X-Message-Number: 30107
Date: Thu, 6 Dec 2007 09:45:56 -0800 (PST)
Subject: types of cryoprotectant toxicity part 2

[Very good results have been obtained with concentrated ethylene glycol
solutions, provided the period of exposure is limited.]

Theriogenology. 1998 Nov;50(7):1001-13.
Effect of cryoprotectants and their concentration on post-thaw survival and
development of expanded mouse blastocysts frozen by a simple rapid-freezing
  Nowshari MA, Brem G. Interuniversit res Forschungsinstitut f r
Agrarbiotechnologie, Tulln, Austria.
  Experiments were conducted to develop a simple rapid-freezing protocol for
expanded mouse blastocyst-stage embryos. The effect of type of
cryoprotectant (ethylene glycol and propylene glycol) and its concentrations
(4.5, 6.0 and 7.0 mol/L each with 0.5 mol/L sucrose) on morphological
survival and development in vitro were studied. The survival and development
of embryos frozen with best concentration of each cryoprotectant pre-exposed
to either a low concentration (1.5 mol/L with 0.25 mol/L sucrose) of the
respective cryoprotectant or ascending concentrations of sucrose were also
compared. The in vivo development of embryos frozen with best protocol
(pre-exposure to 1.5 mol followed by 7.0 mol ethylene glycol) was compared
with nonfrozen embryos. The rate of re-expansion and hatching was influenced
by the type and concentration of the cryoprotectant. A significantly higher
re-expansion and hatching rate was achieved at 7.0 mol of both
cryoprotectants compared with 4.5 and 6.0 mol of the respective
cryoprotectants. When comparing 2 cryoprotectants, a higher (P < 0.05) rate
of hatching was obtained with ethylene glycol at 7.0 mol compared with a
similar concentration of propylene glycol. The highest re-expansion (91%)
and hatching (86%) of expanded blastocysts was achieved with pre-exposure of
embryos to a low concentration of ethylene glycol followed by freezing in
the same cryoprotectant at 7.0 mol. The transfer of embryos frozen using
this protocol resulted in the development of live fetuses. The proportion of
live fetuses in the pregnant recipients with frozen-thawed embryos were not
different from those transferred nonfrozen embryos (49 vs 57%). It may be
concluded that simple rapid-freezing with dehydration in ascending sucrose
concentrations or pre-equilibration in a low concentration of ethylene
glycol or propylene glycol followed by exposure to the respective
cryoprotectant at 7.0 mol resulted in high survival and development of
expanded blastocysts. Ethylene glycol at 7.0 mol with pre-equilibration is,
however, most effective for cryopreservation of this stage in the mouse.
PMID: 10734419

[Prompt treatment by alcohol dehydrogenase inhibitors such as fomepizole or
ethanol dramatically reduce ethylene glycol toxicity.]

Pharmacotherapy. 2002 Mar;22(3):365-72.
Role of fomepizole in the management of ethylene glycol toxicity.
  Druteika DP, Zed PJ, Ensom MH. Division of Clinical Pharmacy, Faculty of
Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
  OBJECTIVE: To systematically review English-language articles on
administration in patients with ethylene glycol poisoning. DATA SOURCES:
MEDLINE, EMBASE, Current Contents, and PubMed. Search terms were fomepizole,
4-methylpyrazole, and ethylene glycol. The search was supplemented with a
bibliographic review of all relevant articles. STUDY SELECTION: All
published reports of fomepizole administration in patients with ethylene
glycol poisoning were reviewed, irrespective of study design. We identified
one clinical trial and subsequent pharmacokinetic study, one case series,
and 13 case reports. RESULTS: Fomepizole has been investigated in 70
patients in open, unblinded studies. Most patients received an intravenous
loading dose, with subsequent variable maintenance doses every 12 hours
until plasma ethylene glycol levels became undetectable. Additional
hemodialysis treatment generally was administered when patients had renal
insufficiency or ethylene glycol levels above 50 mg/dl. Many patients had
detectable ethanol levels either because of coadministration or as a result
of adjunctive treatment at a referring center. Poorer patient outcomes, such
as death and renal insufficiency, were associated with later clinical
presentation time after ingestion. At therapeutic fomepizole levels (> 8.6
mg/ml), the half-life of ethylene glycol was prolonged to over 19 hours.
Fomepizole appeared to be well tolerated by most patients. CONCLUSION:
Fomepizole is an effective alcohol dehydrogenase inhibitor that decreases
production of ethylene glycol metabolites. Reduced mortality and morbidity
are undetermined because of the small number of patients evaluated to date.
Data on comparative efficacy of fomepizole versus ethanol and data on
administration of fomepizole in children are limited.
PMID: 11899949

[Although glycerol by itself gave poor results due to high osmotic toxicity,
the addition of a small amount of rapidly permeating methanol with glycerol
gave the best results in this test. 20% glycerol yielded a 34.9% survival,
while a 20% glycerol & 5% methanol combination yielded a 69.5% survival.
Methanol also dramatically reduced the toxicity of ethylene glycol, probably
by lowering its extrinsic toxicity. 20% ethylene glycol yielded a 0%
survival, while a 20% ethylene glycol & 5% methanol combination yielded a
38.6% survival.]

Theriogenology. 2005 Feb;63(3):763-73.
Toxicity and protective efficiency of cryoprotectants to flounder
(Paralichthys olivaceus) embryos.
  Zhang YZ, Zhang SC, Liu XZ, Xu YJ, Hu JH, Xu YY, Li J, Chen SL. Department
of Marine Biology, Ocean University of China, Qingdao 266003, PR China.
  With the purpose of finding an ideal cryoprotectant or combination of
cryoprotectants in a suitable concentration for flounder (Paralichthys
olivaceus) embryo cryopreservation, we tested the toxicities, at culture
temperature (16 degrees C), of five most commonly used
cryoprotectants-dimethyl sulfoxide (Me2SO), glycerol, methanol (MeOH),
1,2-propylene glycol (PG) and ethylene glycol (EG). In addition,
cryoprotective efficiency to flounder embryos of individual and combined
cryoprotectants were tested at -15 degrees C for 60 min. Five different
concentrations of each of the five cryoprotectants and 20 different
combinations of these cryoprotectants were tested for their protective
efficiency. The results showed that the toxicity to flounder embryos of the
five cryoprotectants are in the following sequence: PG < MeOH < Me2SO <
glycerol < EG (P < 0.05); whereas the protective efficiency of each
cryoprotectant, at -15 degrees C for a period of 60 min, are in the
following sequence: PG > Me2SO approximately MeOH approximately glycerol >
EG (greater symbols mean P < 0.05, and approximate symbols mean P > 0.05).
Methanol combined with any one of the other cryoprotectants gave the best
protection, while ethylene glycol combined with any one of the other
cryoprotectants gave the poorest protection at -15 degrees C. Toxicity
effect was concentration dependent with the lowest concentration being the
least toxic for all five cryoprotectants at 16 degrees C. For PG, MeOH and
glycerol, 20% solutions gave the best protection at -15 degrees C; whereas a
15% solution of Me2SO, and a 10% solution of EG, gave the best protection
at -15 degrees C.
PMID: 15629795

[Formamide, methanol and DMSO are all bad for DNA. It is interesting that in
addition to glycerol, ethylene glycol is also innocuous to DNA.]

Biotechnol Bioeng. 2000 May 5;68(3):339-44.
Structural stability of DNA in nonaqueous solvents.
  Bonner G, Klibanov AM. Department of Chemistry, Massachusetts Institute
of Technology, Cambridge, Massachusetts 02139, USA.
  One of the defining physicochemical features of DNA in aqueous solution is
its ability to maintain a double-helical structure and for this structure to
undergo a cooperative, heat-induced denaturation (melting). Herein we show
that a 21-mer synthetic DNA can form and maintain such a duplex structure
not only in water but even in 99% glycerol; moreover, this double-helical
structure reversibly and cooperatively melts in that solvent, with a T(m)
value of some 30 degrees lower than in water. Two much larger, natural DNAs,
from calf thymus and salmon testes, exhibit similar behavior in glycerol.
All three DNAs can also sustain a double-helical structure in 99% ethylene
glycol, although its thermostability (as reflected by the melting
temperature) is some 20 degrees lower than in glycerol. In contrast, no
duplex structure of any of the DNAs was detected in 99% formamide, methanol,
or DMSO. This solvent trend resembles that previously observed in studies of
protein structure and folding and underscores the importance of hydrophobic
interactions in both protein and DNA structure and stability. Our findings
suggest that water may not be unique as a suitable medium not only for
protein structure but also for that of nucleic acids. Copyright 2000 John
Wiley & Sons, Inc.
PMID: 10745202

[Calcium oxalate is the major toxic metabolite of ethylene glycol.]

Toxicol Lett. 2007 Aug 30;173(1):8-16. Epub 2007 Jun 20.
Calcium oxalate, and not other metabolites, is responsible for the renal
toxicity of ethylene glycol.
    Guo C, Cenac TA, Li Y, McMartin KE. Department of Pharmacology,
Toxicology & Neuroscience, Louisiana State University Health Sciences
Center, 1501 Kings Highway, Shreveport, LA 71130-3932, United States.
    Ethylene glycol (EG) is nephrotoxic due to its metabolism. Many studies
suggest that the toxicity is due to oxalate accumulation, but others have
conversely suggested that toxicity results from effects of metabolites such
as glycolaldehyde or glyoxylic acid on proximal tubule cells. In vivo
studies have indicated that accumulation of calcium oxalate monohydrate
(COM) corresponds closely with development of toxicity in renal tissue. The
present studies were therefore designed to clarify the roles of various
metabolites in the mechanism for EG toxicity in vitro by comparing the
relative cytotoxicity of EG metabolites using three measures of cell death,
ethidium homodimer uptake, lactate dehydrogenase (LDH) release and the
conversion of the tetrazolium salt XTT to a colorimetric dye. Human proximal
tubule cells in culture were incubated in physiologic buffers for 6h at 37
degrees C with COM (147-735mug/ml, an oxalate equivalence of 1-5mM),
glycolate (5-25mM), glyoxylate (0.2-5mM) and glycolaldehyde (0.2-2mM). To
assess the effects of acidity on the cytotoxicity, incubations were carried
out at pH 6-7.4. The results show that COM dose-dependently increased LDH
release and ethidium homodimer uptake, while the other metabolites did not.
Conversely, COM had no effect on the XTT assay, while high concentrations of
glycolaldehyde and glyoxylate decreased XTT activity, but the latter only at
acidic pH. The correlation between the uptake of ethidium homodimer and the
release of LDH suggest that COM is cytotoxic to human kidney cells in
culture, while the XTT assay does not validly measure cytotoxicity in this
system. These results indicate that COM, and not glyoxylate or
glycolaldehyde, is the toxic metabolite responsible for the acute tubular
necrosis and renal failure that is observed in EG-poisoned patients.
PMID: 17681674

[It is interesting that chronicly administered fucoidan can apparently
completely block 0.75% ethylene glycol toxicity.]

Life Sci. 2006 Oct 4;79(19):1789-95. Epub 2006 Jun 16.
Renal peroxidative changes mediated by oxalate: the protective role of
    Veena CK, Josephine A, Preetha SP, Varalakshmi P, Sundarapandiyan R.
Department of Medical Biochemistry, Dr ALM Post Graduate Institute of Basic
Medical Sciences, University of Madras, Taramani Campus, Chennai, India.
    Oxalate, one of the major constituents of renal stones is known to
induce free radicals which damage the renal membrane. Damaged epithelia
might act as nidi for stone formation aggravating calcium oxalate
precipitation during hyperoxaluria. In the present study, the beneficial
effects of fucoidan on oxalate-induced free radical injury were
investigated. Male Wistar rats were divided into four groups. Hyperoxaluria
was induced in two groups by administration of 0.75% ethylene glycol in
drinking water for 28 days and one of them was treated with fucoidan from
Fucus vesiculosus at a dose of 5 mg/kg b.wt subcutaneously commencing from
the 8th day of induction. A control and drug control (fucoidan alone) was
also included in the study. The extent of renal injury in hyperoxaluria was
evident from the increased activities of alkaline phosphatase,
gamma-glutamyl transferase, beta-glucuronidase,
N-acetyl-beta-D-glucosaminidase in urine. There was a positive correlation
between plasma malondialdehyde levels and renal membrane damage indicating a
striking relation between free radical formation and cellular injury.
Increased protein carbonyl and decreased thiols further exemplified the
oxidative milieu prevailing during hyperoxaluria. Decreased renal membrane
ATPases accentuated the renal membrane damage induced by oxalate. Renal
microscopic analysis showed abnormal findings in histology as an evidence of
oxalate damage. The above biochemical and histopathological discrepancies
were abrogated with fucoidan administration, indicating its protective role
in oxalate mediated peroxidative injury.
PMID: 16820173

[With calcium oxalate being the primary toxin generated from the metabolism
of ethylene glycol, it is no surprise that a reduced calcium content greatly
lowered ethylene glycol toxicity in vitrification solutions.]

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.
  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
(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

[Reducing extrinsic toxicity is tricky. Using ethanol to reduce calcium
oxalate production, can also increase formaldehyde production from ethylene

Toxicol Lett. 1995 Jun;78(1):9-15.
Increased oxidation of ethylene glycol to formaldehyde by microsomes after
ethanol treatment: role of oxygen radicals and cytochrome P450.
  Kukielka E, Cederbaum AI. Department of Biochemistry, Mount Sinai School
of Medicine, New York, NY 10029, USA.
  The production of ferryl-type oxidants by microsomes from ethanol-fed rats
and pair-fed controls was determined by assaying for the production of
formaldehyde from ethylene glycol. Microsomes from the ethanol-fed rats were
more reactive than controls in oxidizing ethylene glycol. Catalase was a
powerful inhibitor for this reaction, superoxide dismutase was slightly
inhibitory and hydroxyl radical scavengers had no effect. These results
suggest an important role for H2O2, but not O2-. or .OH in the overall
pathway for oxidizing ethylene glycol to formaldehyde. The production of
H2O2 by microsomes was increased after ethanol treatment, the extent of
increase corresponding to the increase in oxidation of ethylene glycol. A
variety of inhibitors and ligands of cytochrome P450, including miconazole,
diethyldithiocarbamate, tryptamine, and 4-methylpyrazole, inhibited
formaldehyde production by both microsomal preparations. Anti-cytochrome
P4502E1 IgG also inhibited the reaction with both microsomal preparations
and prevented the increase caused by ethanol treatment. These results
indicate that microsomes from ethanol-treated rats are more reactive than
pair-fed controls in generating ferryl-type oxidants and that increased
production of H2O2 by cytochrome P4502E1 plays a role in the elevated
oxidation of ethylene glycol to formaldehyde.
PMID: 7604403

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