Despite the current lack of information about the molecular
basis of CTN, a specific molecular example of CTN is available that
may be instructive in future studies. In particular, the inactivation
of membrane Na
+ , K
+
-ATPase by urea is blocked by Me 2 SO [319].
Although the combination of amides and Me 2 SO was originally
proposed to neutralize the toxic effects of Me 2 SO [295, 318], this
effect has not been observed subsequently [295, 320]. Interestingly,
however, recent studies indicate that 30% Me 2 SO can reduce the
thermal denaturation temperatures of lysozyme, but that 5% formamide, but not dimethylformamide, can reverse this effect [321],
results that mirror the protective interaction between Me 2 SO and
formamide and the lack of protective interaction between Me 2 SO
and DMF observed in kidney slice experiments [313]. Short of
blocking Me 2 SO toxicity directly, acetylcholinesterase inhibition
by Me 2 SO can be physiologically blocked by atropine [322],
some oxidative effects of Me 2 SO can be reversed with reducing
agents [312, 313], and there is one still unconfirmed report that
glucose can prevent irreversible binding of Me 2 SO to proteins and
also reduce its toxicity [283].
So far, few systems have been evaluated for their ability to
benefit from CTN. CTN is known to apply to rabbit renal cortical,
liver, and brain slices and to murine osteoblasts [248] and seems
very likely to pertain as well to rat liver slices [61], rat renal cortical
and medullary slices [323], and rat brain [247] slices, and amide
+Me 2 SO mixtures have also been successfully applied to other
systems ([19, 147], and unpublished results). The only tested
systems that so far do not seem to benefit from CTN are human
oocytes and early-stage embryos (S.F. Mullen, unpublished
results).
3.8 Mechanisms
of Cryoprotectant
Toxicity
Cryoprotectant toxicity is defined here as non-osmotic adverse
effects on cellular or tissue viability or functionality induced by
treatment of cells or tissues with cryoprotectants for the purpose
of enabling their cryopreservation. Cryoprotectant toxicity is the
central problem of vitrification, but it has received little relevant
attention from biochemists until very recently, with one notable
exception dating from 1971 that has led to our current knowledge
of cryoprotectant toxicity neutralization [318]. Criteria proposed
in 1990 [295] for showing the causal relevance of specific biochemical responses associated with observed cryoprotectant toxicity are
only now beginning to be applied.
Most examples of cryoprotectant metabolism in vivo or in vitro
at elevated temperatures, such as the phosphorylation of glycerol
leading to ATP depletion [307, 324] or the transformation of
ethylene glycol into toxic by-products [325, 326], have little to
no clear relevance to events at the lower temperatures at which high
concentrations of pCPAs are usually administered, or in tissues or
cells whose metabolic activities are different than those studied.
72
Gregory M. Fahy and Brian Wowk
basis of CTN, a specific molecular example of CTN is available that
may be instructive in future studies. In particular, the inactivation
of membrane Na
+ , K
+
-ATPase by urea is blocked by Me 2 SO [319].
Although the combination of amides and Me 2 SO was originally
proposed to neutralize the toxic effects of Me 2 SO [295, 318], this
effect has not been observed subsequently [295, 320]. Interestingly,
however, recent studies indicate that 30% Me 2 SO can reduce the
thermal denaturation temperatures of lysozyme, but that 5% formamide, but not dimethylformamide, can reverse this effect [321],
results that mirror the protective interaction between Me 2 SO and
formamide and the lack of protective interaction between Me 2 SO
and DMF observed in kidney slice experiments [313]. Short of
blocking Me 2 SO toxicity directly, acetylcholinesterase inhibition
by Me 2 SO can be physiologically blocked by atropine [322],
some oxidative effects of Me 2 SO can be reversed with reducing
agents [312, 313], and there is one still unconfirmed report that
glucose can prevent irreversible binding of Me 2 SO to proteins and
also reduce its toxicity [283].
So far, few systems have been evaluated for their ability to
benefit from CTN. CTN is known to apply to rabbit renal cortical,
liver, and brain slices and to murine osteoblasts [248] and seems
very likely to pertain as well to rat liver slices [61], rat renal cortical
and medullary slices [323], and rat brain [247] slices, and amide
+Me 2 SO mixtures have also been successfully applied to other
systems ([19, 147], and unpublished results). The only tested
systems that so far do not seem to benefit from CTN are human
oocytes and early-stage embryos (S.F. Mullen, unpublished
results).
3.8 Mechanisms
of Cryoprotectant
Toxicity
Cryoprotectant toxicity is defined here as non-osmotic adverse
effects on cellular or tissue viability or functionality induced by
treatment of cells or tissues with cryoprotectants for the purpose
of enabling their cryopreservation. Cryoprotectant toxicity is the
central problem of vitrification, but it has received little relevant
attention from biochemists until very recently, with one notable
exception dating from 1971 that has led to our current knowledge
of cryoprotectant toxicity neutralization [318]. Criteria proposed
in 1990 [295] for showing the causal relevance of specific biochemical responses associated with observed cryoprotectant toxicity are
only now beginning to be applied.
Most examples of cryoprotectant metabolism in vivo or in vitro
at elevated temperatures, such as the phosphorylation of glycerol
leading to ATP depletion [307, 324] or the transformation of
ethylene glycol into toxic by-products [325, 326], have little to
no clear relevance to events at the lower temperatures at which high
concentrations of pCPAs are usually administered, or in tissues or
cells whose metabolic activities are different than those studied.
72
Gregory M. Fahy and Brian Wowk