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Interactions between cryoprotectants and cryosensitizers
ABSTRACTS,
26th ANNUAL
pressed to low temperatures by that concentration. Thus, when polymers are used as cryoprotective agents, cell survival is contingent upon maintenance of osmotic stress within narrow limits. (Supported by NIH Grant GM 17959.) 6. Cryoprotectant Toxicity and Ctyoprotectant Toxicity Reduction: In Search of Molecular Mechanisms. G. M. FAHY, T. H. LILLEY,* H.
M. ST. JOHN DOUGLAS, AND H. T. (Transplantation Laboratory, American Red Cross, Rockville, Maryland; and *University of Sheffield, Sheffield, United Kingdom). LINSDELL,* MERYMAN
Cryoprotectant toxicity is a primary problem of applied cryobiology since it limits cryoprotection and therefore survival after freezing and thawing. One line of research on mechanisms of cryoprotectant toxicity has been derived from the observation in 1971 that dimethyl sulfoxide (D) toxicity in rat renal cortex may derive largely from an interaction between D and protein lysine residues. Compounds such as lysine, urea, formamide, and acetamide prevented changes in enzyme activity. Both the 1971 study and the follow-up studies using rabbit renal cortex indicate that there is specificity of the toxicity blocking effect, but the mechanism of blockade has remained conjectural. Recent experiments have failed to demonstrate consistent reduction of toxicity when blocking compounds are added to rather than substituted for D and have also failed to support the idea that toxicity closely relates to the genera1 protein stabilizing or destabilizing properties of the mixed solvent medium. The implication of the original study was that blocking compounds associate with D so as to prevent its interaction with lysine. We have recently studied the interactions between D and the blocking compounds in aqueous solution using calorimetric techniques. We find that, contrary to expectation, the reaction between D and lysine in aqueous solution is thermochemically repulsive (endothermic), but the more endothermic the reaction between D and the other blocking agents, the less effective the agents are at reducing toxicity. Furthermore, when lysine is added to a mixture of D and formamide, the enthalpy of its interaction with D is not affected by formamide. It would seem that current concepts of cryoprotectant toxicity are in need of major revision. (Supported by NIH Grant GM 17959.) 7. Interactions between Ctyoprotectants and Cryosensitizers. J. KRLNJV, D. J. GLOFCHESKI, AND
(University of Waterloo, Waterloo, Ontario, Canada).
J. R. LEWCK
When cryoprotected (CP) or unprotected mammalian cells are exposed to multiple freeze-thaw (FT) cycles, the survival response is exponential, even if
MEETING
537
combinations of CP agents are used. This enables one to calculate interaction, independence, or synergism between different CP (and/or cryosensitizers) agents. However, theoretical and practical considerations restrict the accuracy and interpretation of these experiments to a limited range of survivals. For example, low concentrations of CPs act independently until the concentration is increased so that competition for “FT targets” occurs. While the standard CP agents, DMSO, propylene glycol (PG), HES, and glycerol interact with each other, glutamine acts independently. If cryosensitizers (CS) are used, some difference between standard CPs are observed. For example, DMSO and PG are better protectants than HES against FT sensitization by protein denaturants like ethanol or guanidine HCi. Some of the CSs (ethanol, A23187, BHT, zinc sulfate) interact with some of the CPs, while others (guanidine HCI, DTNB, europium) act independently with some CPs. These interactions, or lack of interactions, may help us determine how various CPs protect the many different targets of FT damage. PLENARY
SESSION
I
8. Freeze Tolerance in Animals: Variations on the Theme in Nature and Applications for Cryopreservation. KENNETH B. STOREY (Institute of
Biochemistry & Department of Biology, Carleton University, Ottawa, Canada KlS SB6). Natural freeze tolerance occurs in diverse animal groups including insects, marine molluscs and crustatea, and several terrestrially hibernating amphibians and reptiles. Certain basic principles for freezing survival, such as a limitation of about 65% of total body water as ice, are apparently followed in all groups but increasingly we find variation in the adaptive strategies employed by different animal groups. For example, the function of freeze tolerance in garter snakes is questionable. Snakes readily survive short exposures (
26th ANNUAL
pressed to low temperatures by that concentration. Thus, when polymers are used as cryoprotective agents, cell survival is contingent upon maintenance of osmotic stress within narrow limits. (Supported by NIH Grant GM 17959.) 6. Cryoprotectant Toxicity and Ctyoprotectant Toxicity Reduction: In Search of Molecular Mechanisms. G. M. FAHY, T. H. LILLEY,* H.
M. ST. JOHN DOUGLAS, AND H. T. (Transplantation Laboratory, American Red Cross, Rockville, Maryland; and *University of Sheffield, Sheffield, United Kingdom). LINSDELL,* MERYMAN
Cryoprotectant toxicity is a primary problem of applied cryobiology since it limits cryoprotection and therefore survival after freezing and thawing. One line of research on mechanisms of cryoprotectant toxicity has been derived from the observation in 1971 that dimethyl sulfoxide (D) toxicity in rat renal cortex may derive largely from an interaction between D and protein lysine residues. Compounds such as lysine, urea, formamide, and acetamide prevented changes in enzyme activity. Both the 1971 study and the follow-up studies using rabbit renal cortex indicate that there is specificity of the toxicity blocking effect, but the mechanism of blockade has remained conjectural. Recent experiments have failed to demonstrate consistent reduction of toxicity when blocking compounds are added to rather than substituted for D and have also failed to support the idea that toxicity closely relates to the genera1 protein stabilizing or destabilizing properties of the mixed solvent medium. The implication of the original study was that blocking compounds associate with D so as to prevent its interaction with lysine. We have recently studied the interactions between D and the blocking compounds in aqueous solution using calorimetric techniques. We find that, contrary to expectation, the reaction between D and lysine in aqueous solution is thermochemically repulsive (endothermic), but the more endothermic the reaction between D and the other blocking agents, the less effective the agents are at reducing toxicity. Furthermore, when lysine is added to a mixture of D and formamide, the enthalpy of its interaction with D is not affected by formamide. It would seem that current concepts of cryoprotectant toxicity are in need of major revision. (Supported by NIH Grant GM 17959.) 7. Interactions between Ctyoprotectants and Cryosensitizers. J. KRLNJV, D. J. GLOFCHESKI, AND
(University of Waterloo, Waterloo, Ontario, Canada).
J. R. LEWCK
When cryoprotected (CP) or unprotected mammalian cells are exposed to multiple freeze-thaw (FT) cycles, the survival response is exponential, even if
MEETING
537
combinations of CP agents are used. This enables one to calculate interaction, independence, or synergism between different CP (and/or cryosensitizers) agents. However, theoretical and practical considerations restrict the accuracy and interpretation of these experiments to a limited range of survivals. For example, low concentrations of CPs act independently until the concentration is increased so that competition for “FT targets” occurs. While the standard CP agents, DMSO, propylene glycol (PG), HES, and glycerol interact with each other, glutamine acts independently. If cryosensitizers (CS) are used, some difference between standard CPs are observed. For example, DMSO and PG are better protectants than HES against FT sensitization by protein denaturants like ethanol or guanidine HCi. Some of the CSs (ethanol, A23187, BHT, zinc sulfate) interact with some of the CPs, while others (guanidine HCI, DTNB, europium) act independently with some CPs. These interactions, or lack of interactions, may help us determine how various CPs protect the many different targets of FT damage. PLENARY
SESSION
I
8. Freeze Tolerance in Animals: Variations on the Theme in Nature and Applications for Cryopreservation. KENNETH B. STOREY (Institute of
Biochemistry & Department of Biology, Carleton University, Ottawa, Canada KlS SB6). Natural freeze tolerance occurs in diverse animal groups including insects, marine molluscs and crustatea, and several terrestrially hibernating amphibians and reptiles. Certain basic principles for freezing survival, such as a limitation of about 65% of total body water as ice, are apparently followed in all groups but increasingly we find variation in the adaptive strategies employed by different animal groups. For example, the function of freeze tolerance in garter snakes is questionable. Snakes readily survive short exposures (