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Calcium phosphate granules: A trap for transuranics and iron in crab hepatopancreas
Comp. Biochem. Physiol. Vol. 68A, pp. 423 to 427 0 Pergamon Press Ltd 1981. Printed in Great Britain
0300-9629/81/0301-0423802.00/O
CALCIUM PHOSPHATE GRANULES: A TRAP FOR TRANSURANICS AND IRON IN CRAB HEPATOPANCREAS J. C. GUARYand R.
N~~GREL
International Laboratory of Marine Radioactivity, IAEA MusCe Odanographique, Monaco and Centre de Biochimie, Pacult des Sciences, Part Valrose, Nice, France (Received 22 May 1980) Abstract-l.
Subcellular fractionation of crab hepatopancreas from animals fed with labelled food shows localization of plutonium, americium and iron mainly in a dense nonmitochondrial mineral fraction composed of calcium phosphate granules. 2. Plutonium, and particularly iron, are also associated with calcium phosphate microgranules sequestered in the microsomal fraction. 3. This situation, quite different from that observed in mammals, suggests that accumulated transuranits and other heavy metals could be partly extruded afterwards into the lumen of the hepatopancreas with the calcium granules.
In vivo contamination of crabs Male crabs Cancer pagurus (L.) at intermolt stage C4 and weighing about 600 g, were each fed for 10 days with 10-12 mussels labelled with “‘Pu, 24’Am and 59Fe respectively by injection into the digestive gland. The crabs were then fed for 1 week with uncontaminated mussels before dissection.
INTRODUCTION Marine organisms are implicated in the biogeochemical cycle of transuranic elements (Guary, 1980). We have shown previously that plutonium was absorbed from ingested food and incorporated mostly into the hepatopancreas of crabs (Fowler & Guary, 1977) and that, in the cytosolic fraction of the hepatopancreas, it is mainly associated with proteins of low molecular weight (Guary & Ntgrel, 1980). Knowledge of localization of transuranics helps understand both assimilation and excretion pathways and intracellular irradiation effects (Pentreath, 1975). Studies dealing with the subcellular distribution of transuranic elements in mammalian liver have been carried out (Boocock et al., 1970; Popplewell et al., 1971; Bruenger et al., 1971, 1976) and similar studies involving “‘PO and neutron activation products in the digestive gland and other tissues of marine invertebrates have been reported (Romeril, 1971; Pentreath, 1975; Cherry et al., 1979). To our knowledge, no such data are available for transuranics in marine species. It was therefore of interest to compare the subcellular distribution of two naturally ingested transuranic elements to that of iron in the hepatopancreas of the edible crab Cancer pagurus (L.) and to the situation observed previously in mammals.
Subcellular fractionation of hepatopancreas
After washing with ice-cold isotonic saline, the minced tissue was homogenized (seven strokes) with a teflon-glass homogenizer in 4 volumes of 0.01 M N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonate (HEPES) buffer, pH 7.5, containing 0.15 M NaCl. The resulting homogenate was then fractionated at 4°C by differential centrifugation at 960 g (10 min), 25,000 g (10 min), 31,300 g (30 min) and 125,000g (70 min) to yield four pellets and a supernatant. The first, second and fourth pellet were divided in two parts during resuspension in the same buffer as above: the less dense upper portion was called “light”, the lower “heavy”. Analysis of subcellular fractions
Protein content of each fraction was estimated according to Lowry et al. (1951). DNA determinations were carried out as described by Burton (1968). (K+)-p-nitrophenylphosphatase, as a part of (Na+, K+)-ATPase (Balerna et al., 1975), cytochrome C oxidase (Hogeboom & Schneider, 1952) and p-nitrophenylacetate hydrolase (Ntgrel et al., 1976) were assayed spectrophotometrically as marker enzymes for plasma membranes, mitochondria and endoplasmic reticulum respectively.
MATERIAL AND METHODS Radioisotopes and counting techniques 237Pu (half life 46 days, specific activity 1.1 x lo4 mCi/mg) in 0.1 N HCI was purchased from Oak Ridge National Laboratory (U.S.A.). The characteristic X-rays of 237Np which result from the decay of the present z3’Pu are 01 sufficiently high energy (100 keV) to be measured with NaI (Tl) scintillation crystals with 40% efficiency. 24’Am (half iife 433 years, specific activity 3.4j mCi/mgj in 0.1 N HNOq. from the C.E.A.. Gif-sur-Yvette (France) and S9Fe Cl3 (half life 45 days, s&fic activity 30‘mCi/m& in 0.1 N HCl, from the Radiochemical Centre, Amersham (G.B.) were counted in the same manner with 30% and 34% efficiency respectively.
RESULTS AND DISCUSSION
As shown in Table 1, plasma membranes are enriched mainly in the P2 light fractions (five-fold with 48% yield) but also in the P1 light and P, pellets. Mitochondria are enriched in the P2 heavy fraction (17-fold) and endoplasmic reticulum in the P3 (sixfold) and P4 light pellets (four-fold). Most 237Pu and 241Am (50-60%) is observed in the fastest sedimenting fraction (P1 heavy); in this fraction, the specific activities, also expressed on a protein basis (cpm/mg pro-
423
J. C. GUARY and R.
N&~~;REL
tein), are higher than those in the homogenate by factors of 3.3 and 5.7 for ‘j’Pu and 241A respectbimodally: J sides the ively. s9Fe is distributed enrichment (2.4) and recovery (37’:,) again observed in the PI heavy fraction, about 22”, is recovered in the P4 petiet with a higher enrichment (4.9) in the P* heavy fraction. Enrichment of between 2.4 and 5.7 fold is thus observed for all three radionuclides in the Pr heavy fraction” Interestingly, the Pr heavy fraction contains no marker enzymes of plasma membranes, mitochondria or endoplasmic reticulum: it does. however, contain nuclei, as indicated by the presence of DNA. This low speed pellet (PI heavy) consists of dense, white and chalky material. Moreover, microscopic examination show small refractile bodies stainable with Alizarin Red S, a fact suggesting a high calcium content (Ciurr, 1962). These spherules are very similar to those observed in cells of the crab hepatopancreas with an electron microscope (Fig. f f. They vary in diameter from I to Spm and show the characteristic concentric layered structure observed previously in some other decapod crustaceans (Becker et ul., 1974; Gibson & Barker, 1979) and in other invertebrates (Simkiss, 1976). All properties of this pellet presented in Tabfe 2 agree with those for the calcium phosphate granule fraction described by Becker et ui. (1974) in the hepatopancreas of the blue crab. During purification of the crude PI heavy pellet by repeated solubilizations and precipitations at acidic and neutral pH, most proteins are lost but all of the Pu, Am or Fe radioactivity is retained in the purified granule fraction. When this fraction is filtered through Sephadex G-25 or G-75 gel in 0.1 N HCf, radionuclides. calcium phosphate and UV-absorbing material are coeluted. The apparent molecular weight of this complex is around 3000. The LJV-absorbing material, mostly ATP, can be eliminated by repeated washes at alkaline pH without loss of radioactivity in the granule fraction when returned to neutral pH. Thus, neither ATP nor other UV-absorbing comTable 2. Miscellaneous properties of the first heavy pellet isolated from crab hepatopancreas Weight loss (“;): By drying By asking Sdubitity properties: At neutral or alkaline pH At acidic pH (1 N-2 N HCI) In 0.1 M EGTA, pH 7.5 Chemical composition: (kitatom or pmotjdry weight) Ca P M% ATP
40 22 No Yes Yes 3 4 0.4 0.1
Samples were dried (105°C) and ashed (450°C) after several washes of the crude fraction at neutral pH. Calcium, magnesium and inorganic phosphate content were determined by atomic absorption spe~iropbotometry. ATP was measured by a specific enzymatic assay (Lamprecht Br Trautschold, 1965) after solubilization in an excess of 0.1 M EGTA, pH 7.5. The ATP content agreed well with that determined by UV absorption based on the ATP extinction coefficients.
Pu, Am and Fe in crab hepatopancreas
425
Fig. 1. Electron micrographs of calcium phosphate granule(s) in cells of crab hepatopancreas. (A) General view of a cell with several granules enclosed in vacuoles ( x 8800); (B) Detail of a granule showing the concentric layered structures ( x 25,800). Sections were fixed with glutaraldehyde and osmium tetroxide, and stained with uranyl acetate and lead citrate following the procedure described by Reynolds (1963) ponents are directly involved in the interaction between radionuclides and calcium phosphate. The occurrence of 59Fe, and to a lesser degree, of 237Pu in the P4 pellets led us to examine the association of these radionuclides with components of the microsomal fraction. The 59Fe radioactivity of the P4 light pellet can be recovered in a soluble form after neutral detergent solubilization (3 hr in 0.3% Triton X-100). In this fraction, iron probably binds to high
molecular weight proteins since it is eluted in the void volume when solubilized and submitted to a Sephadex G-200 gel filtration in presence of the detergent. In the case of the P4 heavy pellet, where a large enrichment in 59Fe and 237Pu is observed (five- and three-fold respectively), no solubilization of the radionuclides occurs after two successive 15 hr treatments with 1% Triton X-100 and 2% deoxycholate respectively. After such treatment, leading to lipid and pro-
426
J. C. GUARY and R. N&REL
tein loss, the radioactive particulate fraction becomes easily sedimentable at a low centrifugal force (2500 g. 15 min). Repeated solubilizations and precipitations of this newly sedimented material shows that it has the same solubility properties as the PI heavy pellet (see Table 2). This suggests that “37Pu and 5gFe are also associated with calcium phosphate microgranules sequestered in the microsomal fraction of the crab hepatopancreas. The occurrence of calcium microgranules, which are often rich in iron, was in fact demonstrated in small vesicles (Berkaloff, 1958) or in cisternae of the endoplasmic reticulum from the cells of the midgut wall of terrestrial arthropods (Gouranton, 1968; Waku & Sumimoto, 1974). These microgranules could be the first stage of the mineral phase formation. Our results show clearly that the bulk of plutonium and americium retained in the hepatopancreas after ingestion is in the low speed sedimenting nonmitochondrial fraction, which is composed essentially of calcium phosphate granules. Therefore, their localization is completely different to that of transuranic elements in the liver of mammals, where they occur mostly in the mitochondrial-lysosomal fraction (Boocock et ul., 1970; Popplewell et u/., 1971; Bruenger er a/., 1971. 1976). This could however be due to a difference in the route of absorption, since studies related to mammals were conducted after intravenous injection of the isotopes. It is also noteworthy that iron, a metal involved in enzymatic and respiratory processes, is found, although to a lesser degree, in the same PI heavy fraction as the artificial nuclides plutonium and americium. The occurrence of these three elements in this fraction is probably due to their wellknown chemical affinity for calcium phosphate. A common absorption and excretion pathway is thus given support. Using cytochemical methods or X-ray microanalysis electron microscopy, iron has been found previously to be associated with spherites in the midgut wall of insects (Ehrhardt, 1965; Gouranton, 1968), and annelids (Boilly & Richard, 1978), as well as in viscera of a marine gastropod (Bryan et ul., 1977). Moreover, the occurrence of various other heavy metals in granules generally composed of calcium phosphate is well established (see the review by Coombs & George, 1978). For example, zinc, copper, manganese, magnesium and lead have been shown to be associated with or included in phosphorite concretions of mollusc viscera (Bryan et ul., 1977), kidney (Bryan, 1973; Doyle et ul., 1978), gill and mantle (George et ul.. 1978) and midgut of barnacles (Walker rt al., 1975a,b). Calcium phosphate granules, which could be involved in shell calcification (Travis, 1955, 1957) are more probably implicated in excretion processes by trapping excess metals (Simkiss, 1976; Coombs & George, 1978). Moreover, it has been stated that, in the majority of cases, the intracellular granules are extruded from the cell into the lumen of the excretory or digestive system (Simkiss, 1976). In the case of a marine crustacean, in particular, Becker et al. (1974) have observed extruded granules in the lumen of the hepatopancreas and have suggested that the intraluminal granules could reflect a mechanism of Ca’+
mobilization in the crab. Nonetheless, it seems likely that the metals associated with calcium phosphate granules can, at least partially, be excreted in the lumen of the hepatopancreas at the time of granule extrusion. The suggestion that calcospherites play a role in metal detoxication processes in invertebrates is thus supported. Acknow/edgements~---The International Laboratory of Marine Radioactivity operates under a tripartite agreement between the International Atomic Energy Agency. the Government of the Principality of Monaco and the Oceanographic Institute at Monaco. We thank Drs S. W. Fowler, R. D. Cherry and G. Ailhaud for their support and critical comments, and Mr Pagliardini and Mr Marichy for
performing the analysis of hepatopancreas and the electron microscopy respectively.
mineral content
REFERENCES
BALERNA M., FOSSET M., CHICHEPORTICHE R., ROMEY G. & LAZDUNSKI M. (1975) Constitution and properties of axonal membranes of crustacean nerves. Biochemistr) 14, 55o(r5511. BECKER G. L., CHEN C. H., GREENAWALT J. W. & LEHNINGER A. L. (1974) Calcium phosphate granules in the hepatopancreas of the blue crab Callinectes sapidus. J. cell Biol. 61, 316-326. BERKALOFF A. (1958) Les grains de sCcretion des tubes de Malpighi de Gr$lus domesticus (Orthoptkre). C.r. hebd. SPanc. Acad. Sci. Paris 246, 2807-2809. BOILLY B. & RICHARD A. (1978) Accumulation de fer chez une Anntlide Polychete: Magelona papillicornis F. Miiller. C.r. hebd. S&c. Acad. Sci. Paris 286, 1005-1008. BABCOCK G., DANPURE C. J., POPPLEWELL D. S. & TAYLOR D. M. (1970) The subcellular distribution of plutonium in rat liver. Radiation Res. 42, 381-396. BRUENGER F. W., STOVER B. J. & STEVENSW. (1971) 239Pu (IV): its subcellular distribution and association with feriit& in the canine liver. Health PhVs. 21, 679487. BRUENGER F. W., GRUBE B. J., ATH~RTON D. R., TAYLOR G. N. & STEVENS W. (1976) Subcellular distribution of curium in beagle liver. Radihrion Res. 66, 443452. BRYAN G. W. (1973) The occurrence and seasonal variation of trace metals in the scallops Pecten maximus (L.) and Chlamys opercularis (L.). J. mur. biol. Ass. U.K. 53, 145-166. BRYAN G. W., POTTS G. W. & FORSTER G. R. (1977) Heavy metals in the gastropod mollusc Huliotis tuberculara (L.). J. mar. biol. Ass. U.K. 57, 379-390. BURTON K. (1968) Determination of DNA concentration with diphenyl amine. In Merhods in Enzymology, Vol. 12. part B, pp. 163-165. Academic Press, New York. CHERRY K.-D., DOWDLE E. B. D. & TODD G. (1979) Intracellular sites of natural “(‘PO in the lobster heuatouan. . treas. S. Afr. .l. Sri. 15, 39. CRUMBS T. L. & GEORGE S. G. (1978) Mechanisms of immobilization and detoxication of metals in marine organisms. In Physio/ogy and Behaviour of Marine Organisms, pp. 179-187. Pergamon Press, Oxford. DOYLE L. J., BLAKE N. J., Woo C. C. & YEVICH P. (1978) Recent biogenic phosphorite: concretions in mollusc kidneys. Science 199, 1431-1433. EHRHARDT P. (1965) Speicherung anorganischer substanzen in den Mitteldarmzellen von Aphis fabae Stop. und ihre Bedeutung fiir die Ernlhrung. Z. t:ergl. Physiol. 50, 293. FOWLER S. W. & GUARY J. C. (1977) High absorption efficiency for ingested plutonium in crabs. Nature, Land. 266, 827-828. GEORGE S. G., PIRIE B. J. S., CHEYNE A. R., CRUMBS T. L. & GRANT P. T. (1978) Detoxication of metals by marine
Pu, Am and Fe in crab hepatopancreas bivalves: an ultrastructural study of the compartmentation of copper and zinc in the oyster Ostrea edulis. Mar. Biol. 45, 147-156. GIBSON R. & BARKERP. L. (1979) The decapod hepatopantreas. Oceanog. mar. Biol. Ann. Rev. 17, 285-346. GOURANTON J. (1968) Composition, structure et mode de formation des concrttions minerales dans l’intestin moyen des Homoptirres Cercopides. J. cell Biol. 37, 316-328. GUARYJ. C. (1980) Recherches sur les transferts et la fixation du plutonium, de l’amtricium et du neptunium dans le milieu marin. Thtse Doctorat-&s-Sciences, UniversitC Aix-Marseille II. GUARYJ. C. & NBGRELR. (1980) Plutonium and iron association with metal-binding proteins in the crab Cancer pagurus (L.). J. exp. mar. Biol. Ecol. 42, 87-98. GURR E. (1962) Staining. Practical and Theoretical. Williams & Wilkins, Baltimore. HOGEB~~MG. H. & SCHNEIDER W. C. (1952) Cvtochemical studies. IV. Physical state of certain ;espi;at&y enzymes of mitochondria. J. biol. Chem. 194, 513-519. LAMPRECHT W. & TRAUTSCHOLD I. (1965) Adenosine-5’-triphosphate. Determination with hexokinase and glucose6-phosphate dehydrogenase. In Methods of Enzymatic Analysis, 2nd edn, pp. 543-551. Academic Press, New York. LOWRY0. H., ROSEBROUGH N. J., FARR A. L. & RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. NBGRELR., SERREROG., FERNANDEZ-LOPEZ V. & AILHAUD G. (1976) Esterolytic activities of rat intestinal mucosa. I. Characterization, cellular distribution and subcellular localization of a glycerol-ester hydrolase. Eur. J. Biothem. 71, 249-258. PENTREATH R. J. (1975) Subcellular techniques in radioecological studies. In Design of Radiotrachr Experiments in Marine Biology Systems, Tech. Rep. Ser., 167, pp. 171-183. IAEA, Vienna.
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POPPLEWELL D. S., Boococ~ G., TAYLORD. M. & DANPUREC. J. (1971) The subcellular distribution of americium and curium in rat liver. In Radiation Protection Problems Relating to Transuranium Elements, pp. 205-222. Centre for Information and Documentation, Luxembourg. REYNOLDS E. S. (1963) The use of lead citrate of high pH as an electron opaque stain in electron microscopy. J. cell Biol. 17, 208-212. ROMERILM. G. (1971) The uptake and distribution of 65Zn in oysters. Mar. Biol. 9, 347-354. S~MKISS K. (1976) Intracellular and extracellular routes in biomineralization. In Calcium in Biological Systems, Symp. Sot. Exp. Biol., 30, pp. 423444. University Press, Cambridge. TRAVISD. F. (1955) The molting cycle of the spiny lobster, Pant&us argus Latreille. II. Pre-ecdysial histological and histochemical changes in the hepatopancreas and integumental tissues. Biol. Bull. mar. biol. Lab., Woods Hole 108, 88-112. TRAVISD. F. (1957) The molting cycle of the spiny lobster, Panulirus argus Latreille. IV. Post-ecdysial histological and histochemical changes in the hepatopancreas and integumental tissues. Biol. Bull. mar. biol. Lab., Woods Hole 113, 451479. WAKUY. & SUMIMOTO K. (1974) Metamorphosis of midgut epithelial cells in the silkworm (Bombyx mori) with special regard to the calcium salt deposits in the cytoplasm. II. Electron microscopy. Tissue Cell 6, 127-136. WALKERG., RAINBOWP. S., FOSTERP. & CRISP D. J. (1975a) Barnacles: Possible indicators of zinc pollution’? Mar. Biol. 30, 57-65. WALKERG., RAINBOWP. S., FOSTER’ P. & HOLLANDD. L. (1975b) Zinc phosphate granules in tissue surrounding the midgut of [he barnacle Balanus balanoides. Mar. Biol. 33, 161-166.
0300-9629/81/0301-0423802.00/O
CALCIUM PHOSPHATE GRANULES: A TRAP FOR TRANSURANICS AND IRON IN CRAB HEPATOPANCREAS J. C. GUARYand R.
N~~GREL
International Laboratory of Marine Radioactivity, IAEA MusCe Odanographique, Monaco and Centre de Biochimie, Pacult des Sciences, Part Valrose, Nice, France (Received 22 May 1980) Abstract-l.
Subcellular fractionation of crab hepatopancreas from animals fed with labelled food shows localization of plutonium, americium and iron mainly in a dense nonmitochondrial mineral fraction composed of calcium phosphate granules. 2. Plutonium, and particularly iron, are also associated with calcium phosphate microgranules sequestered in the microsomal fraction. 3. This situation, quite different from that observed in mammals, suggests that accumulated transuranits and other heavy metals could be partly extruded afterwards into the lumen of the hepatopancreas with the calcium granules.
In vivo contamination of crabs Male crabs Cancer pagurus (L.) at intermolt stage C4 and weighing about 600 g, were each fed for 10 days with 10-12 mussels labelled with “‘Pu, 24’Am and 59Fe respectively by injection into the digestive gland. The crabs were then fed for 1 week with uncontaminated mussels before dissection.
INTRODUCTION Marine organisms are implicated in the biogeochemical cycle of transuranic elements (Guary, 1980). We have shown previously that plutonium was absorbed from ingested food and incorporated mostly into the hepatopancreas of crabs (Fowler & Guary, 1977) and that, in the cytosolic fraction of the hepatopancreas, it is mainly associated with proteins of low molecular weight (Guary & Ntgrel, 1980). Knowledge of localization of transuranics helps understand both assimilation and excretion pathways and intracellular irradiation effects (Pentreath, 1975). Studies dealing with the subcellular distribution of transuranic elements in mammalian liver have been carried out (Boocock et al., 1970; Popplewell et al., 1971; Bruenger et al., 1971, 1976) and similar studies involving “‘PO and neutron activation products in the digestive gland and other tissues of marine invertebrates have been reported (Romeril, 1971; Pentreath, 1975; Cherry et al., 1979). To our knowledge, no such data are available for transuranics in marine species. It was therefore of interest to compare the subcellular distribution of two naturally ingested transuranic elements to that of iron in the hepatopancreas of the edible crab Cancer pagurus (L.) and to the situation observed previously in mammals.
Subcellular fractionation of hepatopancreas
After washing with ice-cold isotonic saline, the minced tissue was homogenized (seven strokes) with a teflon-glass homogenizer in 4 volumes of 0.01 M N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonate (HEPES) buffer, pH 7.5, containing 0.15 M NaCl. The resulting homogenate was then fractionated at 4°C by differential centrifugation at 960 g (10 min), 25,000 g (10 min), 31,300 g (30 min) and 125,000g (70 min) to yield four pellets and a supernatant. The first, second and fourth pellet were divided in two parts during resuspension in the same buffer as above: the less dense upper portion was called “light”, the lower “heavy”. Analysis of subcellular fractions
Protein content of each fraction was estimated according to Lowry et al. (1951). DNA determinations were carried out as described by Burton (1968). (K+)-p-nitrophenylphosphatase, as a part of (Na+, K+)-ATPase (Balerna et al., 1975), cytochrome C oxidase (Hogeboom & Schneider, 1952) and p-nitrophenylacetate hydrolase (Ntgrel et al., 1976) were assayed spectrophotometrically as marker enzymes for plasma membranes, mitochondria and endoplasmic reticulum respectively.
MATERIAL AND METHODS Radioisotopes and counting techniques 237Pu (half life 46 days, specific activity 1.1 x lo4 mCi/mg) in 0.1 N HCI was purchased from Oak Ridge National Laboratory (U.S.A.). The characteristic X-rays of 237Np which result from the decay of the present z3’Pu are 01 sufficiently high energy (100 keV) to be measured with NaI (Tl) scintillation crystals with 40% efficiency. 24’Am (half iife 433 years, specific activity 3.4j mCi/mgj in 0.1 N HNOq. from the C.E.A.. Gif-sur-Yvette (France) and S9Fe Cl3 (half life 45 days, s&fic activity 30‘mCi/m& in 0.1 N HCl, from the Radiochemical Centre, Amersham (G.B.) were counted in the same manner with 30% and 34% efficiency respectively.
RESULTS AND DISCUSSION
As shown in Table 1, plasma membranes are enriched mainly in the P2 light fractions (five-fold with 48% yield) but also in the P1 light and P, pellets. Mitochondria are enriched in the P2 heavy fraction (17-fold) and endoplasmic reticulum in the P3 (sixfold) and P4 light pellets (four-fold). Most 237Pu and 241Am (50-60%) is observed in the fastest sedimenting fraction (P1 heavy); in this fraction, the specific activities, also expressed on a protein basis (cpm/mg pro-
423
J. C. GUARY and R.
N&~~;REL
tein), are higher than those in the homogenate by factors of 3.3 and 5.7 for ‘j’Pu and 241A respectbimodally: J sides the ively. s9Fe is distributed enrichment (2.4) and recovery (37’:,) again observed in the PI heavy fraction, about 22”, is recovered in the P4 petiet with a higher enrichment (4.9) in the P* heavy fraction. Enrichment of between 2.4 and 5.7 fold is thus observed for all three radionuclides in the Pr heavy fraction” Interestingly, the Pr heavy fraction contains no marker enzymes of plasma membranes, mitochondria or endoplasmic reticulum: it does. however, contain nuclei, as indicated by the presence of DNA. This low speed pellet (PI heavy) consists of dense, white and chalky material. Moreover, microscopic examination show small refractile bodies stainable with Alizarin Red S, a fact suggesting a high calcium content (Ciurr, 1962). These spherules are very similar to those observed in cells of the crab hepatopancreas with an electron microscope (Fig. f f. They vary in diameter from I to Spm and show the characteristic concentric layered structure observed previously in some other decapod crustaceans (Becker et ul., 1974; Gibson & Barker, 1979) and in other invertebrates (Simkiss, 1976). All properties of this pellet presented in Tabfe 2 agree with those for the calcium phosphate granule fraction described by Becker et ui. (1974) in the hepatopancreas of the blue crab. During purification of the crude PI heavy pellet by repeated solubilizations and precipitations at acidic and neutral pH, most proteins are lost but all of the Pu, Am or Fe radioactivity is retained in the purified granule fraction. When this fraction is filtered through Sephadex G-25 or G-75 gel in 0.1 N HCf, radionuclides. calcium phosphate and UV-absorbing material are coeluted. The apparent molecular weight of this complex is around 3000. The LJV-absorbing material, mostly ATP, can be eliminated by repeated washes at alkaline pH without loss of radioactivity in the granule fraction when returned to neutral pH. Thus, neither ATP nor other UV-absorbing comTable 2. Miscellaneous properties of the first heavy pellet isolated from crab hepatopancreas Weight loss (“;): By drying By asking Sdubitity properties: At neutral or alkaline pH At acidic pH (1 N-2 N HCI) In 0.1 M EGTA, pH 7.5 Chemical composition: (kitatom or pmotjdry weight) Ca P M% ATP
40 22 No Yes Yes 3 4 0.4 0.1
Samples were dried (105°C) and ashed (450°C) after several washes of the crude fraction at neutral pH. Calcium, magnesium and inorganic phosphate content were determined by atomic absorption spe~iropbotometry. ATP was measured by a specific enzymatic assay (Lamprecht Br Trautschold, 1965) after solubilization in an excess of 0.1 M EGTA, pH 7.5. The ATP content agreed well with that determined by UV absorption based on the ATP extinction coefficients.
Pu, Am and Fe in crab hepatopancreas
425
Fig. 1. Electron micrographs of calcium phosphate granule(s) in cells of crab hepatopancreas. (A) General view of a cell with several granules enclosed in vacuoles ( x 8800); (B) Detail of a granule showing the concentric layered structures ( x 25,800). Sections were fixed with glutaraldehyde and osmium tetroxide, and stained with uranyl acetate and lead citrate following the procedure described by Reynolds (1963) ponents are directly involved in the interaction between radionuclides and calcium phosphate. The occurrence of 59Fe, and to a lesser degree, of 237Pu in the P4 pellets led us to examine the association of these radionuclides with components of the microsomal fraction. The 59Fe radioactivity of the P4 light pellet can be recovered in a soluble form after neutral detergent solubilization (3 hr in 0.3% Triton X-100). In this fraction, iron probably binds to high
molecular weight proteins since it is eluted in the void volume when solubilized and submitted to a Sephadex G-200 gel filtration in presence of the detergent. In the case of the P4 heavy pellet, where a large enrichment in 59Fe and 237Pu is observed (five- and three-fold respectively), no solubilization of the radionuclides occurs after two successive 15 hr treatments with 1% Triton X-100 and 2% deoxycholate respectively. After such treatment, leading to lipid and pro-
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J. C. GUARY and R. N&REL
tein loss, the radioactive particulate fraction becomes easily sedimentable at a low centrifugal force (2500 g. 15 min). Repeated solubilizations and precipitations of this newly sedimented material shows that it has the same solubility properties as the PI heavy pellet (see Table 2). This suggests that “37Pu and 5gFe are also associated with calcium phosphate microgranules sequestered in the microsomal fraction of the crab hepatopancreas. The occurrence of calcium microgranules, which are often rich in iron, was in fact demonstrated in small vesicles (Berkaloff, 1958) or in cisternae of the endoplasmic reticulum from the cells of the midgut wall of terrestrial arthropods (Gouranton, 1968; Waku & Sumimoto, 1974). These microgranules could be the first stage of the mineral phase formation. Our results show clearly that the bulk of plutonium and americium retained in the hepatopancreas after ingestion is in the low speed sedimenting nonmitochondrial fraction, which is composed essentially of calcium phosphate granules. Therefore, their localization is completely different to that of transuranic elements in the liver of mammals, where they occur mostly in the mitochondrial-lysosomal fraction (Boocock et ul., 1970; Popplewell et u/., 1971; Bruenger er a/., 1971. 1976). This could however be due to a difference in the route of absorption, since studies related to mammals were conducted after intravenous injection of the isotopes. It is also noteworthy that iron, a metal involved in enzymatic and respiratory processes, is found, although to a lesser degree, in the same PI heavy fraction as the artificial nuclides plutonium and americium. The occurrence of these three elements in this fraction is probably due to their wellknown chemical affinity for calcium phosphate. A common absorption and excretion pathway is thus given support. Using cytochemical methods or X-ray microanalysis electron microscopy, iron has been found previously to be associated with spherites in the midgut wall of insects (Ehrhardt, 1965; Gouranton, 1968), and annelids (Boilly & Richard, 1978), as well as in viscera of a marine gastropod (Bryan et ul., 1977). Moreover, the occurrence of various other heavy metals in granules generally composed of calcium phosphate is well established (see the review by Coombs & George, 1978). For example, zinc, copper, manganese, magnesium and lead have been shown to be associated with or included in phosphorite concretions of mollusc viscera (Bryan et ul., 1977), kidney (Bryan, 1973; Doyle et ul., 1978), gill and mantle (George et ul.. 1978) and midgut of barnacles (Walker rt al., 1975a,b). Calcium phosphate granules, which could be involved in shell calcification (Travis, 1955, 1957) are more probably implicated in excretion processes by trapping excess metals (Simkiss, 1976; Coombs & George, 1978). Moreover, it has been stated that, in the majority of cases, the intracellular granules are extruded from the cell into the lumen of the excretory or digestive system (Simkiss, 1976). In the case of a marine crustacean, in particular, Becker et al. (1974) have observed extruded granules in the lumen of the hepatopancreas and have suggested that the intraluminal granules could reflect a mechanism of Ca’+
mobilization in the crab. Nonetheless, it seems likely that the metals associated with calcium phosphate granules can, at least partially, be excreted in the lumen of the hepatopancreas at the time of granule extrusion. The suggestion that calcospherites play a role in metal detoxication processes in invertebrates is thus supported. Acknow/edgements~---The International Laboratory of Marine Radioactivity operates under a tripartite agreement between the International Atomic Energy Agency. the Government of the Principality of Monaco and the Oceanographic Institute at Monaco. We thank Drs S. W. Fowler, R. D. Cherry and G. Ailhaud for their support and critical comments, and Mr Pagliardini and Mr Marichy for
performing the analysis of hepatopancreas and the electron microscopy respectively.
mineral content
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