Transcriptional profiling studies of this kind can identify
changes associated with cryoprotectant toxicity and thus provide
valuable clues to the origins of observed toxic effects, but it is
difficult to disentangle cause and effect from such results. For
example, although HSP expression is triggered by unfolded proteins, this could be an epiphenomenon not directly relevant to the
main cause of cellular injury. This issue was recently investigated by
Ghousifam et al. in human renal epithelial cells exposed to 10%
Me 2 SO or 10–90% M22 at 37 or at 4
C [301]. Exposure to 10%
Me 2 SO and to 10 or 30% M22 (6.5% or 19.4% w/v cryoprotectants) did not lower cellular viability after 1 h at 37
C and, at this
temperature, 10% Me 2 SO and 10% M22 increased expression of
HSP27, and 30% M22 elevated expression of HSP47, HSP60, and
HSP70. Intriguingly, when exposure was at 4
C, HSP70 and
HSP27 induction was lessened in the presence of 10% Me 2 SO
(suggesting traditional protection by lower temperatures) but augmented in the presence of 30% M22 (suggesting a possible contribution of cold denaturation) and particularly in the presence of 90%
M22. Most significantly, pretreatment of the cells with geranylgeranylacetone (GGA, or teprenone), a drug that induces heat shock
proteins [343, 344], protected the cells against exposure to 90%
M22 when combined with ATP pretreatment to compensate for
ATP depletion presumably caused by the ATP-dependent protein
refolding process (viability with GGA + ATP greater than viability
with either of these two interventions alone). These preliminary
studies may open up new avenues for both understanding and
mitigating cryoprotectant toxicity.
It is possible that the proteins requiring protection against
cryoprotectant-induced destabilization or denaturation are a small
subset of the total [227]. Cryoprotectants decrease the solubility of
tyrosine, leucine, alanine, cystine, and glycine [227, 332], which
should have a generally inhibitory effect on denaturation, although
20% Me 2 SO increases the solubility of tryptophan by about 40%
[332]. Different proteins respond differently to cryoprotectants
[306, 345], suggesting that the most susceptible proteins may be
selectively involved in toxic responses. More globally, and dramatically, closely related tissues as a whole also react differently to
cryoprotectants despite the fact that the same basic “housekeeping”
enzymes are essentially common to all cells. For example, guinea
pig uteri [346] and intestinal smooth muscle [120] can tolerate the
levels of Me 2 SO required for metastable cooling to dry ice temperature without freezing, whereas rabbit renal cortex cannot
[347]. More strikingly, frog hearts [116, 117], but not rat hearts
[118], can tolerate 10–11 M ethylene glycol.
Recently, an entirely new way of probing and correcting cryoprotectant toxicity has been introduced [302]. By screening a
library of 12,000 different transposon-mutated embryonic stem
cells (ESCs) for their ability to resist M22 toxicity, Cypser et al.
were able to isolate single gene mutants that could withstand M22
Principles of Vitrification
75
changes associated with cryoprotectant toxicity and thus provide
valuable clues to the origins of observed toxic effects, but it is
difficult to disentangle cause and effect from such results. For
example, although HSP expression is triggered by unfolded proteins, this could be an epiphenomenon not directly relevant to the
main cause of cellular injury. This issue was recently investigated by
Ghousifam et al. in human renal epithelial cells exposed to 10%
Me 2 SO or 10–90% M22 at 37 or at 4
C [301]. Exposure to 10%
Me 2 SO and to 10 or 30% M22 (6.5% or 19.4% w/v cryoprotectants) did not lower cellular viability after 1 h at 37
C and, at this
temperature, 10% Me 2 SO and 10% M22 increased expression of
HSP27, and 30% M22 elevated expression of HSP47, HSP60, and
HSP70. Intriguingly, when exposure was at 4
C, HSP70 and
HSP27 induction was lessened in the presence of 10% Me 2 SO
(suggesting traditional protection by lower temperatures) but augmented in the presence of 30% M22 (suggesting a possible contribution of cold denaturation) and particularly in the presence of 90%
M22. Most significantly, pretreatment of the cells with geranylgeranylacetone (GGA, or teprenone), a drug that induces heat shock
proteins [343, 344], protected the cells against exposure to 90%
M22 when combined with ATP pretreatment to compensate for
ATP depletion presumably caused by the ATP-dependent protein
refolding process (viability with GGA + ATP greater than viability
with either of these two interventions alone). These preliminary
studies may open up new avenues for both understanding and
mitigating cryoprotectant toxicity.
It is possible that the proteins requiring protection against
cryoprotectant-induced destabilization or denaturation are a small
subset of the total [227]. Cryoprotectants decrease the solubility of
tyrosine, leucine, alanine, cystine, and glycine [227, 332], which
should have a generally inhibitory effect on denaturation, although
20% Me 2 SO increases the solubility of tryptophan by about 40%
[332]. Different proteins respond differently to cryoprotectants
[306, 345], suggesting that the most susceptible proteins may be
selectively involved in toxic responses. More globally, and dramatically, closely related tissues as a whole also react differently to
cryoprotectants despite the fact that the same basic “housekeeping”
enzymes are essentially common to all cells. For example, guinea
pig uteri [346] and intestinal smooth muscle [120] can tolerate the
levels of Me 2 SO required for metastable cooling to dry ice temperature without freezing, whereas rabbit renal cortex cannot
[347]. More strikingly, frog hearts [116, 117], but not rat hearts
[118], can tolerate 10–11 M ethylene glycol.
Recently, an entirely new way of probing and correcting cryoprotectant toxicity has been introduced [302]. By screening a
library of 12,000 different transposon-mutated embryonic stem
cells (ESCs) for their ability to resist M22 toxicity, Cypser et al.
were able to isolate single gene mutants that could withstand M22
Principles of Vitrification
75