X-Message-Number: 30297
Date: Fri, 11 Jan 2008 19:41:42 -0800 (PST)
From:
Subject: calcium ion homeostasis may mediate some cryoprotectant toxic...
[Here's another attempt at elucidating cryoprotectant toxicity. Below is a
table which quickly summarizes the relative contribution of four different
types of toxicity. I assume vitrification solutions are gradually ramped up,
so that of the intracellular cryoprotectants only glycerol would pose a
significant osmotic risk. I've subdivided intrinsic toxicity into
denaturing, and calcium poisoning. These two types of toxicity appear to be
associated with each other, possibily because release of intracellular
calcium from sarcoplasmic reticulum stores is aided by some denaturation of
membrane proteins. I wonder how great would be the reduction in toxicity if
an intracellular calcium chelator like BAPTA were to be added to low calcium
vitrification solutions?
Although some cryoprotectants show a very high extrinsic toxicity related
to the production of toxic metabolites, low perfusion temperatures would
reduce tissue metabolism, and the short time of application of
cryoprotectants yields some doubt about the robustness of any extrinsic
contributions to toxicity. Only cryopreservation experiments using pyrazole,
and disulfiram to complately block metabolism could measure the contribution
of extrinsic toxicity. Surprisingly such experiments appear to never have
been published.
This is not the last word on cryoprotectant toxicity. I'm not convinced
that anybody has produced a convincing full explanation of this phenomina.
For example, one question which remains largely unanswered is the nature of
glycerol toxicity.]
Types of Toxicity
Cryoprotectant Osmotic Denaturing Calcium poisoning Extrinsic
DMSO low high high low
Ethylene glycol low low high medium
Glycerol high none none none
Propylene glycol medium high high low
Methanol none medium high medium
[Below the toxicity of ethylene glycol (EG) is convincingly demonstrated to
be due almost entirely to calcium poisoning of cells. With all cells dead
within two minutes upon exposure to 40% EG, with no blocking extracellular
solutes, no other conclusion is possible. There was simply not enough time
for extrinsic toxicity to be a significant factor. Note that extracellular
solutes cause dehydration of cells, and reduce uptake of both ethylene
glycol and calcium. A calcium free vitrification solution reduces toxicity
due to reduced calcium uptake by cells, so that 67% of cells are still
intact at 2 minutes. Finally the further addition of an intracellular
calcium chelator called BAPTA to a calcium free solution reduces toxicity so
that 100% of cells were still intact at 3 minutes. By comparison the
addition of extracellular solutes alone yielded a 3 minutes survival of 83%.
The addition of all three methods to reduce toxicity (calcium free, BAPTA, &
extracellular solutes) was not attempted, but I suspect would have had a
synergistic effect.]
Mol Reprod Dev. 2004 Jun;68(2):250-8.
Lowering intracellular and extracellular calcium contents prevents cytotoxic
effects of ethylene glycol-based vitrification solution in unfertilized
mouse oocytes.
Takahashi T, Igarashi H, Doshida M, Takahashi K, Nakahara K, Tezuka N,
Kurachi H. Department of Obstetrics and Gynecology, Yamagata University
School of Medicine, Yamagata 990-9585, Japan.
We investigated the characteristics of the changes in intracellular
calcium (Ca2+) concentration ([Ca2+](i)) and the viability of the
unfertilized mouse oocytes exposed to various concentrations of ethylene
glycol (EG)-containing solutions or vitrification solutions. Oocytes exposed
to EG (1, 5, 10, 20, and 40% (v/v)) exhibited a rapid and dose-dependent
increase in [Ca2+](i). The survival rate was 100% when oocytes were exposed
to the EG concentration up to 5% through 5 min, while all oocytes were dead
within 3 min when exposed to 10, 20, or 40% EG. When extracellular Ca2+ was
removed, increase in [Ca2+](i) at 10 and 20% EG was less than that at the
same concentrations of EG with extracellular Ca2+. The survival rates of the
oocytes exposed to 10, 20, and 40% EG at 3 min were 100, 97, and 0%,
respectively. In the presence of 20 microM
1,2-bis(o-aminopheoxy)ethane-N,N,N',N'-tetraacetic acid tetra acetoxymethyl
ester (BAPTA-AM), a Ca2+ chelator, a small increase in [Ca2+](i) exposed to
10, 20, and 40% EG was observed until 4 min. Subsequently prolonged
elevation of the [Ca2+](i) was observed in the oocytes exposed to 40% EG but
not with 10 and 20% EG. The survival rate of the oocytes, in the presence of
20 microM BAPTA-AM, exposed to 10 and 20% EG was 100% throughout 5 min,
while the oocytes exposed to 40% EG were alive only for 3 min. Treatment by
the vitrification solution with various concentrations of EG (10, 20, and
40%) caused a smaller increase in [Ca2+](i), while the survival rates were
higher compared to those without vitrification solution at the same
concentrations of EG. These data suggested that the sustained [Ca2+](i)
rises by EG in unfertilized mouse oocytes resulted in cell death. Therefore,
the lowering of [Ca2+](i) in the oocytes exposed to the cryoprotectant may
improve the viability of cryopreserved unfertilized oocytes. Copyright 2004
Wiley-Liss, Inc.
PMID: 15095347
[Snip: "dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large
transient increase in intracellular calcium concentration".]
Reproduction. 2006 Jan;131(1):53-61.
Calcium-free vitrification reduces cryoprotectant-induced zona pellucida
hardening and increases fertilization rates in mouse oocytes.
Larman MG, Sheehan CB, Gardner DK. Colorado Center for Reproductive
Medicine, 799 East Hampden Avenue, Suite 520, Englewood, 80113, USA.
Despite the success of embryo cyropreservation, routine oocyte freezing
has proved elusive with only around 200 children born since the first
reported birth in 1986. The reason for the poor efficiency is unclear, but
evidence of zona pellucida hardening following oocyte freezing indicates
that current protocols affect oocyte physiology. Here we report that two
cryoprotectants commonly used in vitrification procedures, dimethyl
sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in
intracellular calcium concentration in mouse metaphase II (MII) oocytes
comparable to the initial increase triggered at fertilization. Removal of
extracellular calcium from the medium failed to affect the response exacted
by DMSO challenge, but significantly reduced the ethylene glycol-induced
calcium increase. These results suggest that the source of the DMSO-induced
calcium increase is solely from the internal calcium pool, as opposed to
ethylene glycol that causes an influx of calcium across the plasma membrane
from the external medium. By carrying out vitrification in calcium-free
media, it was found that zona hardening is significantly reduced and
subsequent fertilization and development to the two-cell stage significantly
increased. Furthermore, such calcium-free treatment appears not to affect
the embryo adversely, as shown by development rates to the blastocyst stage
and cell number/allocation. Since zona hardening is one of the early
activation events normally triggered by the sperm-induced calcium increases
observed at fertilization, it is possible that other processes are
negatively affected by the calcium rise caused by cryoprotectants used
during oocyte freezing, which might explain the current poor efficiency of
this technique.
PMID: 16388009
[Snip: "propanediol, ethylene glycol and DMSO, all independently result in
an increase in calcium".]
Theriogenology. 2007 Jan 1;67(1):64-72. Epub 2006 Oct 17.
Analysis of oocyte physiology to improve cryopreservation procedures.
Gardner DK, Sheehan CB, Rienzi L, Katz-Jaffe M, Larman MG. Colorado Center
for Reproductive Medicine, Englewood, CO 80113, USA.
In contrast to the preimplantation mammalian embryo, it has been
notoriously difficult to cryopreserve the metaphase II oocyte. The ability
to store oocytes successfully at -196 degrees C has numerous practical and
financial advantages, together with ethical considerations, and will
positively impact animal breeding programs and assisted conception in the
human. Differences in membrane permeability and in physiology are two main
reasons why successful oocyte cryopreservation has remained elusive. It is
proposed, therefore, that rather than relying on technologies already
established for the preimplantation embryo, the development of
cryopreservation techniques suitable for the mammalian oocyte needs to take
into account the idiosyncratic physiology of this cell. Analysis of
intracellular calcium, for example, has revealed that exposure to
conventional permeating cryoprotectants, such as propanediol, ethylene
glycol and DMSO, all independently result in an increase in calcium, which
in turn has the potential to initiate oocyte activation, culminating in zona
hardening. Quantification of the metabolome and proteome of the oocyte has
revealed that whereas slow freezing has a dramatic effect on cell
physiology, vitrification appears to have limited effect. This is plausibly
achieved by the limited exposure to cryoprotectants. Analysis of meiotic
spindle dynamics and embryo development following IVF, also indicate that
vitrification is less traumatic than slow freezing, and therefore has the
greatest potential for successful oocyte cryopreservation.
PMID: 17049589
[Snip: "PrOH caused a protracted increase in calcium, which was
sufficient to induce zona pellucida hardening and cellular degeneration.
Using 'nominally calcium free' media during PrOH exposure significantly
reduced the detrimental effects".]
Hum Reprod. 2007 Jan;22(1):250-9. Epub 2006 Aug 12.
1,2-propanediol and the type of cryopreservation procedure adversely affect
mouse oocyte physiology.
Larman MG, Katz-Jaffe MG, Sheehan CB, Gardner DK. Colorado Center for
Reproductive Medicine, Englewood, CO 80133, USA.
BACKGROUND: The aim of this work was to examine the effect of
1,2-propanediol (PrOH) and type of cryopreservation procedure (slow freezing
and vitrification) on oocyte physiology. METHODS: Intracellular calcium of
mouse metaphase II (MII) oocytes was quantified by fluorescence microscopy.
The effect of PrOH on cell physiology was further assessed through analysis
of zona pellucida hardening and cellular integrity. Protein profiles of
cryopreserved oocytes were generated by time-of-flight mass spectrometry
(TOF-MS). RESULTS: PrOH caused a protracted increase in calcium, which was
sufficient to induce zona pellucida hardening and cellular degeneration.
Using 'nominally calcium free' media during PrOH exposure significantly
reduced the detrimental effects. Proteomic analysis identified numerous up-
and down-regulated proteins after slow freezing when compared with control
and vitrified oocytes. CONCLUSIONS: Using such approaches to assess effects
on cellular physiology is fundamental to improving assisted reproduction
techniques (ART). This study demonstrates that PrOH causes a significant
rise in intracellular calcium. Using calcium-free media significantly
reduced the increase in calcium and the associated detrimental physiological
effects, suggesting that calcium-free media should be used with PrOH. In
addition, analysis of the oocyte proteome following cryopreservation
revealed that slow freezing has a significant effect on protein expression.
In contrast, vitrification had a minimal impact, indicating that it has a
fundamental advantage for the cryopreservation of oocytes.
PMID: 16905767
[Glycerol does not elevate intracellular calcium. The nature of glycerol
toxicity is still unknown.]
Br J Nutr. 2004 Nov;92(5):751-5.
Various non-digestible saccharides increase intracellular calcium ion
concentration in rat small-intestinal enterocytes.
Suzuki T, Hara H. Division of Applied Bioscience, Graduate School of
Agriculture, Hokkaido University, Japan.
We have previously shown that non-digestible saccharides (NDS) stimulate
intestinal Ca absorption via tight junctions. However, the cellular
mechanisms activated by the NDS are not yet known. We investigated the
effects of four NDS, difructose anhydride (DFA) III, DFAIV,
fructo-oligosaccharides, and maltitol, on intracellular Ca signalling in
isolated rat small-intestinal enterocytes. The changes in intracellular
Ca(2+) concentration were measured before and after the addition of capric
acid (7.5 or 15 mmol/l, a positive control), glycerol, or each NDS (1 or 10
mmol/l) to fura-2-loaded enterocytes. Treatment with capric acid or each NDS
caused an immediate and dose-dependent rise in intracellular Ca(2+)
concentration. Mechanical and osmotic stimulation achieved by adding
glycerol had no effect on intracellular Ca(2+) concentration. The
intracellular Ca(2+) concentration in enterocytes treated with DFAIII and
fructo-oligosaccharides reached a peak level at about 30 s after
stimulation, but those treated with DFAIV and maltitol showed further
increases after the initial rapid rise. The maximum change in intracellular
Ca(2+) concentration obtained by the application of maltitol was higher than
that of DFAIII at 10 mmol/l. These findings suggest that each of the four
NDS directly stimulates rat enterocytes, and increases intracellular Ca(2+)
concentration. Thus, molecular structure may be more important than the size
of the NDS in the induction of Ca signalling in the cells.
PMID: 15533262
[Snip: "intracellular release of Ca2+ is required for methanol-induced
contractions" and curiously enough "Methanol-induced contractions were,
however, completely abolished by pretreatment of tissue with 10 mM
caffeine".]
Toxicol Appl Pharmacol. 1998 Jun;150(2):361-8.
Methanol-induced contraction of canine cerebral artery and its possible
mechanism of action.
Li W, Altura BT, Altura BM. Department of Physiology, State University of
New York, Brooklyn, USA.
In the present report, we investigated the effects of methanol on canine
basilar cerebral arterial rings. Our data indicate that acute methanol
exposure (5-675 mM) induces potent contractile responses of cerebral
arteries in a concentration-dependent manner. Pharmacological antagonists,
such as propranolol, phentolamine, haloperidol, methysergide, naloxone,
diphenhydramine, and cimetidine, did not exert any effects on these
methanol-induced contractions. Likewise, a potent antagonist of
cyclo-oxygenase, and subsequent synthesis of prostanoids (i.e.,
indomethacin), failed to exert any effect on methanol-induced contractions.
No differences in responsiveness to methanol in canine cerebral arteries
were found in vessel segments with or without endothelial cells. Removal of
extracellular Ca2+ ([Ca2+]o) partially attenuated methanol-induced
contractions, while withdrawal of extracellular Mg2+ ([Mg2+]o) potentiated
the contractions. In the complete absence of [Ca2+]o, 10 mM caffeine and 400
mM methanol induced similar, transient contractions followed by relaxation
in K(+)-depolarized cerebral vascular tissues. Methanol-induced contractions
were, however, completely abolished by pretreatment of tissue with 10 mM
caffeine. Our results indicate that (1) methanol causes contractile
responses of cerebral arterial smooth muscle (independent of amine,
prostanoid, or opioid mediation; (2) in addition to a need for [Ca2+]o, an
intracellular release of Ca2+ is required for methanol-induced contractions;
and (3) Mg deficiency potentiates the contractile responses of methanol on
these brain vessels. The data presented in the study suggest that
methanol-induced contractions occur via an sarcoplasmic reticulum-releasable
store of [Ca2+]i; via mediation of either ryanodine-caffeine type receptors
or a caffeine-releasable intracellular store of CA2+.
PMID: 9653067
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