Me 2 SO, whose toxic effects tend to be overstated, has numerous
pharmacological effects at body temperature [327] and can chemically react with tissue sulfhydryl groups [328], but understandably
has no demonstrated pharmacological effects at 0
C, at which
temperature its reaction with sulfhydryl groups may be too slow
to be meaningful for cellular viability [248, 312]. It has been shown
to react with steroids and triterpenoids [329], but never to do so
under biologically meaningful conditions. As summarized elsewhere [295] (references given therein), pCPAs have been shown
to elevate membrane phase transition temperatures; rearrange the
cytoskeleton, including most significantly the meiotic spindle;
cause membrane blistering; fuse cell membranes; change gene
expression; alter RNA polymerase; weaken DNA-nucleosome binding; destabilize nucleic acid duplexes; impair ribosome assembly;
and induce many other adverse changes, but for the most part, the
relevance of these observed changes, if any, to most cells being
prepared for cryopreservation is currently unknown. There seems
to be little or no generalized effect of vitrification solutions on
passive membrane permeability to sodium and potassium under
practical conditions [227]. Some pCPAs can induce differentiation
of leukemia cells [330] and can change their chromatin and DNA
conformation [331], but these are not what would normally be
considered toxic effects. In summary, it seems that the literature on
the biochemical effects of cryoprotectants and the literature on the
toxic effects of CPAs in cryobiological applications are mostly
disconnected from one another.
A proposed protein-altering mechanism of Me 2 SO toxicity
involving specific interaction between Me 2 SO and protein surface
lysine residues [318] has not been supported by subsequent investigations [295, 312] and is not favored by the general observation
that small molecules, including Me 2 SO [332], tend to be preferentially excluded from the hydration layer surrounding protein surfaces, thus stabilizing them against denaturation [316, 333–335]
and even enhancing renaturation after previous denaturation
[336, 337]. Mixtures of pCPAs in vitrification solutions intended
for use at high hydrostatic pressures did show increased protein
destabilization tendencies, but these effects were correlated
inversely with toxicity [295]. In addition, the toxic effects of individual commonly used pCPAs do not appear to be accounted for by
their general protein denaturation tendency [295, 313, 332, 338,
339] or by their ability to increase the permeability of membranes
when used below 15
C [320], and Arakawa et al. have argued that
disruption of the hydration layer surrounding proteins and membranes by pCPAs may account for pCPA toxicity at high temperatures but not at low temperatures [339].
Nevertheless, protein denaturation has not been ruled out in
ambient pressure vitrification solutions composed of mixtures of
pCPAs, which is the most common type of VS in use today. Even
Principles of Vitrification
73
pharmacological effects at body temperature [327] and can chemically react with tissue sulfhydryl groups [328], but understandably
has no demonstrated pharmacological effects at 0
C, at which
temperature its reaction with sulfhydryl groups may be too slow
to be meaningful for cellular viability [248, 312]. It has been shown
to react with steroids and triterpenoids [329], but never to do so
under biologically meaningful conditions. As summarized elsewhere [295] (references given therein), pCPAs have been shown
to elevate membrane phase transition temperatures; rearrange the
cytoskeleton, including most significantly the meiotic spindle;
cause membrane blistering; fuse cell membranes; change gene
expression; alter RNA polymerase; weaken DNA-nucleosome binding; destabilize nucleic acid duplexes; impair ribosome assembly;
and induce many other adverse changes, but for the most part, the
relevance of these observed changes, if any, to most cells being
prepared for cryopreservation is currently unknown. There seems
to be little or no generalized effect of vitrification solutions on
passive membrane permeability to sodium and potassium under
practical conditions [227]. Some pCPAs can induce differentiation
of leukemia cells [330] and can change their chromatin and DNA
conformation [331], but these are not what would normally be
considered toxic effects. In summary, it seems that the literature on
the biochemical effects of cryoprotectants and the literature on the
toxic effects of CPAs in cryobiological applications are mostly
disconnected from one another.
A proposed protein-altering mechanism of Me 2 SO toxicity
involving specific interaction between Me 2 SO and protein surface
lysine residues [318] has not been supported by subsequent investigations [295, 312] and is not favored by the general observation
that small molecules, including Me 2 SO [332], tend to be preferentially excluded from the hydration layer surrounding protein surfaces, thus stabilizing them against denaturation [316, 333–335]
and even enhancing renaturation after previous denaturation
[336, 337]. Mixtures of pCPAs in vitrification solutions intended
for use at high hydrostatic pressures did show increased protein
destabilization tendencies, but these effects were correlated
inversely with toxicity [295]. In addition, the toxic effects of individual commonly used pCPAs do not appear to be accounted for by
their general protein denaturation tendency [295, 313, 332, 338,
339] or by their ability to increase the permeability of membranes
when used below 15
C [320], and Arakawa et al. have argued that
disruption of the hydration layer surrounding proteins and membranes by pCPAs may account for pCPA toxicity at high temperatures but not at low temperatures [339].
Nevertheless, protein denaturation has not been ruled out in
ambient pressure vitrification solutions composed of mixtures of
pCPAs, which is the most common type of VS in use today. Even
Principles of Vitrification
73