Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms.
Cryoprotectant toxicity is a fundamental obstacle to the full potential of artificial cryoprotection, yet it remains in general a poorly understood phenomenon. Unfortunately, most relevant biochemical studies to date have not met the basic criteria required for demonstrating mechanisms of toxicity. A model biochemical study of cryoprotectant toxicity was that of Baxter and Lathe, which demonstrated that alteration of a specific enzyme (fructose diphosphatase, or FDPase) was the cause of impaired glycolysis after treatment with and removal of dimethyl sulfoxide (D). FDPase alteration by D was reported to be preventable by the simultaneous presence of amides. This protection could be due to a "counteracting solute" effect similar to that employed by nature, but we find no meaningful correlation between the general protein stabilizing or destabilizing tendency of the cryoprotectant medium and its toxicity. Baxter and Lathe postulated that the effect of D arises from hydrogen bonding between D and the epsilon amino groups of surface lysine residues on FDPase, and it was found that molecules which resembled this group could block the alteration induced by D, presumably by competing with lysine residues for association with D. However, we find that the interaction between D and lysine in the presence of water is actually thermochemically repulsive, and that the presence of formamide does not affect the interaction between D and lysine, implying no useful complex formation between formamide and D. We were also unable to demonstrate that the blocking compounds consistently reduce toxicity when added to D rather than substituting for D, contrary to predictions based on complex formation between blocking compounds and D. In summary, it seems that present concepts of cryoprotectant toxicity are in need of serious revision.
American Red Cross, Jerome Holland Laboratory for the Biomedical Sciences, Transplantation Laboratory, Rockville, Maryland 20855.
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Fahy
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Lilley
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Linsdell
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Douglas
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Meryman
D000577:Amides / Q000494:pharmacology; D000818:Animals; D003451:Cryoprotective Agents / Q000494:pharmacology / Q000633:toxicity*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology / Q000633:toxicity; D004347:Drug Interactions; D006801:Humans; D011489:Protein Denaturation; D011506:Proteins / Q000378:metabolism
D000577:Amides; D003451:Cryoprotective Agents; D011506:Proteins; D004121:Dimethyl Sulfoxide