X-Message-Number: 30107 Date: Thu, 6 Dec 2007 09:45:56 -0800 (PST) From: 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 procedure. 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 fomepizole 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 fucoidan. 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 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 [Reducing extrinsic toxicity is tricky. Using ethanol to reduce calcium oxalate production, can also increase formaldehyde production from ethylene glycol.] 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 Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=30107