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57. The effects of glycerol and dimethyl sulfoxide on the metabolism of novikoff hepatoma ascites cells
ABSTRACTS served successfully up to 48 hr at, 2°C amd 3 0 H P I~tll: ctlnno~, tolerate pressures above 3 . 0 0 H P , as a tteste(t b.v severe I)ulmonary dqmage after homoIransl)lantation. This sharl:) contrast indicates differem'es in toleran('e of whole org'ms to hyl)ertmric oxygen ~luring Iwlmthermic storage, and m a y relate to 0-, Ioxicity wl~ich is determined by &tration, intensity, and individual organ susceptil~ilily. 55. E n z y m e l t i s t o c t l e m i s t r y of Tissues F r o z e n Slowly with Protective A(hlitives. :~' P. J. MELXlCK aND I)AUI.INE HEIZEII* (Del:)artment of Pathology, University of Californi~t Medical Cent(,r, San Francisco, California, and Laboratory Service, Veterans Administration Hospital, Martinez, California). Previous studies of the effect of freezing rates on tlm his|ochemical demonstration of enzyme activity in tissues showed that tile full range of a('tivitv (.ouh| be seen in rapidly frozen tissues, wlwreas in aliquot blocks fi'ozen slowly, frequently no enzyme activity or only very feeble activity was observed. ThirW more tissues have since been similarly studied, together with additional aliquot blocks pretreated with dimethyl sulfoxide, glycerin, or polyvinylt)yrrolidone. After p r e l r e a t m e n t with prolective additives, histochemieal enzyme activity in the slowly frozen blocks wg~s e,lU:d Io llmt in the rapidly fi'ozen blocks. Dimethyl sulfoxide gave the best results, yiel(ling brilliant, intensely stained, and sharply localized pictures; it also intensified the results in the ral)idly frozen blocks. The mechanisn~ for this l)henomenon is not cle'w, b u t it would appear that the i)rotective ad(litive prcvenls lhe masking of active sites of enzymes during slow fi'eezing. 56. T h e Kinetics of Glycerol and D i m e t h y l S u ! f o x i d e M o v e m e n t in and out of Cells (luring P r e i n c u b a t i o n and Washing.:" I. ~,V. D. HrxDm~SO.,," (McGill University Surgical Clinic, Montre'd General Hospital, Montreal, Canada). The kinetics of glycerol and dimethyl sulfoxide ( D M S O ) m o v e m e n t into cells during incubation prior 1o freezing and m o v e m e n t out of cells during wasliing after freezing has been investigated by the m e a s u r e m e n t of changes in volume of Novikoff h e p a l o m a ascites cells. Glycerol or D M S 0 was added to a cell suspension in T C M 199 to give a final concentration of 10%. T h e changes in cell volmne were estimated in graduated centrifuge tubes after centrifugation at 1200 × g for 1 rain. '~'Supported by U. S. Public Health Service Research Grant CA-07468 and a Veterans Administration research appropriation. "~'Sul)ported b y grants from the National Cancer Inslitute of Canada and T h e Cancer Research Society, Inc., of Montreal.
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Similarly volmne changes were measured after addition of T C M 199 to cells previoizsly equilibrated with glycerol or D M S O . At room t.emperalure (22°C), addition of glycerol caused an initial shr-inkage of cells to 40% of original v o l u m e ; this shrinkage gradually decre'lsed, attaining equilibrimn after 1 hr. D M S O hnd reached equilibrium after 2 rain, when the first m e a s u r e m e n t was made. At; 4°C both compoun(ls have caused 60% shrinkage after 2 rain. With D M S O the cell volume returned 1o the original after 15 rain b u t with glycerol the. cell volmne was still only 60% of original after 1 hr. During the washing phase the same time sequence was observed for disappearanee of the initial swelling (100%) to original volume for both compound~. In view of the toxic effects of glycerol and D M S O on cell met.abolisni, the much faster m o v e m e n t of D M S O even at low temperature indicates that it is a better preservative during freezing of tissues. 57. The Effects of Glycerol and D i m e t h y l Sulfo x i d e on the M e t a b o l i s m o f Novikoff Hepatoma Ascites CellsF" I. J. BmKUS (McGill lYniversity Surgical Clinic, Montreal Gener'd Hospital, Montreal, C a n a d a ) . Suspensions of Novikoff h e p a t o m a ascite~ ~:::~lls were incubated in W a r b u r g manometric ve::~,As, with and without glycerol or dimethyl sulfoxide ( D M S O ) , for 1 hr at 37°C, and the effects on respiration, glycolysis, :rod radioactive tracer incorporation }nt o various cell fractions were measu'ee(l. It. was found that D M S O is more toxic t h a n glycerol. At 10% concenlration D M S O was without effect and glycerol inhibited free amino acid ~q~take by 20%, but both substances inhibited amino acid incorporation into protein from 60% (glyeine-l-C") to 90% (leucine-l-C*4). At the same concentration D M S O inhibited adenine-8-C" incorporation in tile acid-soluble fraction b y 4b-/¢, and into ribo- and deoxyribonucleic acid ( R N A and D N A ) by 70%, while g~?:i.::,~rol was without effect on the acid-soluble fract~tm and R N A , but inhibited D N A by 40%. Both compounds only slightly inhibited respiration and stimulated aerobic glycolysis. A mixture of 5 % glycerol and 5% D M S O had intermediate effe('ts. The percentage effects remained essentially t h e same when the a m o u n t of cells was doubled or tripled. This infornmtion will be lielpful in selecting conditions for optimal preservation of frozen tissues. 58. Rate o f . U p t a k e of Glycerol by H y p o t h e r m i c Rabbit K i d n e y s d u r i n g Perfusion. "~ DELFOaD L. STmK~L ( D e p a r t m e n t of Surgery, Duke Uni"e Supported by grants from The National Cancel" Institute of Canada and T h e Cancer R e s e a r c h Societ.y, Inc., of Montreal. :~'Supported by Grant GM05385 from the U. S. thlblic H e a l t h Service.
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Similarly volmne changes were measured after addition of T C M 199 to cells previoizsly equilibrated with glycerol or D M S O . At room t.emperalure (22°C), addition of glycerol caused an initial shr-inkage of cells to 40% of original v o l u m e ; this shrinkage gradually decre'lsed, attaining equilibrimn after 1 hr. D M S O hnd reached equilibrium after 2 rain, when the first m e a s u r e m e n t was made. At; 4°C both compoun(ls have caused 60% shrinkage after 2 rain. With D M S O the cell volume returned 1o the original after 15 rain b u t with glycerol the. cell volmne was still only 60% of original after 1 hr. During the washing phase the same time sequence was observed for disappearanee of the initial swelling (100%) to original volume for both compound~. In view of the toxic effects of glycerol and D M S O on cell met.abolisni, the much faster m o v e m e n t of D M S O even at low temperature indicates that it is a better preservative during freezing of tissues. 57. The Effects of Glycerol and D i m e t h y l Sulfo x i d e on the M e t a b o l i s m o f Novikoff Hepatoma Ascites CellsF" I. J. BmKUS (McGill lYniversity Surgical Clinic, Montreal Gener'd Hospital, Montreal, C a n a d a ) . Suspensions of Novikoff h e p a t o m a ascite~ ~:::~lls were incubated in W a r b u r g manometric ve::~,As, with and without glycerol or dimethyl sulfoxide ( D M S O ) , for 1 hr at 37°C, and the effects on respiration, glycolysis, :rod radioactive tracer incorporation }nt o various cell fractions were measu'ee(l. It. was found that D M S O is more toxic t h a n glycerol. At 10% concenlration D M S O was without effect and glycerol inhibited free amino acid ~q~take by 20%, but both substances inhibited amino acid incorporation into protein from 60% (glyeine-l-C") to 90% (leucine-l-C*4). At the same concentration D M S O inhibited adenine-8-C" incorporation in tile acid-soluble fraction b y 4b-/¢, and into ribo- and deoxyribonucleic acid ( R N A and D N A ) by 70%, while g~?:i.::,~rol was without effect on the acid-soluble fract~tm and R N A , but inhibited D N A by 40%. Both compounds only slightly inhibited respiration and stimulated aerobic glycolysis. A mixture of 5 % glycerol and 5% D M S O had intermediate effe('ts. The percentage effects remained essentially t h e same when the a m o u n t of cells was doubled or tripled. This infornmtion will be lielpful in selecting conditions for optimal preservation of frozen tissues. 58. Rate o f . U p t a k e of Glycerol by H y p o t h e r m i c Rabbit K i d n e y s d u r i n g Perfusion. "~ DELFOaD L. STmK~L ( D e p a r t m e n t of Surgery, Duke Uni"e Supported by grants from The National Cancel" Institute of Canada and T h e Cancer R e s e a r c h Societ.y, Inc., of Montreal. :~'Supported by Grant GM05385 from the U. S. thlblic H e a l t h Service.