X-Message-Number: 24136
Date: Sat, 22 May 2004 21:00:56 -0700 (PDT)
From: Doug Skrecky <>
Subject: calcium chelation reduces vitrification solution toxicity

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.
  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.

(Below are some other possible pretreatments, which may help block
vitrification toxicity.)

Methods Find Exp Clin Pharmacol. 2002 Mar;24(2):77-9.
Modulatory effect of Cyclea peltata Lam. on stone formation induced by
ethylene glycol treatment in rats. The inhibitory effect of the root of
Cyclea peltata Lam. on nephrolithiasis induced in rats by feeding with
ethylene glycolated water (1%) for 35 days was summarized. Ethylene
glycol administration led to oxalate stone formation, as indicated by its
high level in urine. Complementary to this anion, the cation calcium level
in urine was elevated. These two ions may have contributed to the
formation of calcium oxalate stones. In addition to high serum potassium,
a low serum magnesium level contributed to stone formation. Simultaneous
administration of the powdered root of Cyclea peltata resulted
in decreased urinary oxalate and calcium. Likewise, serum potassium was
lowered and magnesium was elevated. These observations provided the basis
for the conclusion that this plant inhibits the stone formation induced
by ethylene glycol treatment.

Am J Vet Res. 1994 Dec;55(12):1762-70.
Efficacy of 4-methylpyrazole for treatment of ethylene glycol
intoxication in dogs.
  4-Methylpyrazole (4-MP), an alcohol dehydrogenase inhibitor, was
administered to dogs to treat ethylene glycol (EG) intoxication. Eleven
dogs were given 10.6 g of EG/kg of body weight; 5 dogs were treated with
4-MP 5 hours after EG ingestion and 6 dogs were treated with 4-MP 8 hours
after EG ingestion. 4-Methylpyrazole was administered IV as a 50-mg/ml
[corrected] solution in 50% polyethylene glycol: initial dose, 20 mg/kg;
at 12 hours after initial dose, 15 mg/kg; at 24 hours after initial dose,
10 mg/kg; and at 30 hours after initial dose, 5 mg/kg. Physical,
biochemical, hematologic, blood gas, serum and urine EG concentrations,
and urinalysis findings were evaluated at 0, 1, 3, 6, 9, 12, 24, 48, 72
hours, and at 1 week and 2 weeks after EG ingestion. Dogs of both groups
developed clinicopathologic signs associated with EG intoxication,
including CNS depression, hyperosmolality, high anion gap metabolic
acidosis, polydipsia, polyuria, calcium oxalate monohydrate and dihydrate
crystalluria, and isosthenuria. Fractional excretion of sodium was
increased in all dogs between 1 and 9 hours after EG ingestion, but
remained increased beyond 24 hours only in the 2 dogs treated at 8
hours after EG ingestion that developed acute renal failure. All dogs
treated 5 hours after EG ingestion recovered without morphologic,
biochemical, or clinical evidence of renal impairment. Of the 6 dogs
treated 8 hours after EG ingestion, 2 developed acute renal failure. One
of the dogs treated 8 hours after EG ingestion remained isosthenuric for 2
months, but did not manifest any other signs of renal
impairment.(ABSTRACT TRUNCATED AT 250 WORDS)

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