J. Cell Sci. 39, 383-396 (i979) 383Printed in Great Britain © Company of Biologists Limited

INTRACELLULAR DISTRIBUTION OF LEAD

IN TETRAHYMENA DURING CONTINUOUS

EXPOSURE TO THE METAL

JYTTE R. NILSSONInstitute of General Zoology, University of Copenhagen,Universitetsparken 15, DK-2100 Copenhagen 0, Denmark

SUMMARY

Lead acetate (01-0-2%) forms a precipitate with the organic growth medium. The Tetra-hymena cells ingest this lead-containing precipitate and cell growth is resumed after a variablelag period. Ingested lead is observed as electron-dense material in food vacuoles. Soon afterexposure, cytoplasmic lead (preserved with certain fixation only) is revealed as electron-denseparticles in cilia and in a halo around digestive vacuoles. Later the lead particles pervade theentire cell but after the lag period they are confined to membrane-bound spaces. In dilutegrowth medium, high concentrations of lead inhibit food-vacuole formation and cell growth.Under these conditions lead is deposited in alveoli of the pellicle and is also found in autophagicvacuoles and other membrane-limited structures. The study has revealed that lead entersTetrahymena through the membrane of digestive vacuoles and through the cell surface. Thechange in distribution of lead during the lag period indicates that a mechanism is activated forremoval of lead into membrane-bound spaces. The final storage of lead seems to be in lysosomes.

INTRODUCTION

Lead is a common contaminant of the environment. Exposure to inorganic forms ofthis heavy metal takes place through the digestive tract after intake of contaminatedfood or beverage. More than 90% of the ingested lead may be recovered in faeces andurine (Goyer & Chisolm, 1972). The retained lead is found in soft and hard tissues andthe turnover of lead is high in the former tissue whereas it is low in hard tissue whereaccumulation occurs (Castellino & Aloj, 1964; Barry, 1975; Goyer & Chisolm, 1972).

The ciliate Tetrahymena grown in axenic cultures may be exposed to o-i% leadacetate (550 ppm lead) without appreciable effect on cell proliferation (Nilsson, 1978).Cell growth is resumed, however, only after a lag period and the amount of organicmatter present is critical for this tolerance towards lead. Addition of lead salt to theorganic growth medium results in the formation of a precipitate with a high contentof lead; since the cells ingest this precipitate they become exposed to a larger amountof lead than the percentage added to the medium (Nilsson, 1978). Within digestivevacuoles the lead-containing precipitate is converted to refractive debris which isdefaecated. Hence, Tetrahymena is exposed to lead through the digestive system likemost terrestrial animals; however, as an aquatic organism it is subject to an additionalexposure through the general cell surface.

The present investigation, a continuation of the above-mentioned study, aims tofollow the intracellular distribution of lead in Tetrahymena and to determine a possible

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384 J. R. Nilsson

change in this distribution relative to the lag period before cell proliferation is resumed.The cells were exposed continuously to up to 0-2% lead acetate in the normal growthmedium and in dilute growth medium.

MATERIAL AND METHODS

Tetrahymena pyriformis strain GL was grown axenically at 28 °C in 2 % proteose peptoneenriched with 01 % liver extract and salts (Plesner, Rasmussen & Zeuthen, 1964). The mediumwith a low content of organic matter was prepared by dilution of this growth medium withO'S % NaCl to give a 0-5 % proteose peptone medium. Lead was added as acetate from a 1 %(26 IBM lead) stock solution and in most cases EDTA (ethylenediaminetetra-acetic acid) wasadded to reduce the amount of precipitate and to maintain the pH of the medium (pH 7'2), asdescribed previously (Nilsson, 1978). The ratio of 2 rtiM EDTA/o-i % lead acetate was keptconstant in the different concentrations of lead salt; the distribution of lead in solution andprecipitate under these conditions has been reported (Nilsson, 1978). The cells were exposed to0-02, 01 , 015 and o-2 % lead acetate. Cell samples were removed at intervals during the firstS-h exposure and after a prolonged exposure of 20—24 h; a total of 9 independent sets of experi-ments were analysed. Some of the cell samples derive from experiments on which the light-microscopical observations and cell growth characteristics have been published (Nilsson, 1978).

The cells were fixed for electron microscopy at room temperature in o-i M cacodylate-buffered solutions (pH 7-4) of 2 % glutaraldehyde for 10 min and 1 % osmium tetroxide for1 h, sequentially or separately, or in a freshly prepared mixture of equal amounts of the 2fixatives for 1 h. After fixation the cells were washed in buffer and dehydrated in a gradedseries of ethanols followed by propylene oxide before embedding in Epon according to Luft(1961). The sectioned material was examined unstained or after contrasting with zinc-uranylacetate (Weinstein, Abbiss & Bullivant, 1963) for 20 min and/or with lead acetate (Venable &Coggeshall, 1965) for 3 min. The sections were examined in a Zeiss EM9 electron microscopeat primary magnifications between 1800 and 20000 times.

Cell motility was measured on light micrographs of a i-s exposure of cells moving in a cellchamber having a depth of 220 fim. The length of the paths of moving cells was measured usinga chartometer, and the mean speed of about 100 cells per sample expressed as/tm/s. The measure-ments are based on fast-moving cells only and may be somewhat biased since many lead-treated cells cluster around the precipitate.

RESULTS

A conspicuous feature of lead-treated Tetrahymena is the electron-dense contents ofdigestive vacuoles (Fig. 1). This material, evenly distributed within newly-formedfood vacuoles, resembles in structure the lead-containing precipitate outside the cells,whereas it forms a compact mass in the older, smaller food vacuoles (Fig. 1) and indefaecation balls (Fig. 2). Incidentally, no crystal-like structure could be detected inthe defaecation balls to explain their refractive appearance in the light microscope(Nilsson, 1978). Other aspects of the fine structure of lead-treated cells vary with themethod of fixation, the duration of exposure, the concentration of lead salt and thetype of growth medium.

Electron-dense material in the cytoplasm outside the digestive vacuoles is observedin cells fixed sequentially in glutaraldehyde and osmium tetroxide, but not in cellsfixed in glutaraldehyde alone (compare Figs. 6 and 7). With the former fixation cyto-plasmic lead appears as small, electron-dense particles which are absent from controlcells. These particles are seen both in stained (Fig. 3) and in unstained (Fig. 4)

Intracellular distribution of lead in Tetrahymena 385

sections; typically they form a halo around lead-containing food vacuoles. The distri-bution of cytoplasmic lead varies with duration of the exposure and with the type ofgrowth medium, as will be described below. An analysis of the preservation of cyto-plasmic lead using different fixation procedures is presented in Table 1. The partialpreservation of cytoplasmic lead in cells fixed in the mixture of glutaraldehyde andosmium tetroxide may be ascribed to fixation being carried out at room temperature,since several cells show ruptured sites in the pellicle. The following description appliesto cells fixed sequentially in glutaraldehyde and osmium tetroxide.

Normal growth medium

In this high concentration of organic material, addition of up to 0-2% lead acetatehas little effect on the growth rate of Tetrahymena, apart from inducing a variable lagperiod; food-vacuole formation is not inhibited (Nilsson, 1978). In agreement withthis observation the fine structure of the cells resembles that of growing cells; thenucleoli are small and evenly distributed in the macronucleus, mitochondria andperoxisomes are both of the 'loose' type (see Nilsson, 1976), and no glycogen or lipiddroplets are seen. However, the cells contain electron-dense particles in the cytoplasmand within membrane-bound structures, some of which are not seen in normallygrowing cells.

The distribution of cytoplasmic lead varies with the time of exposure to lead saltbut is largely independent of the concentration of lead salt within the range 0-02-0-2 %;the relative amount of electron-dense particles may, however, be dose-dependent.Within the first hour of exposure cytoplasmic lead is confined to electron-denseparticles forming a gradient radiating from the food vacuoles (Fig. 6) and to similarparticles present in axonemes of cilia; in o-i, 0-15, and 0-2% lead salt electron-denseparticles are also seen within the pellicular alveoli. During the following 2 h the leadparticles pervade the entire cell and all its organelles, including the nucleus, mito-chondria, peroxisomes, and small vacuoles or vesicles with electron-opaque or lami-nated contents (Figs. 6, 8, 9). The size of the electron-dense particles is smaller in thelatter organelles than it is in the general cytoplasm (Figs. 6, 8). The concentration ofcytoplasmic lead may be high in the oral region, around the site of fusion of foodvacuoles, and in the region of the cytoproct during defaecation; in the latterinstance lead particles are also present in the pellicle adjacent to the cytoproct, whereasthey are not abundant elsewhere (Fig. 5). By the end of the lag period, around 4-5 hof exposure, cytoplasmic lead is largely confined to membrane-bound spaces, especiallyto the vesicles and small vacuoles, the membrane of which resembles that of foodvacuoles in thickness. No apparent increase in the number of these vesicles andvacuoles is seen, not even of the vacuoles with an electron-opaque matrix (Fig. 8)previously identified as the refractile granules seen in phase-contrast optics (Nilsson &Coleman, 1977). Cytoplasmic lead around food vacuoles is observed throughout theexposure to lead salt; however, the phenomenon appears to correlate with the stage ofdigestion. The earliest stage at which lead is seen is 0-5 h after uptake of the lead-containing precipitate and typically an electron-translucent zone is seen between theelectron-dense content and the limiting food-vacuolar membrane (Figs. 3, 4, 6). On

386 J. R. Nilsson

Figs. 1-5. For legends see facing page.

Intracellular distribution of lead in Tetrahymena 387

prolonged exposure (up to 24 h) to lead acetate the distribution of intracellular leadremains as described above when EDTA has been added to the medium. In theabsence of EDTA, a 24-h exposure to o-i % lead salt results in the presence of leadparticles in mitochondria and in the perinuclear space (Fig. 10). This distribution isnot observed in the presence of EDTA, even in 0-2% lead acetate (Fig. n ) .

Dilute growth medium

In this low concentration of organic matter the control cells have a generation timecorresponding to that of control cells in the normal growth medium; however, theformer cells are smaller and the final cell density is lower than that found in the normalgrowth medium. The fine structure of these control cells does not differ from that ofgrowing cells in the normal growth medium.

Addition of lead acetate to cells in the dilute growth medium affects cell parameters.In o-i% lead salt the rate of food-vacuole formation is normal but after the lagperiod cell proliferation decreases; in 0-15 and 0-2% lead acetate few and no foodvacuoles are formed, respectively, and cell proliferation ceases (Nilsson, 1978). Thesechanges are reflected in altered fine structure of the lead-treated cells.

After 1 h in o-i% lead salt the cells contain lipid droplets and some autophagicvacuoles (Fig. 1). The intracellular distribution of cytoplasmic lead in these cells(Figs. 3, 4) follows the time-dependent pattern described above for cells in the normalgrowth medium but lead particles within the pellicular alveoli are more numerous inthe dilute growth medium and are a constant feature (Fig. 12). Lead particles within

Unless stated otherwise Tetrahymena pyriformis was fixed sequentially in glutaralde-hyde and osmium tetroxide and the sections were contrasted with zinc-uranyl acetateand lead acetate.Fig. 1. Tetrahymena with food vacuoles (Jv) of varying electron density after ingestionof lead-containing precipitate (arrow). Note the loose texture of ingested material inthe forming food vacuole (Jfv), the even distribution of the material in newly formedfood vacuole (i), and its compactness in older food vacuoles(2). Oral region {pa).Autophagic vacuole (cy). Exposure to o-i % lead acetate for 1 h in dilute growthmedium, x 5400.Fig. 2. Extruded defaecation ball (db) among lead-containing precipitate. From cellsample exposed to 0-2 % lead acetate for 2 h in normal growth medium, x 9000.Fig. 3. Cytoplasmic lead (arrows) is confined to a halo around lead-containing foodvacuole (Jv). Note the clear zone between vacuolar contents and the limiting mem-brane. Macronucleus with nucleoli, n; mitochondrion, m. Exposure to 01 % leadacetate for 1 h in dilute growth medium, x 18000.Fig. 4. Unstained section of lead-containing food vacuole (Jv). Note the electron-denseparticles (cytoplasmic lead) on the cytoplasmic side (arrows) and the absence of leadin zone between vacuolar contents and the limiting membrane. Exposure to 01 %lead acetate for 2 h in dilute growth medium, x 18000.Fig. 5. Unstained section of cytoproct region on completion of defaecation. Note theelectron-dense particles around cytoproct (arrows) and in pellicle (p) extending somedistance from the cytoproct, but the absence of lead particles in pellicle in general(large arrow). Defaecation balls (db). Cilium (c) with lead particles. Lead-containingfood vacuoles (Jv) with little cytoplasmic lead. Exposure to 0-2 % lead acetate for20 h in normal growth medium, x 18000.

388 jf. R. Nilsson

v - •

• • • *•* •

fv

n

10 11 ;Figs. 6—II. For legends see facing page.

Intracellular distribution of lead in Tetrahymena 389

the alveoli appear bound to material of low electron density; such material is also seenin the control cells. The nucleoli become aggregated after the first hour and remainunchanged, in agreement with the prolonged generation time of the cells. Leadparticles occur within small vacuoles and vesicles (Fig. 19) in a manner similar to thatdescribed above for cells in the normal growth medium.

Table 1. Preservation of cytoplasmic lead in Tetrahymena

Fixation In food Halo around In alveoli In cellprocedure vacuoles food vacuoles of pellicle organelles

Sequential glutaraldehyde + + + +and osmium tetroxide

Mixture of glutaraldehyde + + — —and osmium tetroxide

Glutaraldehyde alone + — — —Osmium tetroxide alone + — — —

Fixation at room temperature. Cells exposed to o-i % lead acetate in dilute growth mediumfor 2 h.

In the 2 high concentrations of lead salt marked effects are seen on the fine structureof the cells. Cells in 0-15% lead acetate have food vacuoles with lead-containingprecipitate surrounded by cytoplasmic lead as described above, but cells in 0-2% leadsalt do not contain such food vacuoles. In both concentrations a time- and dose-dependent effect is seen on aggregation of nucleoli and on formation of autophagicvacuoles; furthermore, the mitochondria and peroxisomes are both of the 'dense'

Fig. 6. A gradient of cytoplasmic lead (arrows) extending from food vacuole (Jv)containing lead. Vesicle (lysosome) with laminated content and small lead particles (v).Exposure to 0-2 % lead acetate for 2 h in normal growth medium, x 60000.Fig. 7. Lead-containing food vacuole (Jv) in cell fixed in glutaraldehyde alone. Notethe absence of cytoplasmic lead (electron-dense particles) around food vacuole andof small lead particles in vesicle (v) with laminated contents (lysosome). Compare withFig. 6. Mitochondrion, in. Exposure to 0 2 % lead acetate for 2 h in normal growthmedium, x 60000.Fig. 8. Two small vacuoles limited by a membrane corresponding in thickness to thatof food vacuoles (Jv). Both vacuoles contain electron-dense particles smaller in sizethan the cytoplasmic lead (arrows). The more electron-opaque vacuole is a typicalelectron-dense granule. Exposure to 0-02 % lead acetate for 1 h in normal growthmedium, x 60000.Fig. 9. Small electron-dense structure (arrow) with extending tubular structureadjacent to lead-containing food vacuole (Jv). Exposure to o-i % lead acetate for 1 hin normal growth medium, x 60000.Fig. 10. Lead particles accumulated within mitochondrion (m) and in perinuclearspace (arrow) in cell exposed to o-i % lead acetate in absence of EDTA for 24 h innormal growth medium. Fraction of macronucleus (n). x 60000.Fig. 11. Absence of lead particles in mitochondrion (m) in cell exposed to 0-2 % leadacetate for 20 h in normal growth medium (in presence of EDTA). Compare withFig. 10. Vesicle with laminated contents, v. x 60000.

390 J. R. Nilsson

type (see Nilsson, 1976). These features resemble those observed in starved cells andcells in which food-vacuole formation is inhibited (Nilsson, 1976). In 0-15% leadsalt, lead particles are numerous in the pellicular alveoli after a i-h exposure and theyform aggregates after a 4-h exposure. In 0-2% lead acetate such deposits of leadparticles are seen after a i-h exposure (Fig. 13). The lead deposits appear at regularintervals in the pellicle (Fig. 14) near cilia and at septa between adjacent alveoli. Leadparticles are also present in axonemes of cilia and in the adjacent cytoplasm where theymay accumulate within membrane-limited spaces associated with kinetosomes (Fig.13). These latter membranous structures correspond to the membranous sac system

13 . \\

\ t

Fig. 12. Lead particles within pellicular alveole (a), in axoneme of cilium (c) andkinetosome, and in cytoplasm (arrows). Membranous sac (v), associated with kineto-some (k); note possible accumulation of lead particles in this structure. Exposure too-i % lead acetate for 2 h in dilute growth medium, x 60000.Fig. 13. Deposits of lead particles within pellicular alveoli (large arrows). Notesequestered lead particles within membranous sacs (small arrows) associated withkinetosome (k). Parasomal sac (ps). Exposure to 0-2 % lead acetate for 1 h in dilutegrowth medium, x 60000.

described as interconnecting kinetosomes in Paramecium (Patterson, 1978), where anassociation was seen with the parasomal sacs; however, in Tetrahymena no specialconnexion was observed between lead particles and the parasomal sacs (Fig. 13).Within a i-h exposure lead particles may be found in debris vacuoles (Fig. 15), inautophagic vacuoles (Figs. 15, 18), and in small vacuoles with electron-opaquematerial (Fig. 16); these particles are generally small, like those found in small

Intracellular distribution of lead in Tetrahymena 391

vacuoles in cells forming food vacuoles (Figs. 6, 8, 19). Furthermore, lead binds tosome material in the cytoplasm, for example, in the region of the contractile vacuole(Fig. 17); these structures resemble the electron-dense structures seen in the above-mentioned debris vacuole and the electron-opaque structure (Figs. 15, 16). Occa-sionally, a cell with drastically altered structure was seen in 0-2% lead acetate samples;here the mitochondria were greatly swollen, the nucleoli formed large aggregates, andthe cytoplasm in general was much altered in appearance. These cells are undoubtedlymoribund, in agreement with an increasing cell mortality under these conditions.

Observation of cells exposed to 0-15 and 0-2% lead acetate indicated an initialeffect on cell movement which was not discernible after a 4-h exposure. Since thesecells accumulate lead in the pellicular alveoli, as mentioned above, it was of interestto measure their motility. These measurements revealed mean motilities of 263, 208,and 88 /im s"1 of cells exposed to o-i, 0-15, and 0-2% lead salt, respectively; the corres-ponding control value was 344/im s"1. After a 4-h exposure to o-i, 0-15, and 0-2%lead salt the cells moved at speeds corresponding to 101, 138, and 149%, respectively,of the control value. Since the cells in 0-15 and 0-2% lead salt become smaller after a4-h exposure, their increased cell motility could be due to this factor. For comparison,untreated cells were starved for 1 and 4 h and their cell motilities determined; theseresults showed mean speeds corresponding to 41 and 39%, respectively, of the un-starved control cells. Hence, the increased cell motility of the lead-treated cells cannotbe ascribed to the influence of starvation induced by the partial or total inhibition offood-vacuole formation in 0-15 and 0-2% lead salt, respectively.

DISCUSSION

Tetrahymena is exposed to lead mainly through the digestive system under thepresent experimental conditions since, as mentioned in the Introduction, the formedprecipitate is ingested. The defaecated debris has a higher content of lead than thelead-containing precipitate, a finding interpreted as a retention of lead within thefood vacuoles during digestion of the organic component of the precipitate (Nilsson,1978). This assumption is an approximation, since small, electron-dense particles(cytoplasmic lead) are observed around digestive vacuoles in the present study; thusthe food-vacuole membrane is impervious to lead. The amount of lead entering thecytoplasm is unknown, but presumably it corresponds to the ca. 10% of ingestedlead retained in mammals (Goyer & Chisolm, 1972). Not all food vacuoles are surround-ed by cytoplasmic lead, thus indicating that lead escapes at certain stages of digestion.The earliest time at which cytoplasmic lead is seen is about 0-5 h after uptake ofthe precipitate, and food vacuoles of this age have acquired a low pH value (Nilsson,1977). Liberation of lead may thus be a result of an increased solubility of the metalin the acidic environment, hydrolysis of the organic matter to which it is bound,and/or an increased permeability of the food-vacuolar membrane to the ion.

Lead also enters Tetrahymena through the cell surface, as observed early duringexposure, before lead particles have pervaded the entire cell. Entry occurs through themembrane of cilia but also through the general body surface where, however, lead is

J. R. Nilsson

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cy

15

m

17

i/a• 1 /

. * • ' < •

•1i

18

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Figs. 14-19. For legends see facing page.

Intracellular distribution of lead in Tetrahymena 393

retained within the membrane-limited alveoli in the pellicle as revealed in particularunder extreme conditions during which lead deposits are seen. The function of thepellicular alveoli has been suggested to be an osmotic barrier (Pitelka, 1965) or aregulator of the intracellular calcium concentration in connexion with ciliary move-ment, analogous to the sarcoplasmic reticulum in muscle cells (Allen & Eckert, 1969).The latter interpretation is interesting in view of the correlation observed betweenlead deposits in the alveoli and the increased cell motility of Tetrahymena. Lead maysubstitute for calcium in ciliary movement, since lead interacts with calcium in variousbiological systems (Six & Goyer, 1970; Parr & Harris, 1976; Sandstead, 1977).Another candidate for ion-regulation in ciliary movement is the membranous sacsystem associated with the kinetosomes (Patterson, 1978); this suggestion is supportedby the high calcium affinity of these membranes as demonstrated in another speciesof Paramecium (Fisher, Kaneshiro & Peters, 1976). In Tetrahymena lead is also seenwithin these membranous sacs; however, the present study does not permit anyconclusion in favour of either suggestion. The importance of calcium and theregulation of this ion in ciliary movement in ciliates is discussed by Eckert, Naitoh &Machemer (1976).

Cytoplasmic lead in Tetrahymena is observed only after sequential fixation inglutaraldehyde and osmium tetroxide, and not after fixation in glutaraldehyde alone.Although the present results cannot guarantee complete preservation in situ ofintracellular lead, the observations indicate that the material binding lead is lipo-protein in nature, in which respect it resembles proteins with calcium-bindingproperties (Koenig, 1974; Feldman & Weinhold, 1977; Basu, Anderson, Goldstein &Brown, 1977). Lead has a high affinity for SH- and phosphate groups (Vallee &

Fig. 14. Deposits of lead (arrows) at regular intervals in pellicle. Small vacuole,debris or autophagic, with electron-dense structures (va). Note the condensed mito-chondria (in) ('dense' type). Exposure to 0 2 % lead acetate for 4 h in dilute growthmedium, x 18000.Fig. 15. Part of a debris vacuole (va) with electron-dense structures (arrow) and smalllead particles. Fraction of an autophagic vacuole (cy) containing a mitochondrion andsmall lead particles. Exposure to 0-2 % lead acetate for 1 h in dilute growth medium.Section contrasted with zinc-uranyl acetate only, x 60000.Fig. 16. Small vacuole (arrow) containing electron-dense structures and small leadparticles, a typical small electron-dense granule. Exposure to 02 % lead acetate for1 h in dilute growth medium. Section contrasted with zinc-uranyl acetate only,x 60000.

Fig. 17. Electron-dense structures (arrows) in cytoplasm near contractile vacuole.Compare with structure in vacuoles in Figs. 15, 16. Mitochondrion, m. Exposure to0'2% lead acetate for 1 h in dilute growth medium. Section contrasted with zinc-uranyl acetate only, x 60000.Fig. 18. Two autophagic vacuoles (cy) containing small lead particles (arrows).Exposure to ori % lead acetate for 1 h in dilute growth medium. Section contrastedwith zinc-uranyl acetate only, x 60000.Fig. 19. Vesicle (v) with small lead particles near food vacuole (Jv) containing lead.Cytoplasmic lead (arrows). Exposure to o-i% lead acetate for 1 h in dilute growthmedium. Section contrasted with lead acetate only, x 60000.

394 3- R- Nilsson

Ulmer, 1972) and different macromolecules may be involved in the binding of lead inthe cytoplasm and in the membrane-bound spaces as judged by the difference in sizeof the lead particles at the 2 sites; the macromolecules in the cytoplasm may bind leadand other cations unspecifically, whereas the macromolecules at the other sites playinga role in ion regulation may have more specific binding properties.

It has been suggested that intracellular ion regulation in Tetrahymena involves thesmall refractile granules which appear under unfavourable growth conditions. Thesegranules contain an apatite-like material (calcium, magnesium, potassium, andphosphorus) in an organic matrix (Coleman, Nilsson, Warner & Batt, 1972; Nilsson &Coleman, 1977). Evidence for this proposal was obtained when strontium, added tothe growth medium, was also found to be incorporated into the granular contents(Coleman, Nilsson, Warner & Batt, 1973). By analogy with these results and becauselead-treated cells contain refractile granules, lead was assumed to accumulate in suchgranules (Nilsson, 1978). The present study has demonstrated lead within thesegranules; however, the granules are not very numerous, although there must be aconstant entry of lead into the cells. Assuming that the refractile granules representthe storage site for accumulated lead then their number is kept low either by constantdilution through cell divisions or alternatively by constant elimination of the granulesor their contents from the cells. A possible mode by which accumulated lead may beeliminated is by fusion of the granules with debris vacuoles, especially as the refractilegranules may be considered as a type of lysosome. The membrane of the granulesresembles that of food vacuoles in structure and some of the granules stain with neutralred (Nilsson & Coleman, 1977) as do lysosomes (Holtzman, 1976). Furthermore, indifferent cellular material lysosomes have been found to accumulate lead and othermetals (Koenig, 1963, 1974; Bran & Brunk, 1970, 1974; Barltrop, Barrett & Dingle,1971; Brunk & Brun, 1972; Dean & Barrett, 1976). Moreover, lysosomes have beenfound to contain cationic proteins (MacRae & Spitznagel, 1975; Brown & Wood,1978) and a membrane-bound ATPase, the activity of which is influenced by calciumand magnesium ions (Schneider, 1977; Bunce & Li, 1977). This ATPase has beenproposed as a regulator of the intracellular concentration of calcium by pumping theions into lysosomes (Schneider, 1977) and the efficiency of lysosomes in accumulatingions is indicated by their inhibitory effect on mitochondrial uptake of calcium (Bunce& Li, 1977). The heterogenous structure of the refractile granules in Tetrahymena(Nilsson & Coleman, 1977) also indicates that they may be small, secondary lysosomesgradually losing their enzyme complement whereby they become terminal, non-functional lysosomes ('heterolysosomes'; see Holtzman, 1976) to which group somepigment granules seem to belong.

The distribution of cytoplasmic lead changes during the first hours of exposure,indicating that a mechanism for sequestration of lead is activated in Tetrahymenaduring this lag period before cell proliferation is resumed. The present study has notproduced any detailed information on this 'mechanism'; however, it has demonstratedthe pattern and relative time sequence in this adaptation process. After the lag periodlead still enters the cells, since even on prolonged exposure, cytoplasmic lead is seenaround food vacuoles with lead-containing precipitate and at sites of membrane fusion

Intracellular distribution of lead in Tetrahymena 395

such as during defaecation, coalescence of digestive vacuoles, and at the oral regionduring vacuole formation; apparently membrane fusion temporarily alters thepermeability of the membrane for lead. The adaptation of Tetrahymena to highconcentrations of lead (up to about 1100 ppm) depends to a large extent on the amountof organic matter present as has been demonstrated in the present study. This pointmay also explain the discrepancy in tolerance of Tetrahymena towards lead as reportedby other investigators (Ruthven & Cairns, 1973; Bovee et al. 1977) and the presentauthor (Nilsson, 1978).

My sincere thanks to Dr Cicily Chapman-Andresen for critical reading of the manuscriptand to Mrs Karen Meilvang and Miss Aase Madsen for excellent technical assistance. Thefinancial support of the Carlsberg Foundation and of the Danish Natural Science ResearchCouncil is gratefully acknowledged.

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(Received 6 April 1979)