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Cytotoxic effect of dimethylsulfoxide on the ultrastructure of cultured Rhesus kidney cells
Cytotoxic
Effect of Dimethylsulfoxide on the Ultrastructure of Cultured Rhesus Kidney Cells1
Dimc~thylsulfoxiclc ( DbfSO) is used as a cryoprotcctivc agent in a relatively wide rang’ of concentrations ( I4), although 515% solutions arc’ used most fwqnently ( l-3). \Vhilc the optimal concentrations of DMSO have been established empirically for a variety of cell types, as cviclcmcecl by satisfactoiy post-freeze yielcls of viable cells, the mwhanism of DMSO interaction with wlls and cellular components remains unresolved. Among the problrms pertaining to this question, the extent of DMSO tosicity on the wllular lwcl merits attention. The exwllcmt cryoprotwtiw propcrtics of DMSO should not obscurcl the, fact that it may bc toxic undcar wrtaiu conclitioi~s. Inclc~~l, it was reportccl that DPIISO whew used in less than cytociclal conwntrations inhibits cell growth ( 1) , inclnws ccllulnr necrosis ancl the formation of abnormal cells (6), and is instrumental in the loss of vast qiiantitics of cellular nucleic acids and proteins (4). Such a wide range of DMSO incluccd alterations in cellular function and integrity must be pwcwlc~cl by, or lw ccl1 comitant with, marked uhangcs on the) ultrastructural lewl of organization. Invwtigation of the cytotoxic effect of DMSO on wllular ultrastructurc~ may serw a uwful purpow in defining pathological CO~Iclitions induwcl by an important cryoprotcctant. The purpose of the prcwwt con-
mimic2~tioil is to clcwribc ultrastructural lesions in culturc~cl Rhesus kichwy cells incubatc~cl in DMSO solutions at progressively incrcwing time intcwals and at cliffcwnt tcwipcraturcs.
Explant tc~chniquc was usccl to establish primary cultuws with kidneys from a Rl~sus monkey embryo. The cultures wcrc grown on covcrslips in nutrient medium 199 (ll), supplemented with 10% v/v fetal bovine wrum. The confluent cultures were washed twice with Earle’s l~alanced salt solution ( RSS ) to rcmovc rc,siclual mulium. \Vashc~l cultures wcw thrn diviclc~cl into two scrics consisting of eight cxpc~rinicntal and four control lots each. The rcspc~ctivc lots of clach series were then incubated for 10, 20, 40, and 60 min in 7.ri and lFjc/c v/v DMSO dissolved in Earlc’s balancccl salt solution. To assess a possible tcmperaturc factor in DMSO cffeet on the wlls, the: incubation of wch scrims was carriccl out at 4” C amd 2.5” C. Following l”“l”tc,rmincd~ecl incubation intervals, solutions of DMSO were dccantcd, and ccl1 monolayclrs disassociatrcl with 0.025% tctraacetate c~tliyl~neclianiii~e clisocliuni (EDTA) as clescribccl clsewhcrc ( 15). The clisassociatc~cl ~11s were pt,lletcd in a tentrifugcl at 4” C, and the supwnatant rcmovc~l by suction. They were then fixed in 1%) (v/v) gl~~taralclc~hyclc dissolved in Siirenscn’s phosphate buffer ( pH 7.3). TO
insure uniform fixation, the vials coutainillg cells were agitated at frqwnt intcwals. Fixed cells were washed thrice with thcl same bufkr and thm postfiscd for 1 hr iri l’/(, (w/v) osnrilim tc~troxidc~ dissol\~c~l ill phosphatc~ buffc7 at Iiuitrd pI1. Thc~ postfiscd ~~~11s\vcm’ proCcwcd ~olr\-~lltiollalI!, and cw~Ixddcd ill Epo’l 812. The> ultwtllill scdioils wc’w stained with Icd citrate ant1 uranyl acctatc~ for sulxcclucwt study \\.itlr a Sicmcns 1A dcctrorl microwopc~. HESI’I,TS Controls Large cpithelioitl ~11s of tubular origin fornicd a numerically prdoniinant fraction of culturw. Endotheliul wlls, \vhiIc~ OCGsionally present, \ver(a infrqwnt. Thrl formcr \vc’re clistinguishccl by a largc~ WIItrally Iocatcd nucleus, caiitaining one or inorcb nucleoli and anrplC cytoplasm. The distinct nuclear m~nilxinc~ of all culls ap pcxud to Ix intact aiicl containd nui~ic~rous nuclear porm Similarly, the cytoplasnl was I~ounclcd by distinct ~~11 niwilmnw forming nunic~rous folds, but otlic~rwisc frw of waginations, rupturc~s, or the mtwllxxlc~ bound cxtraccllular niatcrial. The cytoplasnlic matrix containd nuincrous mitochondria. owasional niicrobodies, well-developed rough surfacul cmdoplasmic retidunl ( RER), i~lfrqumt lipid inclusions, as well as ;i few lvsosomcs and \mx~olcx Microtubules mtl ~nicrofilanwnts fornwl an cxtcasi\7~ wtnwrk iii all cells. No autophagic activity \KIS Ilotd. Typical cells of control cdturw ;w shon~ll in Figs. 1 and 2. Experimental
5kricJ.e
A. Cells incubntccl in 7..5:,; D,\fSO solrrtifJ)l c/t 4” C. In gcwd, ~11s inculxbcd in DMSO cww for as Iittlo ;IS 10 mill pxscntcd a iiunibu of strwtural diffrrcmcc~s as compared with controls. Thrw consisted primarily of lipid acciimulation, s\lYclIing of mito~lioirtl~ia. tI;lmagc\ of ~r~ito~I~oiiclri~11 iIic~~~ilm~~~~salrtl cristncl. p;irtial loss oi
-‘? . .
tcrations of practically all ~1~11 components. Damage to ccl1 111~~1nl~r;111cs with the cm1comitant cscapc of iirtracc,llular material into cxtracclhilar space was noted repcatedly (Fig. 7). These events wcrc accompanied by the concomitant obliteration of the internal structure of numerous mitochondria and the loss of structural integrity of mitochondrial membranes. The cxtensivc nuclear damage was represented by the karyorrhexis of numerous cells. As in the preceding series, the extent of cellular lesions appeared to be directly related to the incubation time. The final stages of incubation were charactcrizcd by the prcsence of some cells with such cxtensivc clamagc that probability of their survival, or much less their normal function, could bc seriously questioned (Fig. 8). D. Cel1.s incul?aterZ in 15c/,, DMSO ut 25” C. Total obliteration of cell membrane segments (Fig. 9) was observed in some cells following a lo-min incubation period. As in the cells of the preceding series, protracted incubation resulted in the progressivc increase in the number of damaged cells. Qualitatively, the cellular damage was rcpresentcd by the dilation of RER and its degranulation (Fig. 10). Thr hyaloplasm began to low homogeneous appearance, and the foci consisting of sphcriical cytoplasmic aggregates ww found with an increasing frequency. The number of lysosomes seemed to increase, and the mitochondria appeared to suffer an ever increasing damage (Fig. 11). The network of microtubules and microfilaments embedded in the areas of intact hyaloplasm appeared to be well-drveloped. At the end of 60 min incubation, a number of ceIIs showed irregular protrusions of cytoplasm, swollen mitochondria lacking any internal organization, foci of lipid accumulation, and coarsely granular cytoplasm ( Fig. 12). The chromatin in the nuclei of such cells also appeared quite granular and far less tlcnse than that in oithcr normal cells or
cells incubatctl irl DhISO for only brief iiitcrvals. 1:. General o/~.sarxxrtiot~s. \2’1ic11 comparing the cells of two expcrimcntal scrics, the cells incubated in 15% DYISO tcncled to show greater damage at the early stages of incubation. With increased incubation time, these diffcrcnccs tended to be obliterated. On the other hand, while the wlatively mild manifestations of DMSO toxicity (mitochondrial swelling, dilation of ER. etc.) seemed to converge in both series, the upper limit of serious ccl1 damagc~ (ruptnw of cell nwmbranes, loss of hyalopl~lsm, karyorrhcsis, cytolysis, etc. ) occurring at the early stage was noted mow fwqucntly in cells incubated in EC/, DMSO. During early stagc,s of incubation the tcmpcrature of 25” C, as compared with 4? C appeared to contribute more to the delctorious effect of DMSO. Howcvcr, the conccn t&ion factor seemed to predominate in the initial maiiifcstatioiis of DMSO toxicity. UISCUSSION
In interpreting results wportcd hcrc, it is necessary to consider the cffcct of DhlSO-induced lesions on cellular viability, and, following this, to establish appropriatc> corwlatcs bctnw~n morphological exprcssioii of DI4SO iiitcraction with against the bnckcellular components ground of some known physiological effects. It has been well documented that most cells tend to retain their viability after exposure to DMSO in the 7-G’& conccntration range (1-3, 6), particularly if the duration of exposure is rclativcly brief (1, 2, 6). Thus, there is no reason to assume that the cellular lesions described in this report, except in the cstrrmc cnscs such as karyorrhexis, are incompatible with cc:11survival and eventual repair. Howcvcr, owing to recurrent difficult& inherent in the attempts to define cellular viability in strictly ~norphoIogica1 terms (7), IN) structlual via-
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\lito~hontlrial tlut~g~ \xricd from the \w’y light to total ol~litcration of structiuxl or~aiiization of thcw Organc~llrs ant1 \\x in\xial,ly acwiripai~iul 1)). a sharp incrwsc in clc~position of intracc~llular lipids. O\ving to ;I coirsistc,lrt lability of mitochonclria tci DYISO. it scowls logical to sqgc\st that tlicl cc~lliilar c~lcctron transport chain ma!’ 1)~s tllasticall~~ affcckcl l)!; DhlSO. Apart froni niito~liolltlri~i. rough surfawtl l,ranq orgallc4c~s. and tlw llyaloplaslll. The alteration of structwal integrity of cwdoplawiic r~ticiiluni ( RElZ ) \vas still ~11 memlxaws by DMSO appears to lw anothcxr cytoplasmie c+on~pon~wtccnrsistcwtl~ of a particular significancr. IVhile thcl affinatktc~cl l))r D1ISO. I~c,grnnulatioll of HEK it!, of DMSO for ccl1 twn~1~raw.s WIS tcwls to siiggwt that, ilr tlicx prfwmc’c: of shown to exist under a \wicty of cspcriDIiSO, the ratcl of protein synthesis may mental conditions (5, 8, 9. 12). their struclx, altcwd and that a si,qnificant fraction tural lahilit)- to DMSO has not lwcn dcw~of cxtracc~llular lWLd is probably rc’prc’onstratcd prwiously. This findiq sccw~s to provide the structural basis for rc~portctl scntc~cl by the rihosomal RNA. Arcas of hyaloplasntic tlc~plc+oli found in niimuwiis cscapc~ of cclliilar nuckic acids ant1 proteins from ~11s incubated in DJISO SOIII- cxllls socwi to suggest that h~~iloplasmic ;1lx c~Ytractc~t1from ~11s lg, tions (4) aid swmingly iwccssitaks tlrc ro- Colllpo1Kwts appraisal of a widely h&l view that the D?rlSO. As notclcl in tllis stwly, scwningly traiisnic~iiil)r~iti~~ transport of D\ISO itwlf. progrcwi\-ct incwasc in l!w~somul and autoas \wlI as tlicx 1)~1SO-IIlc,tlintc,cl trawport plingic ac*ti\.ity iii tlw chills of tlw cywriinvariably OC‘CII~S inc2ital scric~s is in qgrcwiwiit of other co~npo~~ncls, \vith im across an intact ~11 cnvc~lopc~. cwlic$r rrport that DhlSO activatcls lysoIn contrast to cell nicw~l~riincs and menlSOmcs ifz oitw ( 10). Olwr~xtions by Perbranw of mitochondria, strwtllrally analsidskv (X3) that lysoson1cs may 1~ one of ogous damage of nuclear mcwrbrancs has the> primary targets of cr)winjiq~ in the JlOt bwn cstablishccl conclusivc~ly. This wlls frozen iii prcwnw of this c’ryoproobservation, hornever. does not riilc: ont tf~ct~lnt. and the finc1iiiji.s rc>portcd hcrc a prolxible labilit)- of nuclear nicwil,ranc~s sc’c’nl to iulplicatc D51SO as an important to DMSO. In fact, mitotic abnormalities factor ilk 1ys0s01nal activation. The lvidc drscrilwxl to occur in soiiif: cells following range of striicturnl nltcwtions indiicccl 1~ their cxposurc~ to DIISO (6) ant1 sul~scDMSO, (ww \vli~w this cqwprotc~ctant is qwntly propag:atccl as cultiiws, ;IS ~~11 ~1s uwd in “optimal” ~on~c~ntlatioiis aid fol the rclcaw of DNA into DSISO-contai~lil~g bric\f pclriotls only, Sf’(‘IllS to s11gpt that solutions ill qwintitics which cannot 11~iltthis c~oiiipo~~iid is toxiv to tlic irl uitw cc>11 tributcd solely to mitochondria aii(l lysc(l systcwls ant1 thcwforc~ cannot 1~ rc~garclcd dls (4), tfwd to support tlw notion thnt ai1 ;Ls iiinoc~iiot~s cqoprotfy$i\~~ ag:c~~rt. As shown ill this investigation, morphological mailifcstations of D1ISO cffcct \7wx~ charactcrizcd by the di\,cuity of affcctcd cc~lliilnr conipownts and hy their early onsrt. To facilitate discussion, thr fornwr may lw con~wiic~ntly groiipcd to iwluclc thrw broad CI;ISSCYof conq~oiic7rts, ix., lllf‘l11-
Cultured Rhesus kidnry ccxlls wore illcubatcd in the 7.5 and 15% solutions at 4 and 25” C and for periods ranging from 10 to 60 min. The ultrastructural alterations, while varying from ccl1 to cell, were evident even following the lo-min incubation interval. The most frequent and least scvcre structural changes included lipid accumulation, increased nurnbcr of lysosomes, dilation and clegranulation of rough surfaced endoplasmic reticulum, as well as the swelling of mitochondria and damage to mitochondrial membranes. The more severe instances of cellular lesions were rcprcsented by the extensive damage to ~11 membranes, karyorrhexis, total obliteration of the internal structure of mitochondria, and areas of complete loss of hyaloplasm. The extent and frequency of cellular lesions appeared to be dctcrmined primarily by the concentration of DMSO with the temperature and duration of incubat:on being important ancillary determinants. REFERENCES 1. Bouroncle, B. A. lreser\atiou of living cells ;lt -79” C lvith dilllethylsulfoaidr. Proc. Sof,. Exp. Bid. Med. 119, 953-961 (1965). 2. Bouroncle, B. A. Preservation of human normal and leukemic cells with dimethylsulfoxide at -80” C. Cryobiology 6, 445-455 (1967). 3. Brown, B. L., and Nagle, S. C., Jr. Preservation of mammalian cells in a chemically defined medium and dimethylsolfoxide. Scionce 149, 1266-1267 ( 1965 ).
5. hlalinin, G. I., F’ontana, D. J., and Brarmgart, D. C. Distribution of C” labeled dimethylsulfoxide in tissues of intact aninxlls. Cryobiology 5, 328-335 ( 1969). of 6. ?rlalinin, T. I., and Perry, V. P. Toxicity dimethylsulfoxide on IIeLa cells. Cryobiology 4, 90-96 ( 1967 ) 7. Malinin, T. I., and Perry, \‘. P. A review of tissne anld organ viability assay. Cryohiology 4, 104-115 ( 1967). 8. hlalinin, T. I., &tontes de Oca, H., Gollan, I;., and !blalinin, G. I. Cell membrane affinity of dimetl~ylsr~lfoxidc. Proc. Fifth Int’l Congrets of PIurnl., San Francisco ( 1972) (Alx.). 9. \\Ialinin, T. I. Preservation of isolated org;ms at hypothenllia ;md by freezing. PTOC. 13th lnt’l Congress of Hf~frigerution, Wdington, D. C. (1971) (In press). 10. Slisch, U. W., and hlisch, hi. S. Dimethylsulfoxide: activation of lysosomes in vitro. Proc. Nut. Acad. Sci. USA 58, 2462-2467 ( 1967). 11, Morgan, T. J., Morton, H. J., and Parker, R. C. Nutrition of animal cells in tissue cllltrlrc. I. Initial studies of synthetic medium. Proc. Sm. Exl~. Bid. Med. 73, l-8 (1950). 12. Olferijns, F. G. J., and Krijnen, II. W. ExperiInents in the prrservution of hexrts at snbzcrn tcw~prratllres. Cryobiology 6, ,589 ( 1970) (Ah.). 1:i. I’crsidsky, \I. 1). l,yso~om~s ils prinlilry targets of cryoilrjllry. Cr!/obio/og~/ 8, 482-488 (1971). 1.1. \‘os, O., and Kaalen, hl. C. A. C. Prevention of freezing damage to prol’fer;l:ing cells in tisslle culture. A qllnntitative stndy of a munber of agents. Cryobiology 2, 90-96 (1965). l,5. Wilmer, E. N. “Cells and tisslles in culture.” Vol. 1, p, 52, Academic Press, New York, 196ri.
Effect of Dimethylsulfoxide on the Ultrastructure of Cultured Rhesus Kidney Cells1
Dimc~thylsulfoxiclc ( DbfSO) is used as a cryoprotcctivc agent in a relatively wide rang’ of concentrations ( I4), although 515% solutions arc’ used most fwqnently ( l-3). \Vhilc the optimal concentrations of DMSO have been established empirically for a variety of cell types, as cviclcmcecl by satisfactoiy post-freeze yielcls of viable cells, the mwhanism of DMSO interaction with wlls and cellular components remains unresolved. Among the problrms pertaining to this question, the extent of DMSO tosicity on the wllular lwcl merits attention. The exwllcmt cryoprotwtiw propcrtics of DMSO should not obscurcl the, fact that it may bc toxic undcar wrtaiu conclitioi~s. Inclc~~l, it was reportccl that DPIISO whew used in less than cytociclal conwntrations inhibits cell growth ( 1) , inclnws ccllulnr necrosis ancl the formation of abnormal cells (6), and is instrumental in the loss of vast qiiantitics of cellular nucleic acids and proteins (4). Such a wide range of DMSO incluccd alterations in cellular function and integrity must be pwcwlc~cl by, or lw ccl1 comitant with, marked uhangcs on the) ultrastructural lewl of organization. Invwtigation of the cytotoxic effect of DMSO on wllular ultrastructurc~ may serw a uwful purpow in defining pathological CO~Iclitions induwcl by an important cryoprotcctant. The purpose of the prcwwt con-
mimic2~tioil is to clcwribc ultrastructural lesions in culturc~cl Rhesus kichwy cells incubatc~cl in DMSO solutions at progressively incrcwing time intcwals and at cliffcwnt tcwipcraturcs.
Explant tc~chniquc was usccl to establish primary cultuws with kidneys from a Rl~sus monkey embryo. The cultures wcrc grown on covcrslips in nutrient medium 199 (ll), supplemented with 10% v/v fetal bovine wrum. The confluent cultures were washed twice with Earle’s l~alanced salt solution ( RSS ) to rcmovc rc,siclual mulium. \Vashc~l cultures wcw thrn diviclc~cl into two scrics consisting of eight cxpc~rinicntal and four control lots each. The rcspc~ctivc lots of clach series were then incubated for 10, 20, 40, and 60 min in 7.ri and lFjc/c v/v DMSO dissolved in Earlc’s balancccl salt solution. To assess a possible tcmperaturc factor in DMSO cffeet on the wlls, the: incubation of wch scrims was carriccl out at 4” C amd 2.5” C. Following l”“l”tc,rmincd~ecl incubation intervals, solutions of DMSO were dccantcd, and ccl1 monolayclrs disassociatrcl with 0.025% tctraacetate c~tliyl~neclianiii~e clisocliuni (EDTA) as clescribccl clsewhcrc ( 15). The clisassociatc~cl ~11s were pt,lletcd in a tentrifugcl at 4” C, and the supwnatant rcmovc~l by suction. They were then fixed in 1%) (v/v) gl~~taralclc~hyclc dissolved in Siirenscn’s phosphate buffer ( pH 7.3). TO
insure uniform fixation, the vials coutainillg cells were agitated at frqwnt intcwals. Fixed cells were washed thrice with thcl same bufkr and thm postfiscd for 1 hr iri l’/(, (w/v) osnrilim tc~troxidc~ dissol\~c~l ill phosphatc~ buffc7 at Iiuitrd pI1. Thc~ postfiscd ~~~11s\vcm’ proCcwcd ~olr\-~lltiollalI!, and cw~Ixddcd ill Epo’l 812. The> ultwtllill scdioils wc’w stained with Icd citrate ant1 uranyl acctatc~ for sulxcclucwt study \\.itlr a Sicmcns 1A dcctrorl microwopc~. HESI’I,TS Controls Large cpithelioitl ~11s of tubular origin fornicd a numerically prdoniinant fraction of culturw. Endotheliul wlls, \vhiIc~ OCGsionally present, \ver(a infrqwnt. Thrl formcr \vc’re clistinguishccl by a largc~ WIItrally Iocatcd nucleus, caiitaining one or inorcb nucleoli and anrplC cytoplasm. The distinct nuclear m~nilxinc~ of all culls ap pcxud to Ix intact aiicl containd nui~ic~rous nuclear porm Similarly, the cytoplasnl was I~ounclcd by distinct ~~11 niwilmnw forming nunic~rous folds, but otlic~rwisc frw of waginations, rupturc~s, or the mtwllxxlc~ bound cxtraccllular niatcrial. The cytoplasnlic matrix containd nuincrous mitochondria. owasional niicrobodies, well-developed rough surfacul cmdoplasmic retidunl ( RER), i~lfrqumt lipid inclusions, as well as ;i few lvsosomcs and \mx~olcx Microtubules mtl ~nicrofilanwnts fornwl an cxtcasi\7~ wtnwrk iii all cells. No autophagic activity \KIS Ilotd. Typical cells of control cdturw ;w shon~ll in Figs. 1 and 2. Experimental
5kricJ.e
A. Cells incubntccl in 7..5:,; D,\fSO solrrtifJ)l c/t 4” C. In gcwd, ~11s inculxbcd in DMSO cww for as Iittlo ;IS 10 mill pxscntcd a iiunibu of strwtural diffrrcmcc~s as compared with controls. Thrw consisted primarily of lipid acciimulation, s\lYclIing of mito~lioirtl~ia. tI;lmagc\ of ~r~ito~I~oiiclri~11 iIic~~~ilm~~~~salrtl cristncl. p;irtial loss oi
-‘? . .
tcrations of practically all ~1~11 components. Damage to ccl1 111~~1nl~r;111cs with the cm1comitant cscapc of iirtracc,llular material into cxtracclhilar space was noted repcatedly (Fig. 7). These events wcrc accompanied by the concomitant obliteration of the internal structure of numerous mitochondria and the loss of structural integrity of mitochondrial membranes. The cxtensivc nuclear damage was represented by the karyorrhexis of numerous cells. As in the preceding series, the extent of cellular lesions appeared to be directly related to the incubation time. The final stages of incubation were charactcrizcd by the prcsence of some cells with such cxtensivc clamagc that probability of their survival, or much less their normal function, could bc seriously questioned (Fig. 8). D. Cel1.s incul?aterZ in 15c/,, DMSO ut 25” C. Total obliteration of cell membrane segments (Fig. 9) was observed in some cells following a lo-min incubation period. As in the cells of the preceding series, protracted incubation resulted in the progressivc increase in the number of damaged cells. Qualitatively, the cellular damage was rcpresentcd by the dilation of RER and its degranulation (Fig. 10). Thr hyaloplasm began to low homogeneous appearance, and the foci consisting of sphcriical cytoplasmic aggregates ww found with an increasing frequency. The number of lysosomes seemed to increase, and the mitochondria appeared to suffer an ever increasing damage (Fig. 11). The network of microtubules and microfilaments embedded in the areas of intact hyaloplasm appeared to be well-drveloped. At the end of 60 min incubation, a number of ceIIs showed irregular protrusions of cytoplasm, swollen mitochondria lacking any internal organization, foci of lipid accumulation, and coarsely granular cytoplasm ( Fig. 12). The chromatin in the nuclei of such cells also appeared quite granular and far less tlcnse than that in oithcr normal cells or
cells incubatctl irl DhISO for only brief iiitcrvals. 1:. General o/~.sarxxrtiot~s. \2’1ic11 comparing the cells of two expcrimcntal scrics, the cells incubated in 15% DYISO tcncled to show greater damage at the early stages of incubation. With increased incubation time, these diffcrcnccs tended to be obliterated. On the other hand, while the wlatively mild manifestations of DMSO toxicity (mitochondrial swelling, dilation of ER. etc.) seemed to converge in both series, the upper limit of serious ccl1 damagc~ (ruptnw of cell nwmbranes, loss of hyalopl~lsm, karyorrhcsis, cytolysis, etc. ) occurring at the early stage was noted mow fwqucntly in cells incubated in EC/, DMSO. During early stagc,s of incubation the tcmpcrature of 25” C, as compared with 4? C appeared to contribute more to the delctorious effect of DMSO. Howcvcr, the conccn t&ion factor seemed to predominate in the initial maiiifcstatioiis of DMSO toxicity. UISCUSSION
In interpreting results wportcd hcrc, it is necessary to consider the cffcct of DhlSO-induced lesions on cellular viability, and, following this, to establish appropriatc> corwlatcs bctnw~n morphological exprcssioii of DI4SO iiitcraction with against the bnckcellular components ground of some known physiological effects. It has been well documented that most cells tend to retain their viability after exposure to DMSO in the 7-G’& conccntration range (1-3, 6), particularly if the duration of exposure is rclativcly brief (1, 2, 6). Thus, there is no reason to assume that the cellular lesions described in this report, except in the cstrrmc cnscs such as karyorrhexis, are incompatible with cc:11survival and eventual repair. Howcvcr, owing to recurrent difficult& inherent in the attempts to define cellular viability in strictly ~norphoIogica1 terms (7), IN) structlual via-
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\lito~hontlrial tlut~g~ \xricd from the \w’y light to total ol~litcration of structiuxl or~aiiization of thcw Organc~llrs ant1 \\x in\xial,ly acwiripai~iul 1)). a sharp incrwsc in clc~position of intracc~llular lipids. O\ving to ;I coirsistc,lrt lability of mitochonclria tci DYISO. it scowls logical to sqgc\st that tlicl cc~lliilar c~lcctron transport chain ma!’ 1)~s tllasticall~~ affcckcl l)!; DhlSO. Apart froni niito~liolltlri~i. rough surfawtl l,ranq orgallc4c~s. and tlw llyaloplaslll. The alteration of structwal integrity of cwdoplawiic r~ticiiluni ( RElZ ) \vas still ~11 memlxaws by DMSO appears to lw anothcxr cytoplasmie c+on~pon~wtccnrsistcwtl~ of a particular significancr. IVhile thcl affinatktc~cl l))r D1ISO. I~c,grnnulatioll of HEK it!, of DMSO for ccl1 twn~1~raw.s WIS tcwls to siiggwt that, ilr tlicx prfwmc’c: of shown to exist under a \wicty of cspcriDIiSO, the ratcl of protein synthesis may mental conditions (5, 8, 9. 12). their struclx, altcwd and that a si,qnificant fraction tural lahilit)- to DMSO has not lwcn dcw~of cxtracc~llular lWLd is probably rc’prc’onstratcd prwiously. This findiq sccw~s to provide the structural basis for rc~portctl scntc~cl by the rihosomal RNA. Arcas of hyaloplasntic tlc~plc+oli found in niimuwiis cscapc~ of cclliilar nuckic acids ant1 proteins from ~11s incubated in DJISO SOIII- cxllls socwi to suggest that h~~iloplasmic ;1lx c~Ytractc~t1from ~11s lg, tions (4) aid swmingly iwccssitaks tlrc ro- Colllpo1Kwts appraisal of a widely h&l view that the D?rlSO. As notclcl in tllis stwly, scwningly traiisnic~iiil)r~iti~~ transport of D\ISO itwlf. progrcwi\-ct incwasc in l!w~somul and autoas \wlI as tlicx 1)~1SO-IIlc,tlintc,cl trawport plingic ac*ti\.ity iii tlw chills of tlw cywriinvariably OC‘CII~S inc2ital scric~s is in qgrcwiwiit of other co~npo~~ncls, \vith im across an intact ~11 cnvc~lopc~. cwlic$r rrport that DhlSO activatcls lysoIn contrast to cell nicw~l~riincs and menlSOmcs ifz oitw ( 10). Olwr~xtions by Perbranw of mitochondria, strwtllrally analsidskv (X3) that lysoson1cs may 1~ one of ogous damage of nuclear mcwrbrancs has the> primary targets of cr)winjiq~ in the JlOt bwn cstablishccl conclusivc~ly. This wlls frozen iii prcwnw of this c’ryoproobservation, hornever. does not riilc: ont tf~ct~lnt. and the finc1iiiji.s rc>portcd hcrc a prolxible labilit)- of nuclear nicwil,ranc~s sc’c’nl to iulplicatc D51SO as an important to DMSO. In fact, mitotic abnormalities factor ilk 1ys0s01nal activation. The lvidc drscrilwxl to occur in soiiif: cells following range of striicturnl nltcwtions indiicccl 1~ their cxposurc~ to DIISO (6) ant1 sul~scDMSO, (ww \vli~w this cqwprotc~ctant is qwntly propag:atccl as cultiiws, ;IS ~~11 ~1s uwd in “optimal” ~on~c~ntlatioiis aid fol the rclcaw of DNA into DSISO-contai~lil~g bric\f pclriotls only, Sf’(‘IllS to s11gpt that solutions ill qwintitics which cannot 11~iltthis c~oiiipo~~iid is toxiv to tlic irl uitw cc>11 tributcd solely to mitochondria aii(l lysc(l systcwls ant1 thcwforc~ cannot 1~ rc~garclcd dls (4), tfwd to support tlw notion thnt ai1 ;Ls iiinoc~iiot~s cqoprotfy$i\~~ ag:c~~rt. As shown ill this investigation, morphological mailifcstations of D1ISO cffcct \7wx~ charactcrizcd by the di\,cuity of affcctcd cc~lliilnr conipownts and hy their early onsrt. To facilitate discussion, thr fornwr may lw con~wiic~ntly groiipcd to iwluclc thrw broad CI;ISSCYof conq~oiic7rts, ix., lllf‘l11-
Cultured Rhesus kidnry ccxlls wore illcubatcd in the 7.5 and 15% solutions at 4 and 25” C and for periods ranging from 10 to 60 min. The ultrastructural alterations, while varying from ccl1 to cell, were evident even following the lo-min incubation interval. The most frequent and least scvcre structural changes included lipid accumulation, increased nurnbcr of lysosomes, dilation and clegranulation of rough surfaced endoplasmic reticulum, as well as the swelling of mitochondria and damage to mitochondrial membranes. The more severe instances of cellular lesions were rcprcsented by the extensive damage to ~11 membranes, karyorrhexis, total obliteration of the internal structure of mitochondria, and areas of complete loss of hyaloplasm. The extent and frequency of cellular lesions appeared to be dctcrmined primarily by the concentration of DMSO with the temperature and duration of incubat:on being important ancillary determinants. REFERENCES 1. Bouroncle, B. A. lreser\atiou of living cells ;lt -79” C lvith dilllethylsulfoaidr. Proc. Sof,. Exp. Bid. Med. 119, 953-961 (1965). 2. Bouroncle, B. A. Preservation of human normal and leukemic cells with dimethylsulfoxide at -80” C. Cryobiology 6, 445-455 (1967). 3. Brown, B. L., and Nagle, S. C., Jr. Preservation of mammalian cells in a chemically defined medium and dimethylsolfoxide. Scionce 149, 1266-1267 ( 1965 ).
5. hlalinin, G. I., F’ontana, D. J., and Brarmgart, D. C. Distribution of C” labeled dimethylsulfoxide in tissues of intact aninxlls. Cryobiology 5, 328-335 ( 1969). of 6. ?rlalinin, T. I., and Perry, V. P. Toxicity dimethylsulfoxide on IIeLa cells. Cryobiology 4, 90-96 ( 1967 ) 7. Malinin, T. I., and Perry, \‘. P. A review of tissne anld organ viability assay. Cryohiology 4, 104-115 ( 1967). 8. hlalinin, T. I., &tontes de Oca, H., Gollan, I;., and !blalinin, G. I. Cell membrane affinity of dimetl~ylsr~lfoxidc. Proc. Fifth Int’l Congrets of PIurnl., San Francisco ( 1972) (Alx.). 9. \\Ialinin, T. I. Preservation of isolated org;ms at hypothenllia ;md by freezing. PTOC. 13th lnt’l Congress of Hf~frigerution, Wdington, D. C. (1971) (In press). 10. Slisch, U. W., and hlisch, hi. S. Dimethylsulfoxide: activation of lysosomes in vitro. Proc. Nut. Acad. Sci. USA 58, 2462-2467 ( 1967). 11, Morgan, T. J., Morton, H. J., and Parker, R. C. Nutrition of animal cells in tissue cllltrlrc. I. Initial studies of synthetic medium. Proc. Sm. Exl~. Bid. Med. 73, l-8 (1950). 12. Olferijns, F. G. J., and Krijnen, II. W. ExperiInents in the prrservution of hexrts at snbzcrn tcw~prratllres. Cryobiology 6, ,589 ( 1970) (Ah.). 1:i. I’crsidsky, \I. 1). l,yso~om~s ils prinlilry targets of cryoilrjllry. Cr!/obio/og~/ 8, 482-488 (1971). 1.1. \‘os, O., and Kaalen, hl. C. A. C. Prevention of freezing damage to prol’fer;l:ing cells in tisslle culture. A qllnntitative stndy of a munber of agents. Cryobiology 2, 90-96 (1965). l,5. Wilmer, E. N. “Cells and tisslles in culture.” Vol. 1, p, 52, Academic Press, New York, 196ri.