
t00SEC HS:
VS:2500
OC/S 20EV/CH
M I
Os L~
I II
~
~
/
C~K~
~4
~
i ~4~
t~
~
F ~ . 12. (left) ~ p i c ~ X-ray ~ e c t m m from an electron d e n ~ body in the g~l ep~he~um of Cd-loaded My ribs. ' Fig. 13. (right) ~ p i c a l X-ray spectrum from a deposit in the capill~y wall in the gill of Pb-loaded My tilus.
Analyses of the electron dense bodies of the Pb-loaded cells were carried out. Electron dense bodies occur throughout the cells of the gill epithelium b u t the presence of Pb was only detected in large crystalline deposits within the capillary wall and at the bases of the epithelial cells. A typical spectrum from a deposit in the capillary wall is shown in Fig. 13. Calcium appears to be invariably associated with Pb in these deposits in an atomic ratio which closely approaches 1:1 (Table III). These results were obtained from glutaraldehyde fixed specimens analysed in the TEM mode. Treatment of the inorganic residue, from papain-treated gills of Pb loaded mussels, with dilute HC1 resulted in the vigorous evolution of CO:. Vigorous effervescence was also obtained by acid treatment of intact gills from Pb loaded mussles whereas this was not the case with normal mussels. Amino acid analysis of the MBP (Table IV) shows t h a t it is rich in Scontaining cysteine residues. A freeze~lried sample of the MBP was analysed by X-ray microanalysis and found to contain Ag, Cd, Cu, Fe, Hg, Sn and Zn. The presence of these elements was confirmed qualitatively by atomic absorption spectrophotomerry. Fig. 9. Frontal and laterofrontal cells in the gills of Cd loaded Mytilus. Membrane-bound electron dense bodies (arrows) and mitochondrla (M) are seen in a reduced and evidently abnormal hyaloplasm. Fig. 10. Thick section (1 /~m) showing electron dense bodies (arrows) in the epithelial cells and amoebocytes (A) and crystalline deposits (CL) in the thickened basal lamina which forms the capillary wall. Gill of Pb loaded Mytilus. Fig. 11. Crystalline deposits (CL) in the thickened basal lamina which forms the capillary walls. Gill of Pb-loaded My tilus.
120 TABLE III X-RAY MICROANALYSIS OF CRYSTALLINE DEPOSITS IN THE CAPILLARY WALLS OF GILLS OF Pb LOADED MUSSELS Element
peak intensity
Atomic ratio
Pb Ca Pb Ca Pb Ca Pb Ca Pb Ca Pb Ca Pb Ca Pb Ca Pb Ca
17324 5521 14008 4696 18712 980~ 18460 10731 17710 8192 17243 10804 16769 7797 18333 8770 17512 3709
1.0 0.7 1.0 0.7 1.0 1.1 1.0 1.2 1.0 1.0 1.0 1.3 1.0 1.0 1.0 1.0 1.0 0.4
L~ Ks L~ Ks Lo~ Ks L~ K~ I_~ Ks L~ Ks Lo~ Ks L~ Ks L~ Ks
T h e a c c u m u l a t i o n levels o f P b a n d C d are sho~vn i n Fig. 14. T h e o p t i m u m levels o f b o t h P b a a d C d i n s e a w a t e r w e r e u s e d t o o b t a i n m a x i m u m a c c u m u l a t i o n a c c o m p a n i e d b y the m i n i m u m m o r t a l i t y . It can be seen t h a t the accum u l a t i o n p a t t e r n s o f Cd a n d P b are m a r k e d l y d i f f e r e n t . TABLE IV AMINO ACID ANALYSIS OF MBP Amino acid
Residues/mol
Lysine Histidine Arginine Aspartic acid Treonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Cysteine
7 2 3 10 6 6/7 10 6
10 6 5 1 4 5 2 3 9
121
o
o
O
Pb
,oo1/
1000
I00
I
' 10
0
20
40
60
80
100
Days
Fig. 14. A c c u m u l a t i o n rates o f C d a n d P b b y
Mytilus gills.
DISCUSSION Lead, associated with Ca in an equiatomic ratio, was shown to be present in the gills of Mytilus specimens, which had been lead loaded, as crystalline extracellular deposits in the capillary wall. These gills have also been shown to contain carbonate. It is therefore assumed that Pb occurs with Ca either a mixed or a complex carbonate. Lead has also been found associated with Ca in Mytilus kidney [11]. The finding of Pb as carbonate is perhaps not surprising since Zirino and Y a m a m o t o [14] predict, on the basis of thermodynamic stability constants and ionic coefficients for seawater, that the most prominent species of Pb in seawater is PbCO3. Similarly the association of Pb with Ca is noted repeatedly in the literature. For example most of the Pb in Mytilus occurs in the CaCO3 rich valves [7] and in man, it has been shown that 90% of the b o d y burden of Pb occurs in bones [15]. Further, deficiency of Ca in man and other animals increases susceptibility to Pb poisoning [16]. In chemical terms the association of Pb and Ca is not surprising since the ionic radius of Pb 2÷ (1.20 A ) is similar to that o f Ca 2÷ (0.99 A), consequently isomorphic replacement is likely even though their polarizabilities vary considerably. Since no Pb was detected within cells and since little Pb appeared to be lost from the tissues during processing, it may be assumed that Pb is rapidly transported through the cells and deposited extracellularly in an inert form. The effective concentration within the organism is thus kept low and this may well account for the lower toxicity of Pb compared to Cd, Zn, Hg and Cu which all show a preference for S-rich organic molecules [13]. Cadmium, unlike Pb, accumulated within the cells in the gills of cadmium-
122 loaded Mytilus. The evidence suggests that like Fe [12] it is taken up by pinocytosis, becomes associated with lysosomes and is s o m e h o w transferred to a m o e b o c y t e s in the capillary lumen. The association of Cd with S within the epithelial cells and a m o e b o c y t e s is further evidence for the existence of S-rich metal binding complexes. It has been suggested the S-rich MBP [13] is synthesised as a response to the uptake of certain heavy metals. This work shows that S is present in greater molar concentrations than Cd which suggests that the constituents of the MBP m a y be present prior to the uptake o f heavy metals. Elemental S or inorganic S-containing salts were n o t detected in the eluate from Sephadex gel filtration of the water soluble cytoplasmic components of the gills. However, S was detected in the MBP primarily as cysteine. Sulphur-containing taurine has also been recently found (Talbotunpublished data). Cadmium was also found, b y X-ray microanalysis, to be associated with P. No chemical analyses for P were carried out, however, on the eluate which contained metal binding protein. The significance of the association of Cd and P, therefore, is n o t obvious. Phosphorus m a y well be present in phospholipids contained within the membranes of the organelles in which Cd is sequestered. The loss (< 10%) of Cd during processing of the gills suggests the possibility that there may be an u n b o u n d Cd component. It is premature to speculate in detail on this b u t the observation is consistent with unpublished observations (Talbot, unpublished work) on the biological half-time (tl/2) o f Cd which suggest that Cd is present in more than one form or in more than one compartment. The MBP was also shown to be associated with Ag, Cu, Fe, Hg, Sn and Zn as well as Cd. It presumably has the capacity to bind all these elements. The amino acid content is similar to that reported for other aquatic organisms, e.g. {eels [17] and limpets [18], b u t has a lower cysteine content than mammalian MBP [19--23]. The different accumulation rates of Cd and Pb support the concept of different accumulation pathways for these t w o elements in the gills. REFERENCES 1 Anon., Heavy metals in the sediments of Port Phillip Bay and input streams. Environmental Protection A u t h o r i t y of Victoria, Rep. No. 16. 1976. 2 J.D. Smith, Proc. R. Aust. Chem. Inst., 43 (1976) 305. 3 V. Talbot, R. Magee and M. Hussain, Mar. Pollut. Bull., 7 (1976) 53. 4 H.T. French and P.J. Thistlethwaite, Proc. R. Aust. Chem. Inst., 43 (1976) 73. 5 V. Talbot, R. Magee and M. Hussain, Mar. Pollut. Bull., 7 (1976) 84. 6 D.J.H. Phillips, M. Sc. Thesis. Chemistry Dept., Melbourne University, 1975. 7 V. Talbot, R. Magee and M. Hussain, Mar. Pollut. Bull., 7 (1976) 234. 8 Victorian Parliamentary Hansard, 24 (1975) 6884, 6958. 9 Victorian Parliamentary Hansard, 27 (1975) 7881. 10 Victorian Parliamentary Hansard, 25 (1975) 7173. 11 M. Schulz-Blades, R. Lasch and S. Boseck, Micros. Acta, Suppl. 2 (1978) 175. 12 S.G. George, B.J.S. Pirie and T.L. Coombs, J. Exp. Mar. Biol. Ecol., 23 (1979) 71.
123 13 V. Talbot and R. Magee, Arch. Environ. Contamin. Toxicol., 7 (1978) 73. 14 A. Zirino and S. Yamamoto, Limnol. Oceanogr., 17 (1972) 661. 15 L.J. Casarett and J. Doull, Toxicology, The Basic Science of Poisons. Macmillan, NY, 1975, p. 478. 16 K.M. Six, Fed. Proc., 29 (1970) 568. 17 J.M. Bouquegreau, Ch. Gerday and A. Disteche, FEBS Lett., 55 (1975) 173. 18 A.G. Howard and G. Nickless, Chem.-Biol. Interact., 16 (1977) 107. 19 H. Newath, The Proteins, New York Academic Press, 1963, Vol. 1,427. 20 D.R. Winge and K.V. Rajagopalan, Arch. Biochem. Biophys., 153 (1972) 755. 21 T. Suda, N. Horinchi, E. Ogata, I. Ezawa, N. Otaki and M. Kimura, FEBS Lett., 42 (1974) 23. 22 J.H.R. Kagi and B.L. Vallee, J. Biol. Chem., 235 (1960) 3460. 23 P. Puldio, J.H.R. Kagi and B.L. Vallee, Biochemistry, 5 (1966) 1768.
111
ACCUMULATION OF CADMIUM AND LEAD IN THE GILLS OF M Y T I L US EDULIS: X-RAY MICROANALYSIS AND CHEMICAL ANALYSIS
A.T. MARSHALL and V. TALBOTa'b
Department of Zoology and aDepartment of Inorganic Chemistry, La Trobe University, Bundoora, (Melbourne) Victoria 3083 (Australia) (Received December 10th, 1978) (Revision received March 19th, 1979) (Accepted March 21st, 1979)
SUMMARY
The accumulation of Cd and Pb in the gills of the lamellibranch mollusc Mytilus edulis has been studied by electron microscopy, X-ray microanalysis, atomic absorption spectroscopy and radionuclide monitoring. The patterns of accumulation of the two elements differ markedly as do the sites of deposition within the gills. Lead is found extracellularly as crystalline deposits in the basal lamina which forms the capillary walls of the gill lamellae. The Pb is found associated with Ca in equiatomic ratios and occurs either as a mixed or complex carbonate. Cadmium is always associated with S and frequently with P in membrane bound vesicles within the cells of the gill epithelium and in the amoebocytes. The S is probably attributable to the presence of cysteine residues in a metal binding protein which can be extracted from the gills. Analysis of the metal binding protein shows t h a t it binds Ag, Cd, Cu, Fe, Hg, Sn and Zn. Its amino acid composition is similar to that reported for eels and limpets but has a lower cysteine c o n t e n t than mammalian metal binding protein.
INTRODUCTION
Anthropogenic discharges of heavy metals into the marine system adjacent to Melbourne, Australia, have resulted in the pollution of the recent fluvial [1,2] and marine (3--4) sediments and the food chain [5--9]. The mussel Mytilus edulis has been reported to contain levels of Cd in excess of 200 ppm dry wt [10]. Lead levels in the same mussel are consistently above health standard regulations along the same coastline. bpresent address: Department of Environmental Science, Rusden State College, Clayton, Victoria 3168, Australia.
112 The uptake of Pb at the cellular level has been investigated in Mytilus kidney, which is reported as a major storage organ for Pb [11]. Lead has been shown to be present with P and Ca in membrane bound granules in the kidney cells. Cadmium uptake, however, does not appear to have been examined at the cellular level. It has been shown [12] that the gills are a major route of uptake of Fe in Mytilus. Uptake is attributed to pinocytosis in the epithelial cells and the Fe is subsequently transferred to amoebocytes and thence to the byssal gland and kidney. It was considered to be of some interest to examine the gills as sites of uptake of Cd and Pb and to investigate their accumulation pathways. The latter might be expected to differ from each other since it has been shown t h a t Cd is bound to a metal binding protein (MBP) in Mytilus whereas Pb is not so bound [13]. Toxicological experiments have shown that Pb is considerably less toxic to Mytilus than is Cd. The toxicological difference may also reflect different accumulation pathways; certainly accumulation rates of both Cd and Pb in Mytilus suggest that Cd has a much shorter biological half-time (tl/2) than Pb (Talbot, unpublished results). MATERIALS AND METHODS
Accumulation of cations Mussels (Mytilus edulis) 5.5 cm long were collected in Port Phillip Bay, adjacent to Melbourne. They were allowed to normalise in acid-washed glass tanks each containing 8 1 filtered (0.45 gm) seawater at pH 8.1, O2 concentration 6--7.5 ppm, salinity 35.5% and at a temperature between 16--18°C. The seawater was changed daily. Cadmium chloride (1 ppm) was administered to one set of tanks and PbC12 {10 ppm) to another. The mortality rates of mussels in these concentrations of toxin are low [13], consequently mussels could be sampled after 60 days exposure. A further series of tanks were used to monitor the metal uptake of M. edulis. The radionuclide Cd 115 m {approx. 1 t~Ci/1) in the chloride form was added to one set of tanks while stable Pb {10 ppm) was used in the other. Mussels were sampled regularly to construct accumulation graphs. The assimilation of this nuclide by the visceral mass, gills, mantle and foot of Mytilus was monitored using a Packard Tri-card Liquid Scintillation Spectrometer. The accumulation levels of Pb in these tissues were monitored using a Varian Atomic Absorption Spectrometer A.A.5. in conjunction with a HNO3/HC104 acid digestion of the tissues [7].
Sample preparation for electron microscopy (EM) studies After 50 days accumulation of stable Cd and Pb, the gills of the healthiest mussels were excised for electron probe X-ray microanalyses and morphological electron microscopy. At the same time samples were taken and stored in uncontaminated seawater at 2°C for chemical analysis. Gill samples were fixed for morphological electron microscopy in 3% glutaraldehyde {w/v) in
113 seawater for 2 h at 4°C, followed by 1% (w/v) OsO4 in seawater for 2 h at 4°C. Dehydration was carried out via a graded series of acetone solutions. The specimens were embedded in Spurr's resin. Ultrathin sections were stained in uranyl acetate and lead citrate. Thick sections (1 ~m) were m o u n t e d on glass slides and stained with methylene blue for light microscopy. Gills from Pb loaded specimens of M. edulis were fixed in 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) for 2 h at 4°C. Other specimens were fixed in 1% (w/v) OsO~ in seawater for 2 h at 4°C. Dehydration and embedding were carried o u t as before. Ultrathin sections were stained in uranyl acetate and lead citrate for morphological purposes. Thick sections (0.5 pm and 1.0 pm) were placed on titanium or carbon coated nylon grids for analysis. Gills from Cd loaded specimens of M. edulis were fixed in 2.5% (w/v) glutaraldehyde in seawater for 2 h at 4°C, or in a mixture of equal parts of 1% (w/v) OsO4 and 2.5% glutaraldehyde in seawater for 2 h at 4°C. Dehydration and embedding were carried out as before. Ultrathin sections were stained with uranyl acetate and lead citrate for morphological examination and thick (0.5 ~m to 1.0 ~m) sections were placed on carbon coated n y l o n grids for analysis. Analyses were carried out in a JEOL JEM 100B STEM fitted with an energy dispersive X-ray spectrometer (Edax 707A). The spectrometer resolution was 165 eV at Mn Ks. Spectra were processed by means of the Edit 7 EM computer programme (Edax Laboratories) running in a Nova 2 minicomputer. Data are expressed in terms of peak counts (1.2 × FWHM) and atomic ratios. Grids were m o u n t e d in a carbon specimen holder tilted 25 ° from the horizontal. Analyses were made in the TEM and STEM modes with beam currents (measured by Faraday cup) of I × 10 -IU A (STEM mode) and 5 × 10 -9 A (TEM mode). Selection of the TEM or STEM mode was made on the basis of granule size and the degree of contrast obtainable in the section. Thus, Cd-containing granules of relatively low contrast and small size were analysed by STEM, whereas Pb containing crystalline deposits of relatively large size and high contrast were analysed by TEM.
Metal binding protein The MBP fraction which contained Cd were isolated by centrifuging the homogenised gill tissue at 80 × 103 g for 1 h in a Beckman LS-65 ultracentrifuge. The water soluble MBP fractions were separated on a Sephadex G-75 column equilibrated with 0.05 M NH4HCO3 [13]. The MBP was then eluted through a G-50 column. The MBP eluate from this column was freezedried for X-ray microanalysis and amino acid analysis. This yielded a discrete fraction [ 13] which was considered to be sufficiently pure for a preliminary amino acid analysis. Amino acid analyses were made on a Beckman 120 Amino Acid Analyzer with the ion exchange column at a temperature of 55°C.
114
115
Papain digestion Gill samples f r o m Pb-loaded Mytilus were digested r e p e a t e d l y with an a q u e o u s solution o f papain and centrifuged. T h e final inorganic s e d i m e n t was r e t a i n e d . RESULTS T h e n o r m a l gill e p i t h e l i u m is s h o w n in Fig. 1 and consists o f several t y p e s o f ciliated and non-ciliated cells. At t h e level o f the e l e c t r o n m i c r o s c o p e , it can be seen t h a t the m a j o r i t y o f the epithelial cells contain e l e c t r o n dense bodies which are m e m b r a n e b o u n d {Fig. 2). These a p p e a r to be lysosomal in n a t u r e (Fig. 3~4,5) and are f o u n d at every level in the cell b u t are particularly p r o m i n e n t at the apical (Figs. 3 and 6) and t h e basal regions o f the cell. T h e y o c c u r in b o t h ciliated and non-ciliated cells. T h e basal lamina o f the epithelial cells is relatively thick' (2--4 p m ) and c o n t a i n s granular and fibrillar c o m p o n e n t s (Fig. 7). It f o r m s the wall o f the gill capillary. Within t h e capillary l u m e n are a m o e b o c y t e s which c o n t a i n electron-dense bodies (Fig. 8). T h e cells in t h e gills o f Cd l o a d e d M. edulis a p p e a r to be severly disrupted. V a c u o l i s a t i o n is extensive and t h e r e is a m a r k e d r e d u c t i o n in the hyaloplasm. M i t o c h o n d r i a and cilia a p p e a r to be relatively u n d a m a g e d . T h e r e are n u m e r o u s m e m b r a n e b o u n d vesicles o f varying size which c o n t a i n e l e c t r o n dense material {Fig. 9). In gills f r o m Pb l o a d e d M. edulis, the cells are highly-vacuolated b u t the vacuoles t e n d t o be larger t h a n in Cd l o a d e d gills. T h e m i t o c h o n d r i a are very swollen and t h e r e are n u m e r o u s m e m b r a n e b o u n d vesicles with e l e c t r o n dense c o n t e n t s (Fig. 10). In addition, crystalline deposits are f o u n d in the t h i c k e n e d basal lamina which f o r m s the capillary walls (Figs. 10 and 11). In o r d e r to d e t e r m i n e w h e t h e r Pb or Cd was leached f r o m tissues during f i x a t i o n and d e h y d r a t i o n , A.A.S. analyses were p e r f o r m e d o n all fluids used f o r the f i x a t i o n and d e h y d r a t i o n o f Pb and Cd l o a d e d gills. T h e levels o f Pb were e x t r e m e l y low b u t s o m e w h a t higher for Cd {Table I). T h e concent r a t i o n s o f Pb and Cd in u n t r e a t e d samples o f loaded gill tissues were 78 604 and 6 8 2 0 p p m d r y wt respectively. Since the u l t r a s t r u c t u r a l preservation with g l u t a r a l d e h y d e in p h o s p h a t e Fig. 1. Light micrograph of gill filaments seen in horizontal section (C: capillary lumen, L: lateral cilia, PL: post lateral cells). Fig. 2. Normal gill epithelium consisting of several types of ciliated and non-ciliated cells (F: frontal, LF: laterofrontal, I: intermediate, L: lateral cells). Membrane-bound electron dense bodies (arrows) are present in the epithelial cells and in the amoebocytes (A) within the capillary vessel (C). Fig. 3. Membrane-bound electron dense bodies (arrows) in the frontal cells. They are particularly prominent in the apical region of the cells (AM: apical plasma membrane). Fig. 4. Multivesicular body in a post lateral cell.
Fig. 5. M e m b r a n e - b o u n d e l e c t r o n d e n s e b o d i e s ( a r r o w s ) f o u n d at every level o f t h e lateral cells. Fig. 6. E l e c t r o n d e n s e b o d i e s ( a r r o w s ) in t h e l a t e r o f r o n t a l cells. Fig. 7. G r a n u l a r ( G ) a n d fibrillar c o m p o n e n t s ( F ) o f t h e basal l a m i n a o f t h e p o s t lateral cells w h i c h f o r m s t h e capillary wall. E l e c t r o n d e n s e b o d i e s (D) are p r e s e n t in t h e cells. Fig. 8. E l e c t r o n d e n s e b o d i e s (D) in t h e a m o e b o c y t e s (A) in t h e capillary l u m e n .
117 TABLE I QUANTITIES OF Pb and Cd LOST TO PROCESSING FLUIDS AS A PERCENTAGE OF A V E R A G E T O T A L GILL CONTENT Percentage of total gill content Processing Fluids
Pb loaded
Cd loaded
Glutaraldehyde in phosphate buffer 3 0 % Acetone 50% Acetone 7 0 % Acetone 8 0 % Acetone 1 0 0 % Acetone
0.40
7.05
0.00 0.01 0.00 0.06 0.06
I.I0 0.65 0.35 0.20 0.05
Total loss
0.53
9.40
buffer was n o t adequate, glutaraldehyde and OsO4 were subsequently used in seawater. Lead and Cd losses were not measured in these fixatives but it seems probable that there would not be a marked increase. Analyses of electron dense bodies in the Cd-loaded specimens were carried out. These dense bodies are si~ed in the cells of the gill epithelium and in amoebocytes which correspond to those seen in normal cells {Fig. 2). They reveal that Cd is always associated with S and frequently with P. A typical spectrum is shown in Fig. 12 and a series of analyses from laterofrontal cells are given in Table II. It can be seen that the atomic ratio of Cd:S is highly variable as also is P content. It is interesting to note that S also exists in the absence of Cd in similar dense bodies. T A B L E II X-RAY MICROANALYSIS
OF GILL EPITHELIA FROM
Site
Element
Apical granule
Cd S P
I~ Ks Ks
Basal granule
Cd S P
Apical granule
Peak intensity
Cd-LOADED MUSSELS
Atomic ratio
Mode
67 416 86
1.0 17.7 4.5
STEM
L~ Ks Ks
185 285 196
1.0 4.4 3.7
STEM
Cd S P
I~ Ks Ks
326 570 59
1.0 5.0 0.6
STEM
Apical granule
Cd S P
La K{x Ka
27 521 14
1.0 55.1 1.8
STEM
Multi-vesicular body
Cd S
I_~ Ks
1122 2389
1.0 6.1
TEM
118
119 | ¥S:
|||S|C 251 HS:
IC/S ~ 2|E¥/CH
0
t00SEC HS:
VS:2500
OC/S 20EV/CH
M I
Os L~
I II
~
~
/
C~K~
~4
~
i ~4~
t~
~
F ~ . 12. (left) ~ p i c ~ X-ray ~ e c t m m from an electron d e n ~ body in the g~l ep~he~um of Cd-loaded My ribs. ' Fig. 13. (right) ~ p i c a l X-ray spectrum from a deposit in the capill~y wall in the gill of Pb-loaded My tilus.
Analyses of the electron dense bodies of the Pb-loaded cells were carried out. Electron dense bodies occur throughout the cells of the gill epithelium b u t the presence of Pb was only detected in large crystalline deposits within the capillary wall and at the bases of the epithelial cells. A typical spectrum from a deposit in the capillary wall is shown in Fig. 13. Calcium appears to be invariably associated with Pb in these deposits in an atomic ratio which closely approaches 1:1 (Table III). These results were obtained from glutaraldehyde fixed specimens analysed in the TEM mode. Treatment of the inorganic residue, from papain-treated gills of Pb loaded mussels, with dilute HC1 resulted in the vigorous evolution of CO:. Vigorous effervescence was also obtained by acid treatment of intact gills from Pb loaded mussles whereas this was not the case with normal mussels. Amino acid analysis of the MBP (Table IV) shows t h a t it is rich in Scontaining cysteine residues. A freeze~lried sample of the MBP was analysed by X-ray microanalysis and found to contain Ag, Cd, Cu, Fe, Hg, Sn and Zn. The presence of these elements was confirmed qualitatively by atomic absorption spectrophotomerry. Fig. 9. Frontal and laterofrontal cells in the gills of Cd loaded Mytilus. Membrane-bound electron dense bodies (arrows) and mitochondrla (M) are seen in a reduced and evidently abnormal hyaloplasm. Fig. 10. Thick section (1 /~m) showing electron dense bodies (arrows) in the epithelial cells and amoebocytes (A) and crystalline deposits (CL) in the thickened basal lamina which forms the capillary wall. Gill of Pb loaded Mytilus. Fig. 11. Crystalline deposits (CL) in the thickened basal lamina which forms the capillary walls. Gill of Pb-loaded My tilus.
120 TABLE III X-RAY MICROANALYSIS OF CRYSTALLINE DEPOSITS IN THE CAPILLARY WALLS OF GILLS OF Pb LOADED MUSSELS Element
peak intensity
Atomic ratio
Pb Ca Pb Ca Pb Ca Pb Ca Pb Ca Pb Ca Pb Ca Pb Ca Pb Ca
17324 5521 14008 4696 18712 980~ 18460 10731 17710 8192 17243 10804 16769 7797 18333 8770 17512 3709
1.0 0.7 1.0 0.7 1.0 1.1 1.0 1.2 1.0 1.0 1.0 1.3 1.0 1.0 1.0 1.0 1.0 0.4
L~ Ks L~ Ks Lo~ Ks L~ K~ I_~ Ks L~ Ks Lo~ Ks L~ Ks L~ Ks
T h e a c c u m u l a t i o n levels o f P b a n d C d are sho~vn i n Fig. 14. T h e o p t i m u m levels o f b o t h P b a a d C d i n s e a w a t e r w e r e u s e d t o o b t a i n m a x i m u m a c c u m u l a t i o n a c c o m p a n i e d b y the m i n i m u m m o r t a l i t y . It can be seen t h a t the accum u l a t i o n p a t t e r n s o f Cd a n d P b are m a r k e d l y d i f f e r e n t . TABLE IV AMINO ACID ANALYSIS OF MBP Amino acid
Residues/mol
Lysine Histidine Arginine Aspartic acid Treonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Cysteine
7 2 3 10 6 6/7 10 6
10 6 5 1 4 5 2 3 9
121
o
o
O
Pb
,oo1/
1000
I00
I
' 10
0
20
40
60
80
100
Days
Fig. 14. A c c u m u l a t i o n rates o f C d a n d P b b y
Mytilus gills.
DISCUSSION Lead, associated with Ca in an equiatomic ratio, was shown to be present in the gills of Mytilus specimens, which had been lead loaded, as crystalline extracellular deposits in the capillary wall. These gills have also been shown to contain carbonate. It is therefore assumed that Pb occurs with Ca either a mixed or a complex carbonate. Lead has also been found associated with Ca in Mytilus kidney [11]. The finding of Pb as carbonate is perhaps not surprising since Zirino and Y a m a m o t o [14] predict, on the basis of thermodynamic stability constants and ionic coefficients for seawater, that the most prominent species of Pb in seawater is PbCO3. Similarly the association of Pb with Ca is noted repeatedly in the literature. For example most of the Pb in Mytilus occurs in the CaCO3 rich valves [7] and in man, it has been shown that 90% of the b o d y burden of Pb occurs in bones [15]. Further, deficiency of Ca in man and other animals increases susceptibility to Pb poisoning [16]. In chemical terms the association of Pb and Ca is not surprising since the ionic radius of Pb 2÷ (1.20 A ) is similar to that o f Ca 2÷ (0.99 A), consequently isomorphic replacement is likely even though their polarizabilities vary considerably. Since no Pb was detected within cells and since little Pb appeared to be lost from the tissues during processing, it may be assumed that Pb is rapidly transported through the cells and deposited extracellularly in an inert form. The effective concentration within the organism is thus kept low and this may well account for the lower toxicity of Pb compared to Cd, Zn, Hg and Cu which all show a preference for S-rich organic molecules [13]. Cadmium, unlike Pb, accumulated within the cells in the gills of cadmium-
122 loaded Mytilus. The evidence suggests that like Fe [12] it is taken up by pinocytosis, becomes associated with lysosomes and is s o m e h o w transferred to a m o e b o c y t e s in the capillary lumen. The association of Cd with S within the epithelial cells and a m o e b o c y t e s is further evidence for the existence of S-rich metal binding complexes. It has been suggested the S-rich MBP [13] is synthesised as a response to the uptake of certain heavy metals. This work shows that S is present in greater molar concentrations than Cd which suggests that the constituents of the MBP m a y be present prior to the uptake o f heavy metals. Elemental S or inorganic S-containing salts were n o t detected in the eluate from Sephadex gel filtration of the water soluble cytoplasmic components of the gills. However, S was detected in the MBP primarily as cysteine. Sulphur-containing taurine has also been recently found (Talbotunpublished data). Cadmium was also found, b y X-ray microanalysis, to be associated with P. No chemical analyses for P were carried out, however, on the eluate which contained metal binding protein. The significance of the association of Cd and P, therefore, is n o t obvious. Phosphorus m a y well be present in phospholipids contained within the membranes of the organelles in which Cd is sequestered. The loss (< 10%) of Cd during processing of the gills suggests the possibility that there may be an u n b o u n d Cd component. It is premature to speculate in detail on this b u t the observation is consistent with unpublished observations (Talbot, unpublished work) on the biological half-time (tl/2) o f Cd which suggest that Cd is present in more than one form or in more than one compartment. The MBP was also shown to be associated with Ag, Cu, Fe, Hg, Sn and Zn as well as Cd. It presumably has the capacity to bind all these elements. The amino acid content is similar to that reported for other aquatic organisms, e.g. {eels [17] and limpets [18], b u t has a lower cysteine content than mammalian MBP [19--23]. The different accumulation rates of Cd and Pb support the concept of different accumulation pathways for these t w o elements in the gills. REFERENCES 1 Anon., Heavy metals in the sediments of Port Phillip Bay and input streams. Environmental Protection A u t h o r i t y of Victoria, Rep. No. 16. 1976. 2 J.D. Smith, Proc. R. Aust. Chem. Inst., 43 (1976) 305. 3 V. Talbot, R. Magee and M. Hussain, Mar. Pollut. Bull., 7 (1976) 53. 4 H.T. French and P.J. Thistlethwaite, Proc. R. Aust. Chem. Inst., 43 (1976) 73. 5 V. Talbot, R. Magee and M. Hussain, Mar. Pollut. Bull., 7 (1976) 84. 6 D.J.H. Phillips, M. Sc. Thesis. Chemistry Dept., Melbourne University, 1975. 7 V. Talbot, R. Magee and M. Hussain, Mar. Pollut. Bull., 7 (1976) 234. 8 Victorian Parliamentary Hansard, 24 (1975) 6884, 6958. 9 Victorian Parliamentary Hansard, 27 (1975) 7881. 10 Victorian Parliamentary Hansard, 25 (1975) 7173. 11 M. Schulz-Blades, R. Lasch and S. Boseck, Micros. Acta, Suppl. 2 (1978) 175. 12 S.G. George, B.J.S. Pirie and T.L. Coombs, J. Exp. Mar. Biol. Ecol., 23 (1979) 71.
123 13 V. Talbot and R. Magee, Arch. Environ. Contamin. Toxicol., 7 (1978) 73. 14 A. Zirino and S. Yamamoto, Limnol. Oceanogr., 17 (1972) 661. 15 L.J. Casarett and J. Doull, Toxicology, The Basic Science of Poisons. Macmillan, NY, 1975, p. 478. 16 K.M. Six, Fed. Proc., 29 (1970) 568. 17 J.M. Bouquegreau, Ch. Gerday and A. Disteche, FEBS Lett., 55 (1975) 173. 18 A.G. Howard and G. Nickless, Chem.-Biol. Interact., 16 (1977) 107. 19 H. Newath, The Proteins, New York Academic Press, 1963, Vol. 1,427. 20 D.R. Winge and K.V. Rajagopalan, Arch. Biochem. Biophys., 153 (1972) 755. 21 T. Suda, N. Horinchi, E. Ogata, I. Ezawa, N. Otaki and M. Kimura, FEBS Lett., 42 (1974) 23. 22 J.H.R. Kagi and B.L. Vallee, J. Biol. Chem., 235 (1960) 3460. 23 P. Puldio, J.H.R. Kagi and B.L. Vallee, Biochemistry, 5 (1966) 1768.
