234

Biochimica et Biophysica Acta, 580 (1979) 234--244 © Elsevier/North-Holland Biomedical Press

BBA 38284 THE OCCURRENCE OF CADMIUM IN SUB-CELLULAR PARTICLES IN THE KIDNEY OF THE MARINE MUSSEL, M Y T I L U S E D U L I S , EXPOSED TO CADMIUM THE USE OF ELECTRON MICROPROBE ANALYSIS

STEPHEN G. GEORGE and BRIAN J.S. PIRIE N.E.R.C. Institute of Marine Biochemistry, St. Fitticks Road, Aberdeen (U.K.)

(Received February 12th, 1979) Key words: Cd 2+ accumulation, Electron microprobe analysis; Marine mussel kidney; (My tilus edulis)

Summary In mussels ( M y t i l u s edulis) chronically exposed to cadmium, 85% of the Cd 2÷ was found to be associated with membrane-limited granular structures when elemental analyses were carried out on cryo-sectioned tissue by electron probe X-ray microanalysis. These granules also contained high concentrations of sulphur and phosphorus as well as other metalions, including Ca 2÷, iron and Zn 2÷. In contrast, after homogenisation and fractionation by differential centrifugation, the major proportion of the Cd 2÷ was found in the cytoplasmic fraction. However, many lysosomes were also ruptured by this treatment. Gel filtration chromatography of this fraction indicated the presence of a Cd 2÷binding c o m p o n e n t of similar molecular weight to the metallothionein purified from the digestive gland of the same animals. It is therefore proposed that metallothionein may be associated with particulate structures which would thus reduce its cellular toxicity.

Introduction The Cd2+-binding protein, metallothionein, was first identified in the cytosolic fraction of horse kidney by Margoshes and Vallee in 1957 [1]. Since then its presence in the kidney and liver of many vertebrates has been demonstrated [2]. Metallothionein contains about 30% cysteine residues and 7--10 g atom/ mol of metal (usually Zn 2÷, Cd 2÷ and Cu 2÷) which are bound to t h e - S H group of the cysteine. This metal composition is variable and depends upon both the

235 tissues of origin and history of metal exposure. Recent studies have indicated that Cd2÷-binding proteins are also found in marine organisms, both vertebrates and invertebrates such as the limpet, whelk, crab, mussel and oyster [3]. The identification of these as metallothioneins has been reported for the crab [4] and mussel [5]. Metallothionein is generally thought to be a cytoplasmic protein and is isolated from a particle-free supernatant fraction of the homogenate. This has resulted in the substitution of a simple one-step scheme for a conventional subcellular fractionation to obtain a 40 000 × g supernatant. Examples are the studies of marine species such as the whelk, Purpura lapillus, in which Noel-Lambot et al. [6] reported 23--91% of the total Cd in this supernatant and a study of Cd localisation in the kidney of the sea-lion, Zolophus californianus, by Lee et al. [7], who found 38% of the cadmium in the particulate fractions and 62% in the cytosol. In rats, there is also evidence that association of Cd 2÷ with the nucleus is the effector for the induction of metallothionein synthesis [8]. There is therefore strong evidence that Cd 2÷ within the tissues is not exclusively bound to a soluble cytoplasmic protein in the cell but is also associated with particulate structures. The present paper is concerned with the subcellular localisation of Cd 2÷ in the kidney of the marine mussel, Mytilus edulis, since this shellfish accumulates very high concentrations of Cd 2+ after chronic exposure and contains a metallothionein [5]. Because Cd 2÷ is redistributed during conventional specimen preparation for electron probe X-ray microanalysis [9] we have used ultra lowtemperature cryo-procedures for preparation of our samples. This has demonstrated that exposure of mussels to Cd 2÷ results in an accumulation of metal in particulate structures. Materials and Methods

Animals. Mature speciments of Mytilus edulis (6--8 cm shell length) were obtained from Montrose Basin, Kincardineshire, and maintained in a sea water aquarium at 10°C. Exposure to metal. A stock CdC12 solution containing 1 mg/ml Cd was made up in 50 mM Tris, pH 8. Batches of 100 animals in tanks containing 501 aerated sea water were exposed to 100 ug Cd/1 for three months. The water was changed weekly. Specimens were used for subcellular fractionation, preparation of Cd2÷-binding proteins [5] and electron microscopy. Subcellular fractionation and analysis of fractions. All operations were carried o u t at 4°C. A 10% w/v homogenate of freshly dissected kidney tissue was prepared in 15% w/v sucrose 1.1% w/v NaC1 10 mM Tris, pH 7.6, by 5 strokes of a loosely fitting Potter-Elvehjem type homogeniser rotating at 200 rev./min. The crude homogenate was filtered through 106 t~m nylon mesh to remove unbroken tissue and gross debris. A sample of the filtrate (designated homogenate) was reserved for analysis and the remainder fractionated by centrifugation. A nuclear and tissue debris fraction were removed by centrifugation at 200 × g for 5 min; a mitochondrial and lysosomal fraction at 5000 × g for 8 min and a microsomal fraction at 180 000 × g for 60 min. A sample of the supernatant (cytoplasmic fraction) after the last centrifugation was used for Sephadex G-75 gel filtration chromatography. 10 ml was applied

236 to a 12.5 × 85 cm column of G-75 equilibrated and eluted with 20 mM Tris pH 8.6. 10-ml fractions were collected and monitored for Cd 2÷, Zn 2÷, A2~0nm, A2sonm and Na ÷. Particulate subcellular fractions were resuspended in the homogenisation medium and all fractions were stored at --15°C before analysis. Samples for metal analysis were wet ashed in 70% w/v HNO3 at 110°C in sealed teflon pressure vessels for 2 h, diluted to 35% HNO3 and the metals measured in a Varian AA5 atomic absorption spectrophotometer. Fractions from gel chromatography were aspirated directly without prior ashing. Corrections were made for non-atomic absorption and background as necessary. Alkaline phosphatase activity was measured by the method of Bessey et al. [10], acid phosphatase by the fluorimetric procedure of Fernley and Walker [11]. Preparation o f tissues for electron microscopy. Samples of kidney {approx. 1 mm 3) were excised, placed on a silver pin and thrown into liquid nitrogen slush at --216°C, sectioned in a c r y o m i c r o t o m e (LKB Cryokit and Ultrotome III, LKB Productor AB., Bromma, Sweden), with the specimen at --120°C, knife at --100°C, chamber at --90°C, transferred onto aluminium grids, freezedried in the microtome chamber for 5 h and coated with approximately 50-100 ~ carbon. Sections were examined in a JEOL 100-CX electron microscope fitted with an ASID-4D scanning attachment operating at 100KV in the STEM mode. Elemental analyses were performed with a stationary probe of approx. 300 ~ diameter at a tilt angle of 35 °. Emitted X-rays were collected and analysed using an energy dispersive X-ray analyser comprising a Kevex detector and Link Systems multichannel analyser, {High Wycombe, Bucks., England). Standardisation and calibration of the instrument were carried out by the procedures described by Chandler [12] and elemental concentrations were calculated by the continuum method of Hall [13]. Results

Subcellular fractionation A homogenate of kidney tissue from Mytilus edulis exposed to Cd 2. was fractionated by centrifugation. The results of Cd 2÷ and enzyme analyses are shown in Table I. The preliminary filtration step effectively removed the unbroken cells as shown by light microscopy and by the very low proportion of enzyme activities for the plasma membrane marker (alkaline phosphatase) and lysosomal marker (acid phosphatase) found in the first pellet, P1, sedimented at 1000 × g min. This fraction contains many nuclei and about 2% of the total Cd 2÷. Most of the Cd 2÷, 80%, is present in the particle-free supernatant (cytosol) fractions, whilst a significant proportion, 14%, is found in the mitochondrial/lysosomal pellet, P2. The distribution of the lysosomal enzyme, acid phosphatase, shows that approximately half of its activity is present in this 40 000 × g • min pellet, whilst approximately 40% is released into the particlefree supematant by damage and rupture during homogenisation. Cytoplasmic Cd 2÷ speciation A sample of the kidney particle-free supernatant fraction from animals exposed to Cd 2÷ for 3 months was chromatographed on Sephadex G-75 to

237 TABLE

I

DISTRIBUTION

O F C d 2+ A N D M A R K E R

ENZYMES

IN SUBCELLULAR

FRACTIONS

A 10% w/v homogenate in 1 . 1 % N a C I 1 5 % s u c r o s e 1 0 m M T r i s p H 7 . 6 w a s f r a c t i o n a t e d Distributions e x p r e s s e d a s p e r c e n t a g e o f t o t a l r e c o v e r e d . Fraction

C d 2+

Alkaline phosphatase

P1 1 - 1 0 3 X g . m i n P2 4 • 1 0 4 X g - m i n P3 1 1 • 1 0 6 × g ' m i n Supernatant (cytosol) Recovery

2 14 4 80 74

1 43 55 1 62

OF KIDNEY by centriguation.

Acid phosphatase 6 43 6 45 132

determine the proportions of free and bound Cd 2÷. The elution pattern is shown in Fig. 1. Since the microprobe studies show high concentrations of Zn :÷ this was also monitored in the column fractions. Approximately 3% of the total Cd 2÷ and Zn 2÷ is excluded from the gel. The major portion of the Cd :÷, 90%, is eluted between 300 and 500 ml in two overlapping peaks, corresponding with peaks in A250nm and only about 7% was associated with low molecular weight materials. The distribution of Zn 2÷ differs from the Cd 2÷ distribution since the major portion of the Zn 2÷ is eluted in the region of the salt peak between 500 and 700 ml. The Cd 2+, Zn 2÷ and A250nm all show multiple peaks in this low molecular weight region. This chromatography pattern is consistent with Cd 2÷ being bound to a low molecular weight thionein as characterized in the digestive gland of these animals [5] and with the association of Zn 2÷ with low molecular weight nucleotides, amino acids or bases as found in the oyster [14]. Ultrastructural localisation The morphological appearance of sections obtained by cryo-sectioning of unfixed frozen tissue is very much different from the generally familiar electronmicrograph of stained sections of fixed, resin embedded tissue [15].

°

!i

N (9

0

~0

~

300

400

El~.ion ~ n ' ~

500

600

(ml)

F i g . 1. S e p h a d e x G - 7 5 g e l f i l t r a t i o n c h r o m a t o g r a p h y o f M y t i l u s k i d n e y c y t o s o l f r a c t i o n s e x p o s e d t o C d 2+, C o l u m n 2 . 5 × 8 5 c m , e l u a n t 0 . 0 2 M T r i s p H 8 . 6 . C d 2+ ( ), Z n 2+ ( . . . . . . ), N a + ( . . . . . ), A 2 5 0 n m ( ...... ).

Fig. 2. E l e c t r o n m i c r o g r a p h s of M y t i l u s k i d n e y . ( A ) S e c t i o n of fixed, d e h y d r a t e d , resin e m b e d d e d tissue, S t a i n e d o s m i u m t e t r o x i d e , u r a n y l a c e t a t e a n d lead c i t r a t e . T r a n s m i s s i o n e l e c t r o n m i e r o g r a p h . (B) C r y o s e c t i o n of u n f i x e d tissue. No staining. S c a n n i n g 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 g r a p h , g, g r a n u l e ; 1, l u m e n ; rot, m i t o c h o n d r i o n ; n, n u c l e u s ; s, b l o o d sinus ~-- apical microvilli.

239 Fig. 2 shows electronmicrographs of Mytilus kidney prepared by the two procedures. The conventional micrograph (Fig. 2A) shows the characteristic features of the kidney cell, columnar shape, apical microvilli, basally located nucleus and most strikingly, the large number of electron-dense, membrane-limited vesicles [16]. The cryo-section is difficult to photograph and the contrast is only obtained from intrinsic density differences. Since these differences are generally small in biological materials, contrast is very poor and mitochondria often show negative contrast. Use of the image enhancement capabilities of the STEM mode of operation of the electron microscope greatly facilitates interpretation. Elemental analysis in subcellular areas approx. 300 ~ diameter by electron probe X-ray microanalysis of cryo sections shows that the highest concentrations of Cd :÷ are found in the electron dense granules. Elements detected in these granules are Na(1.04 keV), Mg(1.25 keV), P(2.0 keV), S(2.3 keV), C1(2.6 keV), Cd(3.13 keV), K(3.3 keV), Ca(3.7, 4.0 keV), Fe (6.4, 7.05 keV), Cu(8.05, 8.9 keV), Zn(8.6, 9.6 kev, as shown in the spectrum Fig. 3. Al(1.5 keV) is derived from the specimen support grid. The composition of the granules is very variable and not all contain Cd. Cd was detectable in about 80% of the granules analysed; it was not detectable in mitochondria and was just above the minimum detectable limit in nuclei and the cytoplasm. The portion of the X-ray spectra from 2--4.2 keV for a granule and the nucleus are shown in Fig. 4. The calculated elemental concentrations of the major cations and anions of interest expressed as mass fractions in these microareas (300 diameter) of one cell of a cryo-sectioned kidney from Mytilus exposed to Cd 2+ are given in Table II. It should be noted that these represent mass fractions in very small volumes of freeze-dried material and are not absolute, in vivo concentrations. The average concentration of Cd 2÷ in the granules is some 20-fold higher than in the nuclei or cytoplasm. It can be calculated that about 20% of the cell volume is made up of the granules (from Fig. 2); thus from the relative

o I

1

2

3

4 X-roy

5

6

7

8

9

10

e n e r g y , keY

Fig. 3. X-ray s p e c t r u m of g r a n u l e in Mytilus k i d n e y o b t a i n e d by e l e c t r o n m i c r o p r o b e analysis. F r e e z e d r i e d c r y o - s e c t i o n , p r o b e 1 0 0 kV, 3 0 0 A d i a m e t e r .

240

f

T ,

¢-

U

'.f ..

:::" ." .',

.... '

2 I

2

2.5 X-roy

3

3.5

4

e n e r g y , keV

Fig. 4. X-ray s p e c t r u m f r o m 2 - - 4 . 2 k e V o f c r y o - s e e t i o n e d M y l i l u s k i d n e y e x p o s e d t o C d 2+. C o n d i t i o n s as t"ig. 3. S o l i d s p e c t r u m , a r e a o f n u c l e u s ; d o t t e d s p e c t r u m , g r a n u l e .

T A B L E II ELEMENTAL

MASS FRACTIONS

IN M I C R O A R E A S

OF,~IYTILt'S

KIDNEY

EXPOSED

T O C d 2+

D e t e r m i n e d by X-ray m i c r o a n a l y s i s of freeze-dried s e c t i o n s p r e p a r e d by e r y o m i c r o t o m y . C o n c e n t r a t i o n s e x p r e s s e d as a v e r a g e w t . % +- S.E. w i t h i n t h e 3 0 0 A d i a m e t e r p r o b e . N u m b e r s o f e a c h a r e a a n a l y s e d g i v e n in p a r e n t h e s e s . Cellular area

Granules

(18)

E +- S . E .

Element (mg/g) P

S

33 15 20 176 22 9 25 53 31 34 33 32 21 35 33 32 I0 1.5

90 127 161 21 129 181 94 103 138 107 114 113 149 129 119 104 173 112

11 33 1 0 17 58 1 7 21 8 16 0 29 9 28 8 40 12

1 2 6 +- 6

19 ± 4

26 + 3

Cd

Ca

Fe

Zn

10 7 7 162 11 11 6 9 9 7 8 8 13 8 11 7 13 6

62 78 27 12 155 15 229 118 133 129 179 211 81 129 108 91 20 115

52 82 32 147 49 143 43 115 83 92 51 54 103 91 86 71 85 59

9 +- 0 . 6

110 ± 15

7 6 +-7

Nucleus

20

57

2

2

10

12

Mitoehondria (2)

15

60

0

9

3

29

Cytoplasm

34 ± 10

51 ± 3

2 ± 0.75

6.6 ± 1

3 . 8 +- 1.8

2 0 +-4

( 5 ) E +- S . E .

241

masses of the areas (obtained from the white radiation counts), vide infra, approximately 85 and 15% of the Cd 2÷ is present in the granules and cytoplasm respectively. The granules in fact contain metals other than Cd as shown in Table II. These are Fe (average 11% w/w) and Zn (8%) and Ca (1%). They also contain about 13% S and 3% P. Elements of atomic number less than 11 are not detected by this system and due to peak overlaps, Na* and Mg 2÷ cannot be quantified. The concentrations of all other metals, Ca, Cd, Fe and Zn are much lower in all other cellular areas than the granules. Some granules contain an excess of Fe (the X-ray spectrum is shown in Fig. 5A) and others which appear to be more perfectly spheroidal in shape, contain very high concentrations of P, Ca and Zn (Fig. 5B). The fact that very high levels of Ca, Zn and P are found in the same granule is strongly suggestive that these elements are associated. This is corroborated by calculation of correlation coefficients for the whole granule population tabulated in Table II. Between Ca and P, the correlation coefficient, R is 0.94, which is highly significant (P (. 0.01), whereas agreement between Zn and P is less significant (R = 0.53, P = 0.02). However, for the spheroidal granules taken from several cells, R = 0.999, P