C-33: Origins of cryoprotectant toxicity

Abstract

The nature of cryoprotectant toxicity has received little attention. Literature studies are mostly confined to in vivo toxicity data that have little to no relevance to events at low temperatures. Cryoprotectant toxicity in the frozen state suggests a rapid mechanism for toxicity that overwhelms the generally protective effects of lower temperatures. Previous evidence argued against generalized protein denaturation as a mechanism of toxicity because (a) toxicity is observed at lower concentrations than those needed to denature proteins, (b) protein destabilization tendency was associated with less toxicity, not more, and (c) preferential exclusion of cryoprotectants from the protein surface implied protein stabilization at lower temperatures. Newer data, however, have reopened the possibility that toxicity may be related to the denaturation of specific proteins. A microarray study showed that vitrification solution exposure increased heat shock protein gene expression, increased transcription of genes in the ERK and JNK pathways, and induced 8 genes associated with ribosome biogenesis, all suggesting a reaction to protein denaturation. The correlation between toxicity in organized tissue and qv ∗ is also compatible with protein denaturation as the primary mechanism of “non-specific” toxicity. Although individual cryoprotectants may be used at sub-denaturing concentrations in vitrification solutions, their cumulative effect could become denaturing, especially if cryoprotectant-mediated destabilization summates with cold destabilization. Pertinent to this possibility, an additional microarray study suggests that chilling injury may be largely related to protein denaturation as well, which raises the question as to whether chilling injury in systems prepared for vitrification may in large part be induced by the presence of the vitrification solution. If so, chilling injury may be considered a toxic effect of cryoprotectants at deep subzero temperatures, consistent with the manifestation of such toxic effects in frozen-thawed systems.