Lysosomes as primary targets of cryoinjury

482-488 (1971)

CII~~HI~L~GY,~,

Lysosomes

as Primary MAXIM

Institute

of Chemical

Biology,

Targets D. PERSIDBKY2

University

There is increasing realization of the importance of the lysosomal role in the interactions between the cell and its environment. Characterized by structure-linked latency of their hydrolytic enzymes, which is in contrast to a high sensitivity to environmental changes, these organelles are involved directly or indirectly in a great variety of normal or pathological activities in cells. Thus, in certain cases, lysosomes can be expected to play a crucial role in the mechanism of cell cryoinjury. In a previous communication (25) the author reported that activity of free acid phosphatase markedly increases in bone marrow cells aft,er freezing and thawing, even if cryoprotective measures are employed, and that the degree of resulting cellular damage correlates with the degree of this enzyme activity. Furthermore, the addition to the cryoprotective medium of acetylsalicylic acid, which reportedly should have a lysosome-stabilizing effect (21), has indeed reduced acid phosphatase activity, while cell recovery has improved. Although these observations, in general, should strengthen considerably the hypothesis of lysosomal involvement in it st,ill remains unclear whether cryoinjury, lysosomal activation is causally related to cryoinjury or is a secondary event. The present study was undertaken to investigate this problem by employing agents with more specific lysosomal action than that of salicylates which may have a stabilizing effect on all the membrane systems of the cell. Of this class, acid bisazo dyes, especially trypan blue, would be of particular int’erest. Recent evidence showed that trypan blue is select.ively concentrated by the lysosomal system after injection into the animals (17, 28), and acts as a potent inhibitor of acid hydrolysis (2, 17, 19). If cells from the animals treated with trypan blue show substantially 1 Supported by Contract N00014-68-C-0367 from t,he Office of Naval Research. *Present addreas: The Institute of Medical Sciences, Pacific Medical Center, San Francisco, California

94115.

of Cryoinjury’

of San Francisco,

San Francisco,

California

higher resistance to cryoinjury than cells from untreated animals, this could possibly mean that lysosomal activation is a primary phenomenon in the response of cells to the effects of freezing and thawing, and that consequent’ly it may play a major role in the mechanism of cryoinjury. In the present study bone marrow cells obtained from rats treated with t.rypan blue and from control rats were subjected to cryopreservation, and thereafter the degree of cell viability in both of these groups was determined and compared on the basis of 14C-l-glycine uptake and also on the ability of cells to resume growth in tissue cult’ure. MATERIALS

AND METHODS

Treatment with trypan blue (Allied Chemical, Color Index No. 23850) was accomplished as follows: In each experiment one Long-Evans male rat was injected intraperitoneally with 3yo solution of the dye made in Hanks’ medium, at a dose of 100 mg/kg. At the same time, two additional rats were injected with comparable volumes of Hanks’ solution alone. Twenty-four hours later all three rats were killed with chloroform, their femurs excised, and the bone marrow aspirated through the needle by syringe and collected in Hanks’ medium. After centrifuging at 750 rpm for 5 min, decant,ing, and resuspending in Hanks’, cells obtained from t,he trypan bluetreated rat were divided into three samples, while cells from the two untreated rats were divided into six samples. Thereafter, all the cell samples were centrifuged again and decanted. Each of the t,rypan blue-treated cell samples was then resuspended in 2 ml of 10% DMSO. Three samples of the untreated cells were also resuspended in the same volume of 10% DMSO, while the remaining three samples were resuspended in Hanks’ solution alone. Three cell samples with and three without trypan blue treatment, each containing DMSO, and one sample of untreated cells without DMSO, were all frozen. Two untreated cell samples served as unfrozen control. There was approximately the same number of cells in each sample since the 482

rub I’or each experiment were all of the same age :III~ t’he same body n-eight. The comparable nunlber of cells in each sample was also evident from the comparison of packed volumes of bone marrow cells in each tube after centrifugation. Theqr packed cell \-olumes appeared to be ap1”““i~Wlt.d~

f!qUd.

Freezing and thawing of the bone marrow cells ~‘1’: carried out iu accordance xvith the procedure previously described (25). Assessnlrnt, of the viability of bone marrow cells after c,r~oI)reservnt,ioII, based on the use of W-l-glycine technique was as follows: After thawing, all samples, including unfrozen control, were ceentxifuged and decant,ed to remove DMSO. The rell> were t.hen resuspended in 2 ml of the spec%llly prepared medium containing radioactive label. This medium w-as prepared by dissolving o..s mg 14c-1-g~ycirie (2.5 niCi,‘mM) (Cal htomic) in 51) ml of Hanks solution and diluting to 250 ml wit’h a solution of nonradioactive glycine in the same concentration. Fifteen milliliters of this solution mixed with 5 ml of calf serum (Rlicrobiological A after equilibration in the Nuclear-Chicago scintillat,ion counter the vials were read twice for a period of 10 min each time. The numerical values, being an average of the two readings, are expressed on the basis of I-min counting period. Cell vi:lbilit,y of bone marrow aft’er cryopreservation, determined on the basis of cell growth in tissue cult.ure, was performed as above in a separate experimental series. Afterwards the cells mere processed and rat,ed as previously described, the scoring system being from 0 to 4+ (25). RESULTS Results from t’he bone marrow cell viability assessment based on the uptake of 14G-l-glycine are presented in Table 1. The radioactive counts in cell samples obtained from the trypan bluetreated animals indicate that treated cells incor-

U\fSO ilo’!;,)

Contml Ilank

; Untreated

I1

t = 4.62; F = 44.21; p < 0.0005. c Since F = 44.24 >> F,ool[~d, 1;) = 5.85, the Student’s 1 test result, 1 = 4.K2, was verified with t,he Behrells-Fisher teqt with the followillp vallles:

t’.ms= /

Since t’.ooos = 4.4973 < 4.62, the computed I is verified and it. can be concluded that difference between the two means is highly significaatlt. porat’e, on the average, three times as much IdC1-glycine after cryopreservat’ion as cells from the untreated animals. In the two experiments there was an even higher radioactive count, in t,he dyetreated samples than in control which, however, may be due to t’he loss of cells in the controls as a result, of processing. Cells frozen in Hanks’ medium alone had very low survival. In spite of considerable variations between t,he individual

484

PERSIDSKY TABLE

R.ITING CELLS WITH TION

2

IN TISSUE CULTUHE OF BONK ill.~~t~to~ FIIOM RATS TRKITED .IIND UNTRKXTED TRYP.IN BLUE AFTER CRYOPHESERV~\-

Frozen-thawed

Expt. no.

DMSO (10%)

Control Hanks’

UP treated

‘b;‘u treated

__~

1

++++ ++++

2

++++ ++++ ++++ ++++ ++++ ++++ ++++ +++-t ++++ ++++ ++++

3

4

0 0 0 0 0

+ ++

0 0

+ Total

score

53

-

3

++

++++ 0 +-t+ 0 +++ 0 ++-t+ 0 ++++ 0 + ++ +++ 0 ++ ++ ++ 0 ++ t++ ++++ ++ ++++ ++ + 0 +++ 0 ++++

-I

12

45

experiments, statistical comparison of the dyetreated and untreated experimental groups, using Student’s t test, shows that the difference between them is highly significant at p < 0.0005. Cell viability of tissue cultured bone marrow after cryopreservation as determined by phase contrast microscopy is presented in Table 2. Cells from the animals treated with trypan blue appear much more like unfrozen cells, scoring much higher rating than cells from the untreated animals, the average score being 45 and 12, respectively. The unfrozen control cells scored on an average the rating of 53, while cells frozen in Hanks’ alone scored only 3. Thus, these results also indicate three times greater resistance to cryoinjury of cells from the treated than the untreated animals, which is in close agreement with the above experiments based on I%-1-glycine determinations. ObservaGons under the microscope of bone marrow from the rats treated with trypan blue reveal that many of its cells contain small darkblue staining granules in their cytoplasm, which,

as observed by others (28), apparentfly lysosomes that had injested the dye.

ale t’he

DISCUSSION In an attempt to interpret these results and to evaluate t,heir possible significance it will be necessary to consider first the fat,e of administered trypan blue and some of its biological effects. Because of its teratogenic properties, which were reviewed by Beck and Lloyd (I), t.his acid bisazo dye has been t,he subject of an extensive study in recent years (2, 17, 18). After parenteral administration trypan blue strongly binds to plasma protein, chiefly albumin (17, 26) which may act as a carrier for the dye. It is then taken up, presumably as the dye-protein complex, by the cells of the reticuloendothelial system as well as by certain phagocytic epithelial cells, such as those of the kidney proximal convoluted tubules, the liver parenchyma, and certain fetal membranes. According to Beck and Lloyd (3) all anionic dyes of the trypan blue group are subject t’o endocytosis as macromolecular complexes with protein, and it is most likely tQat trypan blue gains access to lysosomes by this mechanism rather than by diffusion through the cell membrane. Thus, the dye apparently is being ingested into the cell by pinocytosis and is carried within phagosomes into cytoplasm. After union of phagosomes with primary lysosomes, and formation of secondary lysosomes, protein, to which the dye is bound, probably undergoes digestion under the proteolytic action of lysosomal enzymes liberat’ing trypan blue as free dye (7). The unbound dye is then concent’rated within these organelles when the latter lose much of their hydrolyzable content by diffusion and become heterolysosomes (8, 17). Apparently the dye can be stored in heterolysosomes or residual bodies indefinitely. The possible ways of elimination of the dye have been st’udied (12, 17, 27) ; however, its ultimat,e fate remains unclear. Observations under the light microscope reveal that the dye is differentiated from the cytoplasm as distinct blue “granules” which electron microscopic and other observations show to be lysosomes (17, 28, 29, 31). Lloyd et al. (17) provided further evidence for the lysosomal specificity of trypan blue by demonstrating electron-dense dye deposits and acid phosphatase activity within the same organelle. Although these observations do not constitute proof that dye is not present

in smaller qua&t! in other parts of t,he cell, localization of t’he highest concentration of the dye in the vacuolar system of Iysosomes has been clearly demonstrated. S(xver:tl studies have been concerned with the l)ossible effects which intralysosomal trypan blue might have on lysosomal fun&ons. Lloyd and hi& colleagues (2, 17) in an estensive st,udy on lysosnnx-ricsh subcellular fracations from rat yolk WC and liver homogenate, demonstrated that trypan blue is a strong inhibitor of acid phosphatase and of several ot.her lysosomal hydrolases. Furi,her evidence of inhibition by trypan blue of lysosomal enzymes comes from the direct demonstration by Davies et al. (5) of a reduced rate of the intra,lysosorttal digest,ion of labeled protein in rat, liver heterolysosomes laden with t’his dye. The proposed explanation of the intralysosomal mode of trypan blue action is that free dye, accumulat.ing within these organelles, presumably combines with the enzyme protein and inactivates it n-hen the concentrat,ion of unbound dye reaches an inhibitory le\-el within heterolysosomes (17, 22). ‘l’lu~s, there is ample evidence available that hetrt,olvsosornes are able to concentrate trypan 1~1~ with resulting inhibition of lysosomal hydrolahes. Therefore, it was felt that the striking improvement in cryopreservation of bone marrow cells cont,aining the dye, observed in this study, as compared t,o those without it, may stem directly from t,he t,rypan blue inhibit,ion of lysosomal enzymes, and prevention thereby of their catabolic a&i&y against. cells. This also &on& sugpsts t,liat lysosomal artivation is a primary event, in the response of cells t,o freezing and t,hawing. Consequently, ci,yoinjury of cells, particularlv of those wit’h high lysosomal content, may be caused, t#oa great, extent,, directly by the cat,abolic n&m of lysosomul hydrolases. If t,his interpretation of the mechanism through which trypan blue mediates its cryoprotective effect, is correct’, then a manipulation of t,he lysosOrd functions in order to control their effects may open a new rational approach t’o the probblems of cryopreservation. ,1s it appears front the available information, there are at least, three different ways in which denira.ble controls of the lysosomal complex could he possibly achieved. From the results of this st,udy, concerning the effects of trypan blue, it seems t,hat inactivation of lysosomal enzymes is,

perhaps, one of the most effective :tpproaches in this respect. The other is st,abilization of the lysosomal membranes, which was report,ed by the author (25) in connection wit,h the rryoprotective effect of acetylsalicylic acid. Finally, the third logically possible and potentially useful, but riot yet tried approach ib depletion of 1ysosomul enzymes (4). Each of these l~sosolllal-dirrc.tetl a]~proaches will be considerecl in more detail, :ik follows: 1. Inhibition of bysosomal Enzymes. This :rsl)cct of lysosomal cont,roI can be apparently effected through a number of agentIs. In a broader >ense of de Duve’s (9) calassification for lysosomedirected drugs, such agents can be (*onGdered as “intralysosonlal modifiers.” The nlain requisite for the action of these agent,s is that they should be pinocyt,osed by cells and concent,rat,cd ill lysosomes, where they could then elicit a variety of effects. Among these substances is trypan blue, which apparently, by operating in this manner, inhibits lysosomal enzymes. The ot,hcr acid bisazo dyes, such as Evans b!ue, Congo red, Niagara blue, etc., operate in a similar manner :rlthc;ugh they are apparent]!. not ah effert,ive as I ryp:m blue (17, 22). Direct inhibit,ion of lysosomnl hydrolases ran be also obtained b>- inorganic gold salts which w:ts reported by Ennis et. al. (1 1) :tnd Lorber (20). They *howed that the interaction of gold salts with sulfhydryl groups of acid &OSphatase, /I-glucuronidase, and cathepsin xcounted for their inhibitory effects. In addition, gold complex, aurothiomnlate, and, likewise, SWamin Jvere found to inhibit lysosomal enzyvrnr (6). Also, such heavy metals as CL?, Ag+, lW+, and Hg2+ inactivate acid phosphatase and &glucuronidase in lysosome-rich fractions from rat liver and brain, when used in concentrations above IO-%I. However, in lower c~oncentrations they act as activators of these enzymes (16, 30). Without, considering the relative potency of each of these agents in inactivating lysosor~~al enzymes, t’heir effectiveness would be cspected to depend to a great extent on their ability to reach the sites of lysosomal enzymes. 111following de Duve’s scheme for the flow of mstt,er into, within, and out of the ~11, advantage can be taken of different traffic mechanisms regulating flow-rate of matter through the four currents postulated by de Duve (9). The agents that could be effective at, each of these key traffic points may be grouped under the common name 01

PERSIDSKY “traffic regulators.” Two such traffic mechanisms can be of particular interest. These are: (1) endocytosis, and (2) phagosome-lysosome interact,ion. Endocytosis can be induced or stimulated by a number of agents, and it has been the eubject of extensive studies in this context (13, 15, 17). By intensifying pinocyt,osis, loading of lysosomes with such agents as trypan blue could be facilitated. Concerning the phagosome-lysosome interaction, there is evidence that when phagosomes fail to fuse with primary lysosomes, which contain enzymes, they then serve simply as temporary intracellular storage sites for the endocytosed material (9, 14). If this fusion process can be influenced, it may possibly provide a useful tactic for enhancing the inactivation of lysosomal enzymes. 2. Xtabilization 0s lysosomal membranes. Variat,ions in stability or permeabi1it.y of lysosomal membranes is an important parameter which controls the escape of enzymes from or the access of substrates to the lysosomal complex. This aspect of protecting cells against the effects of potentially damaging enzymes can be achieved by means of a number of agents which de Duve (9) classified under the general term of “perilysosomal modifiers.” They all have common pharmacological characteristics in that they produce antiinflammatory effects (32). They include some steroids, such as cortisone and cortisol, antirheumatic agents such as sodium salicylates (25), chloroquine, and several other drugs (32). A consideration of the accessibility of most of these agents to lysosomes may be also just as valid here, as in the inhibition of lysosomal enzymes discussed above. Therefore, employment of “traffic regulators” may prove to be in some cases beneficial in facilitating stabilization of lysosomal membranes. 3. Depletion of lysosomal enzymes. Studying the fate of the macrophage secondary lysosome, Cohn and Benson (4) found that placing these cells in a medium which did not support pinocytosis resulted in reduction of the total activity of several lysosomal enzymes. 9 similar effect could be brought about, also, through the use of inhibitors of protein synthesis. They concluded that apparently lysosomal enzymes were themselves being degraded wit.hin secondary lysosomes and that eventually this could lead to complete depletion of the enzymes in these organelles which would become simply residual bodies. Such pretreatment

cells prior to cryopreservation may prove to be effective in reducing cell injury. This discussion was concerned so far with the intracellular aspect, of lysosome-induced injury in the individual cells, which may accompany cryopreserrat’ion. It is, however, known that damage of cells may result. in acute extracellular release of lysosomal hgdrolases (10). By assuming that this event. may take place in such a het’erogeneous cell population as the bone marrow, where some cell types contain many more lysosomes than the others, it could follow t,hat cells in which lysosomes are abundant may become the sites of intensive extracellular hydrolytic activity eliciting injurious effects on the surrounding cell populat’ion. These considerations may also hold true in cryopreservation of blood where damaged white cells may become the sites of hemolytic activity. It may be pertinent in this regard that the opt’ima1 cryopreservation requirements for erythrocytes are different from t,hose for white cells. Finally, considering cryopreservation of organs, and in particular, of those with large lysosomal content, such as kidney, it is quite tempting to speculate that massive intra- and extracellular release of acid hydrolases from their activated lysosomes may be a major factor in prevent,ing successful results in this area of cryopreservation. In conclusion, it should be noticed that the conventional cryopreservation procedures apparently are not effective enough to prevent liberation from lysosomes of significant amounts of acid hydrolases, as was indicated in t’he author’s previous study (25). Therefore, adding lysosomeoriented strategies for improvement of cell protection to conventional ones may provide the necessary st.ep to furt,her progress in the field of cryopreservation. of

SUMMARY This study was designed to test the hypothesis that lysosomes may play a primary role in the mechanism of cell cryoinjury. A lysosome-specific agent, t,rypan blue, n as chosen for these tests because of its known ability to concentrate selectively in lysosomes and to inhibit their enzymes. Bone marrow from rats injected with the dye was compared with the control marrow for its ability to recover after cryopreservation. Based on incorporation of W-1-glycine, and on growth in tissue culture, bone marrow cells from treat>ed

I,Y$0SOiUES

AS PRIMARY

artinlals showed on the average three times greater recovery than cells from the untreated animals. It wax concluded that this striking improvement in (sell preservation may result directly from t,he inhihjt.ion of lysosomal enzymes by trypan blue. Consequently, it may follow that activation of l~~sosomal enzymes by freezing and thawing can be w primary cause of cell cryoinjury. Support.ed by the available literature, three 1)ossible ways to cottt,rol Iysosomal activities were suggestSed. These are: (1) inhibition of lysosomal erizynlew; (2) stabilization of lysosomal membranes; and (3) depletion of lyaoaomal enzymes. It was felt that cornbir~~t~ion of t,hese lysosomeoriented approaches for cell protection w&h convcntiortal methods may provide the necessary C(JnditiOlls

for

more

&eCti\-e

CryOpreserxhOn.

11FFEI:ENCES 2 1. Berk, F., and Llol,d, J. B. The teratogenic efl’rcts of azo dyes. In “Advances in TeratolOF;Y,“ Vol. 1, pp. 131-193. Academic Press, New York, 1966. 2. Beck. F., I,loyd, J. B.. and Grifftths, A. Lysosc,tttal etlzyme itthihition by trppan blue: a theory of teratogellesis. Science 157, 11X011x2 (1967ai. 3. Beck. F.. and Lloyd, .J. B. Histochemistry and electron tnicroscop~ of lysosomes. In “1,ysosomes ill Biology and Pathology,” Vol. 2, pp. 56-599. North-Holland Plthl. Amsterdam, 196!). 4. (:ohtr, Z. A., atld Benson, B. The in uilro diffrretttiatiott of mononuclear phagocytes. III. The reversibility of granule and hydrolytic ettzyntes formation and the turttover of granule cotlstituents. Exp. Med. 122, 155m-166(1965d) 5. I)avi(as, Al.? Lloyd, J. B., and Beck, F. Protein digestion in isolated lysosomes by intralysosontal trypall blue. Science 163, 14541456 (1969). 6. l):tvies, XI., Lloyd, J. B., and Beck, F. Effects of suramin and attrothiomalate on rat liver Igsosomes. Biochent. J. 115, 54P. (1969b). 7. I):tvis, Il. W.. attd Gttttherg, D. L. Trypan blue in the rat embr)-0. TerafoZog!/ 1, 125-129 (1968). 8. de I)uve, C., aud Wattialtx, H. Functions of lysosomes. ~1rtrt’!,. Illve, C. 1,ysosotnes as target for drttg?. In “Interactioll of l>rrtgs and Rubcellular (‘otnponents ill Aninlal Cells” P. N. Camp

TARGETS

OF CRYOINJClt’l

487

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PERSIDSKY

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32.

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