though each individual pCPA in the VS mixture may be at a
nominally non-denaturing concentration, the sum total of all of
the pCPAs might still have a cumulative denaturing effect, especially when cold destabilization of proteins is also factored in, and
some evidence indicates that pCPAs do not protect effectively
against cold denaturation [340]. There are now, in fact, two general
lines of evidence for the possibility that high-concentration VSs
might induce biologically relevant protein denaturation under
low-temperature exposure conditions.
The first line of evidence is indirect and arises from the finding
that the toxicity of a large number of VSs can be correlated strongly
with qv∗ (discussed above) [245]. The evidence indicates that
pCPAs that interact strongly with water [341], such as
1,2-propanediol, are more toxic precisely because they interact
strongly with water, presumably competing with biological molecules for access to water, whereas more weakly bonding solutes,
such as ethylene glycol, are required in higher concentrations to
vitrify water because of their weaker interaction with water, yet
leave the water that remains more free to hydrate biomolecules
despite a lower overall concentration of water in the solution
[245]. It is unclear whether this mechanism of non-specific toxicity
arises as a result of protein denaturation or some other waterdependent effect(s), but it is compatible with a role for protein
denaturation in the manifestation of toxicity.
The second line of evidence is more direct and is based in part
on microarray analysis by Guan et al. of alterations in transcription
following exposure of rat liver slices to two candidate (8.8 and 8.9
molar) VSs [61]. It was published as part of an analysis of chilling
injury (see next section), and so full results were not provided, but
the results that were presented are illuminating.
Although neither VS reduced slice ATP, 1985 transcripts were
changed, of which 92 increased at least 1.5-fold and 49 decreased at
least 1.5-fold. The VSs increased transcripts for 11 heat shock
genes, and 1 particular Hsp70 family member, Hspa1b, was elevated 12.6-fold, although transcription of Dnajc12 (an Hsp40
homolog) was slightly decreased. Also consistent with the loss of
some proteins to denaturation, eight genes associated with ribosome biogenesis were induced as well, especially the 5S rRNA gene,
whose transcripts increased 4.5-fold. VS exposure decreased transcription for genes in the p38 signaling pathway about 25% and
depressed TGFβ-1 and TGFβ-3 transcripts, which normally lead to
stimulation of the p38 pathway. In contrast, the VSs greatly
increased transcription related to the ERK and especially to the
JNK pathway, a third finding consistent with the possibility of
protein denaturation. (Though not clearly related to denaturation,
it was also of note that expression of Hmox-1 [heme oxygenase
(decycling) 1], which functions primarily as a major defense against
oxidative stress and injury [342], was decreased by 1.9-fold.)
74
Gregory M. Fahy and Brian Wowk
nominally non-denaturing concentration, the sum total of all of
the pCPAs might still have a cumulative denaturing effect, especially when cold destabilization of proteins is also factored in, and
some evidence indicates that pCPAs do not protect effectively
against cold denaturation [340]. There are now, in fact, two general
lines of evidence for the possibility that high-concentration VSs
might induce biologically relevant protein denaturation under
low-temperature exposure conditions.
The first line of evidence is indirect and arises from the finding
that the toxicity of a large number of VSs can be correlated strongly
with qv∗ (discussed above) [245]. The evidence indicates that
pCPAs that interact strongly with water [341], such as
1,2-propanediol, are more toxic precisely because they interact
strongly with water, presumably competing with biological molecules for access to water, whereas more weakly bonding solutes,
such as ethylene glycol, are required in higher concentrations to
vitrify water because of their weaker interaction with water, yet
leave the water that remains more free to hydrate biomolecules
despite a lower overall concentration of water in the solution
[245]. It is unclear whether this mechanism of non-specific toxicity
arises as a result of protein denaturation or some other waterdependent effect(s), but it is compatible with a role for protein
denaturation in the manifestation of toxicity.
The second line of evidence is more direct and is based in part
on microarray analysis by Guan et al. of alterations in transcription
following exposure of rat liver slices to two candidate (8.8 and 8.9
molar) VSs [61]. It was published as part of an analysis of chilling
injury (see next section), and so full results were not provided, but
the results that were presented are illuminating.
Although neither VS reduced slice ATP, 1985 transcripts were
changed, of which 92 increased at least 1.5-fold and 49 decreased at
least 1.5-fold. The VSs increased transcripts for 11 heat shock
genes, and 1 particular Hsp70 family member, Hspa1b, was elevated 12.6-fold, although transcription of Dnajc12 (an Hsp40
homolog) was slightly decreased. Also consistent with the loss of
some proteins to denaturation, eight genes associated with ribosome biogenesis were induced as well, especially the 5S rRNA gene,
whose transcripts increased 4.5-fold. VS exposure decreased transcription for genes in the p38 signaling pathway about 25% and
depressed TGFβ-1 and TGFβ-3 transcripts, which normally lead to
stimulation of the p38 pathway. In contrast, the VSs greatly
increased transcription related to the ERK and especially to the
JNK pathway, a third finding consistent with the possibility of
protein denaturation. (Though not clearly related to denaturation,
it was also of note that expression of Hmox-1 [heme oxygenase
(decycling) 1], which functions primarily as a major defense against
oxidative stress and injury [342], was decreased by 1.9-fold.)
74
Gregory M. Fahy and Brian Wowk