X-Message-Number: 30338
Date: Sun, 20 Jan 2008 23:58:15 -0800 (PST)
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Subject: The lowest toxicity cryoprotectant is... (part 2/2)

Cryobiology. 2004 Jun;48(3):283-94.
Thermal properties of ethylene glycol aqueous solutions.
  Baudot A, Odagescu V. CRTBT, CNRS, BP 166, 38042 Grenoble, Cedex 9,
France.
  Preventing ice crystallization by transforming liquids into an amorphous
state, vitrification can be considered as the most suitable technique
allowing complex tissues, and organs cryopreservation. This process requires
the use of rapid cooling rates in the presence of cryoprotective solutions
highly concentrated in antifreeze compounds, such as polyalcohols. Many of
them have already been intensively studied. Their glass forming tendency and
the stability of their amorphous state would make vitrification a reality if
their biological toxicity did not reduce their usable concentrations
often below the concentrations necessary to vitrify organs under achievable
thermal conditions. Fortunately, it has been shown that mixtures of
cryoprotectants tend to reduce the global toxicity of cryoprotective
solutions and various efficient combinations have been proposed containing
ethanediol.
This work reports on the thermal properties of aqueous solutions with 40,
43, 45, 48, and 50% (w/w) of this compound measured by differential scanning
calorimetry. The glass forming tendency and the stability of the amorphous
state are evaluated as a function of concentration. They are given by the
critical cooling rates v(ccr)above which ice crystallization is avoided, and
the critical warming rates v(cwr) necessary to prevent ice crystallization
in the supercooled liquid state during rewarming. Those critical rates are
calculated using the same semi-empirical model as previously. This work
shows a strong decrease of averaged critical cooling and warming rates when
ethanediol concentration increases, V(ccr) and V(cwr) = 1.08 x 10 (10) K/min
for 40% (w/w) whereas V(ccr) = 11 and V(cwr) = 853 K/min for 50% (w/w).
Those results are compared with the corresponding properties of other
dialcohols obtained by the same method. Ethylene glycol efficiency is
between those of 1,2-propanediol and 1,3-propanediol.
PMID: 15157777

Cryo Letters. 2006 Nov-Dec;27(6):341-52.
Cryopreservation of the late stage embryos of Spodoptera exigua
(Lepidoptera: Noctuidae).
  Luo L, Pang Y, Chen Q, Li G. State Key Laboratory of Biocontrol and
Institute of Entomology, Sun Yat-sen University, Guangzhou, Guangdong,
China.
  Genetic devolution, genetic drift and contamination are all threats to
maintain germplasm stability during mass rearing of many insects.
Cryopreservation of beet armworm (Spodoptera exigua) embryos was studied to
provide information to improve mass rearing. A series of experiments was
conducted on
late-stage embryos (45-48 h at 27 degree C) of the beet armyworm, which
included evaluation of cryoprotectants (CPAs), their toxicity and
glass-forming tendency and optimization of experimental procedures. The
results showed that ethylene glycol (EG) was the best CPA with comparatively
low toxicity
compared to the other six CPAs tested (methanol, 1,3-propanediol, glycerol,
2-amino-1-ethanol, 3-amino-1-propanol 3-methoxy-1 and 2-propanediol). The
highest hatching rate of 8.8 degree was attained after freezing with a
3-step loading procedure and a 1-step unloading procedure, but the hatched
larvae from frozen-thawed embryos did not actively feed and could not
develop to a later stage. This was attributed to injuries from freezing in
late stage embryos of S. exigua which had formed midguts.
PMID: 17256068

Inhal Toxicol. 2005 Aug;17(9):487-93.
Inhalation toxicity of 1,3-propanediol in the rat.
  Scott RS, Frame SR, Ross PE, Loveless SE, Kennedy GL. DuPont de Nemours
and Company, Haskell Laboratory for Health and Environmental Sciences,
Newark, Delaware 19714, USA.
  1,3-Propanediol (504-63-2) was studied to determine the potential effects
following repeated inhalation exposures to rats. Rats were exposed 6 hr/day,
5 days/wk for 2 wk (9 exposures) to vapor or vapor/aerosol mixtures of
either 0, 41, 650, or 1800 mg 1,3-propanediol/m(3). In vivo responses were
observed or measured daily. Clinical pathology and tissue pathology analyses
were conducted after the 9th exposure and on half of each group following an
18-day recovery (nonexposure) period. All rats showed normal body weights.
No unusual external signs of response were seen, and no deaths were
encountered. Clinical pathology (blood counts, serum chemical parameters)
and tissue pathology (gross pathology, organ weights, and histopathology)
examinations in the 1,3-propanediol exposed rats were similar to those in
the unexposed controls. The highest concentration tested, 1800 mg/m(3),
which
was the highest concentration that could practically be generated, was the
no-observed-effect level (NOEL) for this study. 1,3-Propanediol does not
appear to pose a significant hazard via inhalation of either the vapor or a
vapor/aerosol mixture.
PMID: 16020043

J Am Chem Soc. 2004 Nov 10;126(44):14330-1.
The effect of molecular crowding with nucleotide length and cosolute
structure on DNA duplex stability.
  Nakano S, Karimata H, Ohmichi T, Kawakami J, Sugimoto N. Frontier
Institute for Biomolecular Engineering Research (FIBER), and Department of
Chemistry, Faculty of Science and Engineering, Konan University, 8-9-1
Okamoto, Higashinada-ku, Kobe 658-8501, Japan.
  The thermodynamics of DNA duplex structures in the presence of high
concentrations of cosolutes in solution were investigated to discern nucleic
acid structures and functions in living cells. In the presence of ethylene
glycol (EG) and poly(ethylene glycol) (PEG) (MW = 200-8000), the stability
of
the oligomer DNA duplexes with differing nucleotide length varied, depending
on the nucleotide length as well as the size of PEG. It was also revealed
that the decrease of water activity is the primary factor for
destabilization of the short (8-mer) duplex by addition of high molecular
weight PEGs
as well as low molecular weight PEGs and other low molecular weight
cosolutes. In addition, the number of water molecules taken up per base pair
formation was the same for all the PEGs and for 1,2-dimethoxyethane, which
was greater than in the cases of glycerol, EG, 1,3-propanediol, and
2-methoxyethanol, suggesting that the solvation of nucleotides may differ,
depending on the cosolute structure. These findings are useful not only for
understanding nucleic acid structures and functions in cells but also for
the design of oligonucleotides applicable for cells, such as antisense
nucleic acids, RNAi, and DNA chips.
PMID: 15521733

Cryobiology. 2004 Jun;48(3):283-94.
Thermal properties of ethylene glycol aqueous solutions.
  Baudot A, Odagescu V. CRTBT, CNRS, BP 166, 38042 Grenoble, Cedex 9,
France.
  Preventing ice crystallization by transforming liquids into an amorphous
state, vitrification can be considered as the most suitable technique
allowing complex tissues, and organs cryopreservation. This process requires
the use of rapid cooling rates in the presence of cryoprotective solutions
highly concentrated in antifreeze compounds, such as polyalcohols. Many of
them have already been intensively studied. Their glass forming tendency and
the stability of their amorphous state would make vitrification a reality if
their biological toxicity did not reduce their usable concentrations
often below the concentrations necessary to vitrify organs under achievable
thermal conditions. Fortunately, it has been shown that mixtures of
cryoprotectants tend to reduce the global toxicity of cryoprotective
solutions and various efficient combinations have been proposed containing
ethanediol.
This work reports on the thermal properties of aqueous solutions with 40,
43, 45, 48, and 50% (w/w) of this compound measured by differential scanning
calorimetry. The glass forming tendency and the stability of the amorphous
state are evaluated as a function of concentration. They are given by the
critical cooling rates v(ccr)above which ice crystallization is avoided, and
the critical warming rates v(cwr) necessary to prevent ice crystallization
in the supercooled liquid state during rewarming. Those critical rates are
calculated using the same semi-empirical model as previously. This work
shows a strong decrease of averaged critical cooling and warming rates when
ethanediol concentration increases, V(ccr) and V(cwr) = 1.08 x 10 (10) K/min
for 40% (w/w) whereas V(ccr) = 11 and V(cwr) = 853 K/min for 50% (w/w).
Those results are compared with the corresponding properties of other
dialcohols obtained by the same method. Ethylene glycol efficiency is
between those of 1,2-propanediol and 1,3-propanediol.
PMID: 15157777

Cryobiology. 2004 Feb;48(1):22-35.
Erratum in:
Cryobiology. 2004 Jun;48(3):365.
Cryobiology. 2004 Sep 1;56(4):845.
Improved vitrification solutions based on the predictability of
vitrification solution toxicity.
  Fahy GM, Wowk B, Wu J, Paynter S. 21st Century Medicine, Inc., 10844
Edison Court, Rancho Cucamonga, CA 91730, USA.
  Long-term preservation of complex engineered tissues and organs at
cryogenic temperatures in the absence of ice has been prevented to date by
the difficulty of discovering combinations of cryoprotectants that are both
sufficiently non-toxic and sufficiently stable to allow viability to be
maintained and ice formation to be avoided during slow cooling to the glass
transition temperature and subsequent slow rewarming. A new theory of the
origin of non-specific cryoprotectant toxicity was shown to account, in a
rabbit renal cortical slice model, for the toxicities of 20 vitrification
solutions and to permit the design of new solutions that are dramatically
less toxic than previously known solutions for diverse biological systems.
Unfertilized mouse ova vitrified with one of the new solutions were
successfully fertilized and regained 80% of the absolute control (untreated)
rate
of development to blastocysts, whereas ova vitrified in VSDP, the best
previous solution, developed to blastocysts at a rate only 30% of that of
controls. Whole rabbit kidneys perfused at -3 degrees C with another new
solution at a concentration of cryoprotectant (8.4M) that was previously
100%
lethal at this temperature exhibited no damage after transplantation and
immediate contralateral nephrectomy. It appears that cryoprotectant
solutions that are composed to be at the minimum concentrations needed for
vitrification at moderate cooling rates are toxic in direct proportion to
the
average strength of water hydrogen bonding by the polar groups on the
permeating cryoprotectants in the solution. Vitrification solutions that are
based on minimal perturbation of intracellular water appear to be superior
and provide new hope that the successful vitrification of natural organs as
well as tissue engineered or clonally produced organ and tissue replacements
can be achieved.
PMID: 14969679

Cryobiology. 2000 Mar;40(2):151-8.
Glass-forming tendency in the system water-dimethyl sulfoxide.
  Baudot A, Alger L, Boutron P. Centre de Recherches sur les Tr s Basses
Temp ratures, C.N.R.S., Grenoble Cedex 9, 38042, France.
  The glass-forming tendency on cooling and the stability of the wholly
amorphous state on warming have been previously reported for many
cryoprotective solutions. However, unlike the other solutions, those of
dimethyl sulfoxide (Me(2)SO) have not been studied on cooling. In this
paper, the
glass-forming tendency of Me(2)SO aqueous solutions has been measured for
solutions containing 40, 43, 45, and 47.5% (w/w) Me(2)SO. At a concentration
of 45% (w/w), the glass-forming tendency decreases in the following order:
levo-2, 3-butanediol, 1,3-butanediol, 1,2-propanediol, 1,2,3-butanetriol,
dimethyl sulfoxide, dimethylformamide, diethylformamide, 1, 4-butanediol,
ethylene glycol, glycerol, 1,3-propanediol. New measurements have also been
made on warming the Me(2)SO solutions. Copyright 2000 Academic Press.
PMID: 10788314

Biochem Med Metab Biol. 1991 Apr;45(2):161-70.
The effect of propane-diols on the intestinal uptake of nutrients and brush
border membrane enzymes in the rat.
  Morshed KM, Desjeux JF, Nagpaul JP, Majumdar S, Amma MK. Department of
Biochemistry, Panjab University, Chandigarh, India.
  The effect on rats of oral doses (38.66 mM/kg body wt) of propane-1,2-diol
(PD) administered daily for 10 (Group 1), 20 (Group 2), and 30 days (Group
3) was investigated. Weight gain was initially retarded (P less than 0.05)
in Group 1, but was later reversed and elevated significantly (P less
than 0.05) in Groups 2 and 3 as compared with their respective controls
receiving an equal volume of saline. PD showed a tendency toward enhancing
the activities of various enzymes involved in terminal digestion, with the
significant effect exerted in few groups on sucrase (P less than 0.05),
lactase (P less than 0.05), and gamma-glutamyl transpeptidase (P less than
0.05) when compared with the respective controls. Absorption of D-glucose,
glycine, L-aspartic acid, L-lysine, and calcium was elevated and was
especially significant in Groups 2 and 3 (P less than 0.001). The structural
integrity of the jejunal surface was retained for the most part. A similar
examination of the effects of PD was also carried out in vitro to ascertain
whether PD itself or its metabolites are involved in its action. The in
vitro effects of propane-1,2-diol were compared with those of the more toxic
compound propane-1,3-diol. The former exerted greater inhibitory action on
the activities of the disaccharidases. The degree of inhibition was in the
order sucrase much greater than lactase greater than maltase. The kinetic
data revealed that inhibition by 1,2-diol in native and detergent
solubilized sucrase is noncompetitive, with Ki values in the range of
0.35-0.41 M. The two diols did not alter the nutrient transport in the brush
border membrane vesicles. The present work on rats indicates that PD may
influence the intestinal digestive and absorptive functions in vivo and that
this in vivo effect of PD is different from that observed in vitro
suggesting that the nutritional and toxicological effect of PD may be
mediated by different mechanisms.
PMID: 1883624

Hum Reprod. 1989 Jan;4(1):77-9.
Successful embryo transfer of cryopreserved and in-vitro fertilized rabbit
oocytes.
  al-Hasani S, Kirsch J, Diedrich K, Blanke S, van der Ven H, Krebs D.
Universit ts-Frauenklinik, Bonn, FRG.
  In-vitro fertilization experiments with frozen/thawed rabbit oocytes were
performed to develop an effective technique to be used for the in-vitro
fertilization of cryopreserved human oocytes. Ovulatory oocytes, collected
from the oviduct of virgin does 13 h after induction of ovulation by HCG
injection, were cryopreserved slowly to -30 degrees C and plunged directly
into liquid nitrogen. A mixture of 1.5 M 1,3-propanediol and 0.1 M sucrose
was used as a cryoprotectant. After thawing, the oocytes were incubated with
in-vitro capacitated sperm for 5 h in defined Brackett's medium.
Fertilized ova were cultured for an additional 20 h until the 4-to-8-cell
stage was reached. These embryos were transferred to pseudopregnant
recipient rabbits which were 'asynchronous' in the sense that they had been
given an injection of HCG 30, 24 and 18 h before starting to do the embryo
transfer. A 32% survival rate of frozen/thawed oocytes was achieved. The
fertilization rate was 74% (181/264) in this study. A total of 53 embryos
was transferred to the oviducts of six recipients of three different
asynchronicity and four young were born. The highest implantation rate
(including
resorptions) of 18% could be achieved in this investigation by using -6 h
asynchronous recipients, while the overall implantation rate was 9.4%.
PMID: 2708506

Chem Biol Interact. 1984 Jun;50(1):87-96.
Cross-linking of DNA in liver and testes of rats fed 1,3-propanediol.
  Summerfield FW, Tappel AL.
1,3-Propanediol (PAD) was fed to rats for 15 weeks, and its effects on
hepatic and testicular DNA were studied. The control rats were fed a
casein-based diet that contained 10% tocopherol-stripped corn oil with 30 IU
of d,l-alpha-tocopherol acetate/kg; the experimental rats were fed the same
diet
with 500 ppm of PAD. Homogenates prepared from the livers of each group of
rats converted 1,3-propanediol to malondialdehyde (MDA) with equal efficacy,
but homogenates of testes did not catalyze this conversion. After 10-15
weeks of feeding the diets, the hepatic DNA of the rats fed PAD had less
template activity, more bound tryptophan and more DNA-protein and
interstrand DNA cross-links than that of the control rats. As measured by
template activity and bound tryptophan, testicular DNA of the experimental
rats was not different from that of the control rats; however, there was
slightly
more cross-linking in the testicular DNA of experimental rats than in that
of control rats. Testes of the experimental rats contained more
lipid-soluble fluorophores than did those of the control rats. The results
are consistent with the conclusion that PAD was converted to MDA in vivo and
that MDA
is the reactive species that caused the observed biological damage.
PMID: 6733805

J Reprod Fertil. 1980 Sep;60(1):247-52.
Cryoprotective effects of some amides on rabbit spermatozoa.
  Hanada A, Nagase H.
Semen was diluted 1:9 with egg yolk-citrate medium containing 0.31--3.1 M
(final concentration) formamide, butyramide, acetamide, propionamide,
dimethylformamide, lactamide, malomide, ethylene glycol, trimethylene
glycol, dimethylsulphoxide (DMSO) or glycerol. After 30 min incubation at 20
degrees
C, sperm motility was superior in hypertonic solutions of acetamide,
lactamide, dimethylsulphoxide, trimethylene glycol and ethylene glycol. Some
of these compounds were added to semen diluted 1:2 in an isotonic
egg-yolk-glucose-lactose-raffinose solution and frozen by the pellet method.
Relatively
good survival of motility was obtained in 1.0 M-DMSO, -lactamide
or -acetamide. Dimethylformamide (0.5 M), ethylene glycol (0.5--1.5 M),
trimethylene glycol (1.5 M) and propionamide (0.75 M) also gave some
protection. Insemination of does with semen frozen and thawed with 1.0
M-DMSO, -lactamide or
acetamide gave fertilization rates of 68--88%, and 84% (38/45) of does gave
birth to an average of 5.3 young.
PMID: 7431324

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