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Evaluation of the effects of methanolic extract of Cucumis melo in ethylene glycol-induced nephrolithiasis on Wistar rats. 0.75% solution of ethylene glycol (EG) in payable water was given to produce nephrolithiasis on Wistar rats. The action of oral intake of methanolic extract of Cucumis melo seed in nephrolithiasis is studied and is matched with the action of oral intake of Cystone (standard) on Wistar rats. EG resulted in hyperoxaluria and deposition of calcium oxalate as well as raised urinary excretion of oxalate and calcium. Supplementation with methanolic extract of Cucumis melo seed decreased the increased renal oxalate, indicating a regulatory effect on oxalate formation endogenously. The outcomes stipulate that the seed of Cucumis melo is endowed with antinephrolithiatic action., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The aim of this study is to investigate whether the filtration barrier is affected by experimental kidney stone formation. Thirty-two rats divided into 4 equally groups (n = 8) at random. Group I control; Group II 1% ethylene glycol; Group III 1% Ethylene glycol + 0.25% Ammonium chloride; Group IV 1% Ethylene glycol + 0.5% Ammonium chloride group. Tissues applied hematoxylin-eosin, periodic-acid-Schiff, Pizzolato's staining. Immunohistochemically stained with integrin α3β1, type IV collagen, laminin, nephrin, CD2-associated protein (CD2AP) and podocin to show the filtration barrier structure. The TUNEL method was used for apoptosis. The amount of calcium, magnesium, creatinine and uric acid in urine and blood samples, also urine microprotein determined. Stones were formed in all experimental groups. Urine calcium, creatinine, uric acid levels decreased, magnesium levels were not changed. No statistically significant change was observed in blood serum results and TUNEL analysis. Immunohistochemical results showed an increase in nephrin, podocin, CD2AP, laminin and a decrease in integrin α3β1 and type IV collagen. Consequently, there is an increase in the expression densities of the proteins incorporated in the structure to prevent loss of functionality in the cellular part supporting the structure against a weakening of the basement membrane structure in the glomerular structure in which urine is filtered.

Urolithiasis is one of the oldest diseases affecting humans, while plants are one of our oldest companions providing food, shelter, and medicine. In spite of substantial progress in understanding the pathophysiological mechanisms, treatment options are still limited, often expensive for common people in most parts of the world. As a result, there is a great interest in herbal remedies for the treatment of urinary stone disease as an alternative or adjunct therapy. Numerous in vivo and in vitro studies have been carried out to understand the efficacy of herbs in reducing stone formation. We adopted PRISMA guidelines and systematically reviewed PubMed/Medline for the literature, reporting results of various herbal products on in vivo models of nephrolithiasis/urolithiasis. The Medical Subject Heading Terms (Mesh term) "Urolithiasis" was used with Boolean operator "AND" and other related Mesh Unique terms to search all the available records (July 2019). A total of 163 original articles on in vivo experiments were retrieved from PubMed indexed with the (MeshTerm) "Urolithiasis" AND "Complementary Therapies/Alternative Medicine, "Urolithiasis" AND "Plant Extracts" and "Urolithiasis" AND "Traditional Medicine". Most of the studies used ethylene glycol (EG) to induce hyperoxaluria and nephrolithiasis in rats. A variety of extraction methods including aqueous, alcoholic, hydro-alcoholic of various plant parts ranging from root bark to fruits and seeds, or a combination thereof, were utilized. All the investigations did not study all aspects of nephrolithiasis making it difficult to compare the efficacy of various treatments. Changes in the lithogenic factors and a reduction in calcium oxalate (CaOx) crystal deposition in the kidneys were, however, considered favorable outcomes of the various treatments. Less than 10% of the studies examined antioxidant and diuretic activities of the herbal treatments and concluded that their antiurolithic activities were a result of antioxidant, anti-inflammatory, and/or diuretic effects of the treatments.

This study was designed to assess the nephroprotective effects of Pleurotus ostreatus and Agaricus bisporus aqueous extracts and carvedilol on hyperoxaluria-induced urolithiasis and to scrutinize the possible roles of NF-κB, p53, Bcl-2, Bax and Bak. Phytochemical screening and GC-MS analysis of mushrooms' aqueous extracts were also performed and revealed the presence of multiple antioxidant and anti-inflammatory components. Hyperoxaluria was induced in Wistar rats through the addition of 0.75% ( v / v ) ethylene glycol in drinking water for nine weeks. The ethylene glycol-administered rats were orally treated with Pleurotus ostreatus and Agaricus bisporus aqueous extracts (100 mg/kg) and carvedilol (30 mg/kg) daily during the last seven weeks. The study showed that Pleurotus ostreatus , Agaricus bisporus and carvedilol all successfully inhibited ethylene glycol-induced histological perturbations and the elevation of serum creatinine, serum urea, serum and urinary uric acid, serum, urinary and kidney oxalate, urine specific gravity, kidney calcium, kidney NF-κB, NF-κB p65, NF-κB p50, p53, Bax and Bak expressions as well as serum TNF-α and IL-1β levels. Moreover, the treatment decreased the reduction in urinary creatinine, urinary urea, ratios of urinary creatinine to serum creatinine and urinary urea to serum urea, Fex Urea and Bcl-2 expression in kidney. In conclusion, although Pleurotus ostreatus and Agaricus bisporus extracts and carvedilol all significantly inhibited the progression of nephrolithiasis and showed nephroprotective effects against ethylene glycol-induced kidney dysfunction, Pleurotus ostreatus and Agaricus bisporus seemed to be more effective than carvedilol. Moreover, the nephroprotective effects may be mediated via affecting NF-κB activation, extrinsic apoptosis and intrinsic apoptosis pathways.

Objective: To investigate the effect of α-lipoic acid (α-LA) in the prevention and treatment of ethylene glycol-induced calcium oxalate deposition in a rat model and preliminary exploration of the mechanism., Methods: Sixty male Wistar rats were divided randomly and equally into six groups including two α-LA prevention groups, two α-LA therapeutic groups, one controlled group and one intervention group. Besides controlled group, other group received 1% glycol solution and 2 ml 2% ammonium chloride for 4 weeks as a stone inducer. The prevention groups received 0.1 and 0.2 mg/kg body weight/day/rat α-LA as food supplement during inducing stone and therapeutic groups received 0.1 and 0.2 mg/kg body weight/day/rat α-LA for 4 weeks after 4 weeks stone inducing., Results: The volume of urine, the pH and magnesium levels in the preventive and therapeutic groups were higher in a dose-independent manner (p < 0.05), and urinary calcium was lower (p < 0.05). Antioxidant stress enzyme activity (glutathione, superoxide dismutase, catalase) in serum and kidney homogenates in the preventive and therapeutic groups underwent significant regeneration (p < 0.05) and malondialdehyde levels and the production of free radical moieties decreased. Pathological observation demonstrated that there was deformation due to renal tubular expansion in the control group, greater visible inflammatory cell infiltration into the interstitial spaces and partial destruction of the glomerular structures. α-lipoic acid improved the lesions to varying degrees. The extent of crystal deposition was lower in the preventive and therapeutic groups compared with the control group (p < 0.05)., Conclusion: The present study indicated that α-LA provides both preventive and therapeutic effects against the deposition of calcium oxalate crystals in rats.

In commercial products such as household deodorants or biocides, didecyldimethylammonium chloride (DDAC) often serves as an antimicrobial agent, citral serves as a fragrance agent, and the excipient ethylene glycol (EG) is used to dissolve the active ingredients. The skin sensitization (SS) potentials of each of these substances are still being debated. Moreover, mixtures of DDAC or citral with EG have not been evaluated for SS potency. The in vitro alternative assay called human Cell Line Activation Test (h-CLAT) and Direct Peptide Reactivity Assay (DPRA) served to address these issues. On three independent runs of h-CLAT, DDAC and citral were predicted to be sensitizers while EG was predicted to be a non-sensitizer and also by the DPRA. Mixtures of DDAC or citral with EG at ratios of 7:3 and 1:4 w/v were all positive by the h-CLAT in terms of SS potential but SS potency was mitigated as the proportion of EG increased. Citral and its EG mixtures were all positive but DDAC and its EG mixtures were all negative by the DPRA, indicating that the DPRA method is not suitable for chemicals with pro-hapten characteristics. Since humans can be occupationally or environmentally exposed to mixtures of excipients with active ingredients, the present study may give insights into further investigations of the SS potentials of various chemical mixtures.

In a previous study investigating the relationships between solute physicochemical properties and solvent effects on skin permeation of solutes under finite dose conditions, urea was found to have the highest percent dose absorbed among the model solutes studied. The objective of this study is to probe the mechanism of the observed high skin permeation of urea at finite dose, in contrast to its permeability coefficient obtained under infinite dose condition. Skin permeation experiments were performed with Franz diffusion cells and human epidermal membrane. Dose-dependence and penetration enhancing effects of urea permeation were investigated. Tape stripping was performed. A small hydrophilic solute ethylene glycol with molecular weight similar to urea was studied for comparison. The results suggest that urea did not have penetration enhancing effect to enhance solute permeation across skin under the finite dose conditions. Tape stripping data are consistent with skin permeation mechanism of solute deposition and diffusion. The skin permeation behavior of urea could be attributed to its small molecular size. This suggests that, under the finite dose conditions examined in this study, solutes with molecular sizes similar to or less than urea and ethylene glycol could lead to high percent of skin absorption despite their hydrophilicities., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

We performed morphological analysis of the effect of the peptide complex from porcine kidneys on the course of experimental urolithiasis modeled in rats by treatment with 1% ethylene glycol solution (in drinking water) for 6 weeks. The peptide complex obtained by acetic acid extraction was administered in a dose of 15 mg. Administration of the peptide complex to animals with experimental kidney stone disease leads to 100% destruction of large and medium stones to the "dust" granularity.

Ethylene glycol is a common alcohol found in many household products such as household hard surface cleaner, paints, varnish, auto glass cleaner and antifreeze. While extremely toxic and often fatal on ingestion, few cases with early presentation by the patient have resulted in death; thus, rapid diagnosis is paramount to effectively treating ethylene glycol poisoning. In this study, we compare two sequential cases of ethylene glycol poisoning in a single individual, which resulted in strikingly different outcomes., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

TGF-β1 is the main mediator of epithelial-to-mesenchymal transition (EMT). Hyperoxaluria induces crystalluria, interstitial fibrosis, and progressive renal failure. This study analyzed whether hyperoxaluria is associated with TGF-β1 production and kidney fibrosis in mice and if oxalate or calcium oxalate (CaOx) could induce EMT in proximal tubule cells (HK2) and therefore contribute to the fibrotic process. Hyperoxaluria was induced by adding hydroxyproline and ethylene glycol to the mice's drinking water for up to 60 days. Renal function and oxalate and urinary crystals were evaluated. Kidney collagen production and TGF-β1 expression were assessed. EMT was analyzed in vitro according to TGF-β1 production, phenotypic characterization, invasion, cell migration, gene and protein expression of epithelial and mesenchymal markers. Hyperoxaluric mice showed a decrease in renal function and an increase in CaOx crystals and Ox urinary excretion. The deposition of collagen in the renal interstitium was observed. HK2 cells stimulated with Ox and CaOx exhibited a decreased expression of epithelial as well as increased expression mesenchymal markers; these cells presented mesenchymal phenotypic changes, migration, invasiveness capability and TGF-β1 production, characterizing EMT. Treatment with BMP-7 or its overexpression in HK2 cells was effective at preventing it. This mechanism may contribute to the fibrosis observed in hyperoxaluria.

The cryopreservation of testicular tissue is presented as the only alternative for the preservation of genetic material from prepubertal animals. However, this biotechnology is still being tested. The objective of this study was to evaluate the effect of different associations of cryoprotectants and the potential of cell proliferation after vitrification of testicular tissue of prepubertal cats. Five testicular pairs from five prepubertal cats were used, and each pair was divided into four fragments. Of these, one fragment composed of the control group (CG) and the rest were distributed in experimental groups according to the associations of cryoprotectants to be tested (dimethyl sulphoxide (DMSO)/glycerol (GLY); ethylene glycol (EG)/GLY) or DMSO/EG) in a final cryoprotectant concentration of 5.6 m. The fragments were submitted to vitrification, and after one week, fragments were heated and processed for histomorphological evaluation and quantification of nucleolar organizer regions (NORs). DMSO/GLY did not differ from CG and was superior to the other vitrified groups, as to cell separation and degree of shrinkage of the basal membrane. Concerning cell differentiation, visibility of the nucleus and nuclear condensation, all the vitrified groups were inferior to CG; however, DMSO/EG was inferior to DMSO/GLY and EG/GLY, which did not differ among themselves. CG was superior to all groups in quantification of NORs. DMSO/EG was inferior to all others, and there was no difference between DMSO/GLY and EG/GLY. The association DMSO/GLY presented the best preservation of tissue integrity and potential of cell proliferation after vitrification of the testicular tissue of prepubertal cats., (© 2016 Blackwell Verlag GmbH.)

Recent studies have shown that L-proline is a natural osmoprotectant and an antioxidant to protect cells from injuries such as that caused by freezing and thawing in many species including plant, ram sperm and human endothelial cells. Nevertheless, this nontoxic cryoprotectant has not yet been applied to mammalian oocyte vitrification. In this study we evaluated the efficiency and safety of the new cryoprotectant in oocyte vitrification. The results indicated that L-proline improves the survival rate of vitrified oocytes, protects mitochondrial functions and could be applied as a new cryoprotectant in mouse oocyte vitrification.

This study evaluated two cryoprotectant (CPA) combinations, ethylene glycol (EG) + DMSO and EG + propylene glycol (PG), used for the vitrification of germinal vesicle (GV) porcine oocytes. In experiment 1, the equilibration of GV with the two CPA combinations increased (P < 0.05) the percentage of oocytes that degenerated after IVM (18.1 ± 2.3% and 19.4 ± 2.6% for EG + DMSO and EG + PG groups, respectively) compared with control oocytes (7.6 ± 1.3%). However, CPAs did not affect the fertilization or developmental parameters of the embryos. In experiment 2, the percentages of live vitrified-warmed GV oocytes at 2 hours after warming (EG + DMSO: 67.0 ± 2.3% and EG + PG: 57.6 ± 2.3%) were lower than those of fresh control GV oocytes (97.3 ± 0.7%). The percentage of degenerated oocytes after IVM was higher (P < 0.001) in vitrified-warmed oocytes (EG + DMSO: 59.8 ± 2.3% and EG + PG: 56.2 ± 2.6%) than in the control (1.6 ± 1.3). Fertilization efficiency was higher (P < 0.05) in the EG + PG (39.6 ± 2.4%) and control (42.0 ± 2.2%) groups than in the EG + DMSO (26.3 ± 7.7%) group. The cleavage and blastocyst formation rates of the EG + DMSO (25.9 ± 3.5% and 6.6 ± 2.5%, respectively) and EG + PG (20.2 ± 5.4% and 4.7 ± 1.6%, respectively) vitrification groups were lower (P < 0.001) than those observed in the control oocytes (53.4 ± 2.7% and 31.9 ± 1.7%, respectively). In conclusion, in the absence of vitrification, the toxic effects of both CPA combinations on the GV oocytes were minimal. Vitrification resulted in important losses in viability at each step of the in vitro embryo production procedure. However, the surviving oocytes were able to mature and be fertilized, although the fertilization efficiency in the EG + DMSO group was lower. Blastocysts formation was similar for both CPA combinations., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Ethylene glycol (EG) exposure caused formation of calcium oxalate crystal that led to renal failure, which is associated with higher prevalence of hypertension. L-Arginine is known to have an antioxidant and nephro-protective potential., Objective: To evaluate the effect of L-arginine against EG-induced urolithiasis in uninephrectomized hypertensive rats., Material and Methods: Uninephrectomized male Wistar rats (180-200 g) were used to induce urinary calculi through oral administration of EG (0.75%) in distilled water. Rats were treated with either distilled water (10 mg/kg, p.o.) or telmisartan (10 mg/kg, p.o.) or Cystone (500 mg/kg, p.o.) or L-arginine (250, 500, and 1000 mg/kg, p.o.) for 28 days. Various hemodynamic, biochemical, molecular, and histological parameters were assessed in kidney and heart., Results: Rats treated with L-arginine (500 and 1000 mg/kg) significantly restored altered relative organ weight, urine output, urine density, urinary pH, and water intake. EG-induced alterations in electrocardiographic (QRS interval, HR, and ST height) and hemodynamic (SBP, DBP, MABP, and LVEDP) abnormalities were significantly restored by L-arginine (500 and 1000 mg/kg) treatment. It also significantly restored alteration in serum and urine biochemical parameters induced by EG. The elevated oxido-nitrosative stress was also significantly decreased by L-arginine (500 and 1000 mg/kg) treatment. It also significantly down-regulated EG-induced up-regulated renal KIM-1, NGAL, eNOS, and iNOs mRNA expressions. Histological aberrations induced in the renal and cardiac tissues were also ameliorated by l-arginine treatment., Conclusion: L-Arginine exerts its nephro- and cardio-protective potential in EG-induced urolithiasis in uninephrectomized hypertensive rats via modulation of KIM-1, NGAL, eNOS, and iNOs mRNA expression.

The objectives of this study were to compare glycerol and ethylene glycol at different concentrations as cryoprotectants and lycopene or cysteamine (with/without) as antioxidants in Tris extender for bull semen. Twenty-four ejaculates were obtained from three bulls. Each ejaculate was split into four equal aliquots and diluted using both of the Tris extenders with glycerol (5% or 7%) or ethylene glycol (3% or 5%). After that, each extenders were split into three equal aliquots and added using both of the cysteamine 5 mm or lycopene 500 μg/ml, and control (without additives). The addition of 7% glycerol with cysteamine, 5% ethylene glycol with cysteamine and 3% ethylene glycol with cysteamine groups gave the lowest CASA motility than the other groups. However, 7% glycerol and 7% glycerol with lycopene resulted in a better rate of CASA progressive motility compared with that of other groups. Generally, all the lycopene groups signed better protective effects on acrosome and total morphology than the other groups. Glycerol 7% and 3% ethylene glycol with lycopene groups yielded to slight higher percentages of membrane integrity assessed by HOST than that of the other groups, but 7% glycerol with cysteamine and 3% ethylene glycol with cysteamine showed the worst percentages of membrane integrity. Glycerol 7% and 5% glycerol with lycopene gave rise to a higher value of VAP, VSL and VCL compared with that of the other groups. On the contrary, adding to 5% glycerol with cysteamine showed negative effect for VAP, VSL, VCL and ALH values. All cryoprotectant groups with lycopene decreased chromatin damage than the other groups. Ethylene glycol 3% led to lower non-return rates of inseminated cows. However, this result was not considered to be statistically important., (© 2014 Blackwell Verlag GmbH.)

Objective: To study the preservation of follicles within ovarian tissue vitrified using only one or a combination of three permeating cryoprotectants., Design: Experimental study., Setting: University hospital., Donor(s): Ovarian tissue was donated by consenting women undergoing elective cesarean section., Intervention(s): Ovarian tissue was vitrified in closed sealed vials using either a combination of dimethyl sulfoxide, 1,2-propanediol, and ethylene glycol (EG), or only EG as permeating cryoprotectants., Main Outcome Measure(s): Ovarian tissue was vitrified with the use of two vitrification methods. Tissue from the same donor was used for comparison of two different solutions. The morphology of the follicles was evaluated after vitrification, warming, and culture by light microscopy and transmission electron microscopy. Apoptosis was assessed by immunohistochemistry for active caspase-3 in fresh and vitrified tissue., Result(s): Light and electron microscopic analysis showed equally well preserved morphology of oocytes, granulosa cells, and ovarian stroma when either of the vitrification solutions was used. No apoptosis was observed in primordial and primary follicles., Conclusion(s): Using only EG as a permeating cryoprotectant in a closed tube gives as good ultrastructural preservation of ovarian follicles as a more complicated system using several cryoprotectants., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Flos carthami (FC), also known as Carthamus tinctorius, is a traditional Chinese herbal plant that has been prescribed since centuries for treating various symptoms related to blood circulation improvement. This study aimed to investigate the effects of FC on calcium oxalate (CaOx) formation in ethylene glycol (EG)-fed rats. A total of 50 male Sprague-Dawley rats were divided into the following 6 groups: group 1, as the normal control (n = 5); group 2 received gastric gavages of starch and 0.75% EG (placebo, n = 5) as a stone inducer; group 3 (n = 10) received EG and potassium citrate as positive controls; group 4 (n = 10) received 0.75% EG and 300 mg/day FC; group 5 (n = 10) was treated with EG and 600 mg/day FC; group 6 (n = 10) received with EG and 1,200 mg/day FC. For all experimental animals, 24-h urine and blood samples were analyzed at the beginning and end of the experiment. Kidney tissue was histopathologically examined using a polarized light microscope, and crystal deposits were evaluated by a semi-quantitative scoring method; these scores were significantly lower in the FC groups (600 and 1,200 mg/day) than in the placebo group. Thus, FC administration appeared to inhibit the deposition of CaOx crystal EG-fed rats. We, therefore, consider that FC may be effective for preventing stone disease, albeit with certain side effects, such as a bleeding tendency. Further clinical trials are needed for evaluating its benefits and possible side effects.

Background: The aim of the conducted studies was to evaluate the effect of 4-methylpyrazole, increasingly used in detoxifying treatments after ethylene glycol poisoning, on the activity of some antioxidant enzymes and lipid peroxidation formation in the liver of rats after experimental co-exposure to ethylene glycol and ethyl alcohol., Methods: The trials were conducted on adult male Wistar rats. Ethylene glycol (EG) at the dose of 3.83 g/kg bw and ethyl alcohol (EA) at the dose of 1 g/kg bw were administered po, and 4-methylpyrazole (4-MP) at the dose of 0.01 g/kg bw was administered ip. Parameters of antioxidant balance were evaluated in hepatic cytosol, including the activity of the following enzymes: glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx) and lipid peroxidation level (TBARS)., Results: The results suggest that evaluation of the effects of administrated 4-MP after co-exposure to EG and EA in the liver revealed statistically significant changes on antioxidant enzyme system and malondialdehyde formation., Conclusion: The changes in biomarkers activity indicate a greater production of free radicals which exceeds the capability of antioxidant system, appearing with oxidative stress in the group of animals treated by 4-MP combined with EG and EA.

Experiments performed on 23 male rats, were divided into 2 groups. Animals in the control received group 1% solution of ethylene glycol (EG) as a drink during 6 weeks. In the test group, EG was also introduced for 6 weeks, and meloxicam was administered in a dose of 2.5 mg/kg from the 4th week. Every 7 days, daily urine was analyzed for the concentrations of oxalate, phosphate, and calcium and for the activity of urothelium injury marker enzymes includng lactate dehydrogenase (LDH), gamma-glutamyl transferase (GGT), and N-acetyl-beta-D-glucose aminidase (NAG). In addition, sections of the rats kidney were used to detect calcium deposits by histochemical Van Koss method. The treatment of experimental nephrolithiasis by meloxicame led to simplification of pathology, as indicated by a significant reduction in the urine oxalate and calcium concentrations and a pronounced decrease in the activity of all marker enzymes (LDH, GGT, NAG).This was confirmed by morphological studies, which detected very significant reduction in both number and size of calcium deposits.

In many biomedical and epidemiological studies, data are often clustered due to longitudinal follow up or repeated sampling. While in some clustered data the cluster size is pre-determined, in others it may be correlated with the outcome of subunits, resulting in informative cluster size. When the cluster size is informative, standard statistical procedures that ignore cluster size may produce biased estimates. One attractive framework for modeling data with informative cluster size is the joint modeling approach in which a common set of random effects are shared by both the outcome and cluster size models. In addition to making distributional assumptions on the shared random effects, the joint modeling approach needs to specify the cluster size model. Questions arise as to whether the joint modeling approach is robust to misspecification of the cluster size model. In this paper, we studied both asymptotic and finite-sample characteristics of the maximum likelihood estimators in joint models when the cluster size model is misspecified. We found that using an incorrect distribution for the cluster size may induce small to moderate biases, while using a misspecified functional form for the shared random parameter in the cluster size model results in nearly unbiased estimation of outcome model parameters. We also found that there is little efficiency loss under this model misspecification. A developmental toxicity study was used to motivate the research and to demonstrate the findings., (Copyright © 2011 John Wiley & Sons, Ltd.)

Purpose: To assess the beneficial effect of different fractions of Cynodon dactylon (C. dactylon) on ethylene glycol-induced kidney calculi in rats., Materials and Methods: Male Wistar rats were randomly divided into control, ethylene glycol, curative, and preventive groups. The control group received tap drinking water for 35 days. Ethylene glycol, curative, and preventive groups received 1% ethylene glycol for induction of calcium oxalate (CaOx) calculus formation. Preventive and curative subjects also received different fractions of C. dactylon extract in drinking water at 12.8 mg/kg, since day 0 and day 14, respectively. After 35 days, the kidneys were removed and examined for histopathological findings and counting the CaOx deposits in 50 microscopic fields., Results: In curative protocol, treatment of rats with C. dactylon N-butanol fraction and N-butanol phase remnant significantly reduced the number of the kidney CaOx deposits compared to ethylene glycol group. In preventive protocol, treatment of rats with C. dactylon ethyl acetate fraction significantly decreased the number of CaOx deposits compared to ethylene glycol group., Conclusion: Fractions of C. dactylon showed a beneficial effect on preventing and eliminating CaOx deposition in the rat kidney. These results provide a scientific rational for preventive and treatment roles of C. dactylon in human kidney stone disease.

Primary hyperoxaluria type I (PH1) is an inborn error of metabolism caused by deficiency of the hepatic enzyme alanine-glyoxylate aminotransferase (AGXT or AGT) which leads to overproduction of oxalate by the liver and subsequent urolithiasis and renal failure. The current therapy largely depends on liver transplantation, which is associated with significant morbidity and mortality. To explore an alternative treatment, we used somatic gene transfer in a mouse genetic model for PH1 (Agxt1KO). Recombinant adeno-associated virus (AAV) vectors containing the human AGXT complementary DNA (cDNA) were pseudotyped with capsids from either serotype 8 or 5, and delivered to the livers of Agxt1KO mice via the tail vein. Both AAV8-AGXT and AAV5-AGXT vectors were able to reduce oxaluria to normal levels. In addition, treated mice showed blunted increase of oxaluria after challenge with ethylene glycol (EG), a glyoxylate precursor. In mice, AGT enzyme activity in whole liver extracts were restored to normal without hepatic toxicity nor immunogenicity for the 50 day follow-up. In summary, this study demonstrates the correction of primary hyperoxaluria in mice treated with either AAV5 or AAV8 vectors.

High-dose bolus exposure of rats to ethylene glycol (EG) causes developmental toxicity mediated by a metabolite, glycolic acid (GA), whose levels increase disproportionately when its metabolism is saturated. However, low-level exposures that do not saturate GA metabolism have a low potential for developmental effects. Toward the goal of developing EG risk assessments based on internal dose metrics, this study examined the differences between fast (bolus) and slow (continuous infusion) dose-rate exposures to EG on developmental outcome and pharmacokinetics. Time-mated female CD rats received sc bolus injections of 0, 1000, or 2000 mg/kg/day of EG on gestation day (GD) 6-15 once daily, whereas three corresponding groups were given the same daily doses as an infusion administered continuously from GD 6-15 via an sc implantable pump. In the sc bolus groups, increases in 11 fetal malformations (major defects) and 12 variations (minor alterations) were seen at the 2000 mg/kg/day dose level, whereas increases in 2 malformations and 2 variations occurred at 1000 mg/kg/day. In contrast, equivalent daily doses of EG given slowly via infusion did not cause any developmental effects. A pharmacokinetics time course was then conducted to compare GD 11-12 kinetics from oral bolus (gavage) exposure versus sc infusion of EG. Although dose rate had a modest impact (8- to 11-fold difference) on peak EG levels, peak levels of GA in maternal blood, kidney, embryo, and exocoelomic fluid were 59, 100, 49, and 56 times higher, respectively, following gavage versus the same dose given by infusion. These data illustrate how high-dose bolus exposure to EG causes a dramatic shift to nonlinear GA kinetics, an event which is highly unlikely to occur following exposures to humans associated with consumer and worker uses.

Purpose: The availability of various transgenic and knockout mice provides an excellent opportunity to better understand the pathophysiology of calcium oxalate stone disease. However, attempts to produce calcium oxalate nephrolithiasis in mice have not been successful. We hypothesized that calcium oxalate nephrolithiasis in mice requires increasing urine calcium and oxalate excretion, and experimentally induced hyperoxaluria alone is not sufficient. To provide evidence we induced hyperoxaluria by administering hyperoxaluria inducing agents in normocalciuric and hypercalciuric mice, and investigating various aspects of nephrolithiasis., Materials and Methods: We administered ethylene glycol, glyoxylate or hydroxyl proline via diet in male and female normocalciuric B6 mice, and in hypercalciuric sodium phosphate co-transporter type 2 a -/- mice for 4 weeks. We collected 24-hour urine samples on days 0, 3, 7, 14, 21 and 28, and analyzed them for pH, creatinine, lactate dehydrogenase calcium and oxalate. Kidneys were examined using light microscopy. Urine was examined for crystals using light and scanning electron microscopy., Results: Hypercalciuric mice on hydroxyl proline did not tolerate treatment and were sacrificed before 28 days. All mice on ethylene glycol, glyoxylate or hydroxyl proline became hyperoxaluric and showed calcium oxalate crystalluria. No female, normocalciuric or hypercalciuric mice showed renal calcium oxalate crystal deposits. Calcium oxalate nephrolithiasis developed in all mice on glyoxylate and in some on ethylene glycol. In all mice the kidneys showed epithelial injury. Male mice particularly on glyoxylate had more renal injury and inflammatory cell migration into the interstitium around the crystal deposits., Conclusions: Results confirm that hyperoxaluria induction alone is not sufficient to create calcium oxalate nephrolithiasis in mice. Hypercalciuria is also required. Kidneys in male mice are more prone to injury than those in female mice and are susceptible to calcium oxalate crystal deposition. Perhaps epithelial injury promotes crystal retention. Thus, calcium oxalate nephrolithiasis in mice is gender dependent, and requires hypercalciuria and hyperoxaluria., (2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to evaluate the role of the inducible nitric oxide synthase (iNOS), selective nuclear factor-kappaB (NF-kappaB) and p38-mitogene-activated protein kinase (p38-MAPK) on oxalate-induced crystal deposition in renal tubules. The rats were divided into three groups; group 1; control group, group 2; ethylene glycol (EG) group, group 3; EG + pyrrolidine dithiocarbamate (PDTC) group. Rats were sacrified on 7, 15 and 45th days. The iNOS expression, p65/NF-kappaB and p38/MAPK activity, and oxidative stress markers were evaluated in the kidney. Crystal depositions were evident on day 7, mild and severe crystallization were observed on day 15 and 45 in EG group, respectively. There was limited or no crystal formation in the EG + PDTC group. While EG stimulates iNOS, p65/NF-kappaB and p38/MAPK activity in renal tubules, PDTC inhibited it. PDTC prevents crystal depositions in renal tubules by reducing oxidative stress, iNOS, NF-kappaB, and p38-MAPK expression.

Purpose: The pathogenesis of kidney stones remains elusive. There is some evidence that hyperoxaluria may effect vascular endothelium and many studies link renal stones to atherosclerosis. Also, renal vascular endothelial cells regulate proximal tubular epithelial cell function. We determined the effect of hyperoxaluria on plasma and tissue levels of asymmetrical dimethylarginine. The secondary aim was to determine the effect of verapamil on asymmetrical dimethylarginine., Materials and Methods: A total of 42 Sprague-Dawley rats were included in the study. In groups 1A, 1B and 1C hyperoxaluria was induced with ethylene glycol for 2 weeks. Groups 2A, 2B and 2C received ethylene glycol for 14 days and verapamil for 28 days. Control group 3 received no specific medication but distilled water. Blood samples were obtained at 24 hours and at study end, and kidney samples were obtained at 24 hours, and 7 and 28 days for histopathological evaluation., Results: Plasma asymmetrical dimethylarginine increased early in the hyperoxaluric group (p = 0.0002). The effect was retained at the end of the study period (p = 0.01). There was no increase in asymmetrical dimethylarginine in the verapamil group on short-term and long-term followup. Hyperoxaluria induced a significantly dense staining pattern in renal tissue asymmetrical dimethylarginine vs controls (p = 0.01). Asymmetrical dimethylarginine staining did not differ in the control and verapamil groups., Conclusions: Increased systemic and local tissue asymmetrical dimethylarginine may help explain the pathogenetic mechanisms of hyperoxaluria induced disorders such as nephrolithiasis and atherosclerosis., (Copyright 2010 American Urological Association. Published by Elsevier Inc. All rights reserved.)

Objectives: The effect on endogenous oxalate synthesis of acute intravenous loading with ethylene glycol or glycine was investigated in rats on a standard or a vitamin B6-deficient diet., Methods: Twenty-four male Wistar rats weighing approximately 180 g were randomly divided into ethylene glycol and glycine groups of 12 animals each. These groups were further divided into two subgroups of six animals each that were fed either a standard or a vitamin B6-deficient diet for 3 weeks. Animals of these two subgroups received an intravenous infusion of 20 mg (322.22 micromol) of ethylene glycol or 100 mg (1332.09 micromol) of glycine, respectively. Urine samples were collected just before intravenous infusion of each substance and at hourly intervals until 5 h after receiving the infusion. Urinary oxalate, glycolate, and citrate levels were measured by capillary electrophoresis., Results: Urinary oxalate and glycolate excretion was significantly increased after ethylene glycol administration. Significant differences between the control and vitamin B6-deficient groups were found. In contrast, there were only small changes of oxalate and glycolate excretion after glycine administration. Recovery of the given dose of ethylene glycol as oxalate in 5-h urine was 0.31% and 7.15% in the control and vitamin B6-deficient groups, respectively, whereas recovery of glycolate was 0.68% and 7.22%, respectively., Conclusions: Ethylene glycol loading has a significant effect on urinary oxalate excretion in both normal and vitamin B6-deficient rats, whereas glycine loading only has a small effect. Oxalate and glycolate excretion after ethylene glycol loading were respectively 23-fold and 11-fold higher in vitamin B6-deficient rats than in controls.

Background: Primary hyperoxaluria type-1 (PH1) is an autosomal recessive disease characterized by excessive oxalate production by hepatocytes caused by the deficiency of peroxisomal alanine-glyoxylate aminotransferase (AGT) activity. Persistent hyperoxaluria causes nephrocalcinosis and urolithiasis, leading to renal failure, followed by tissue oxalosis with life-threatening complications. Combined liver-kidney transplantation is the only definitive treatment of PH1. Hepatocyte transplantation, which is much less invasive, could have offered an attractive alternative. However, because the AGT-deficient hepatocytes overproduce oxalate, a large fraction of the mutant host hepatocytes must be replaced by AGT-competent cells, which is beyond the capacity of current hepatocyte transplantation procedures. Here, we have evaluated a preparative irradiation-based method of liver repopulation in an Agxt-deleted mouse model of PH1 (Agxt-/-)., Materials and Methods: Hepatocytes (10(6) viable cells) isolated from congeneic mice ([ROSA]26 C57BL/6J) expressing Escherichia coli beta-galactosidase were transplanted into Agxt-/- mice by intrasplenic injection. The preparative regimen consisted of X-irradiation of the host liver and mitotic stimulation of the hepatocytes by adenovector-based expression of hepatocyte growth factor., Results: The procedure resulted in progressive replacement of the mutant host hepatocytes with the AGT-competent hepatocytes, leading to correction of urinary oxalate excretion. Oral ethylene glycol challenge (0.7% for 1 week) resulted in nephrocalcinosis and microlithiasis in untreated Agxt-/- mice, but not in the mice after hepatic repopulation., Conclusion: The results indicate that hepatocyte transplantation after appropriate preparative regimens may permit sufficient repopulation of the liver to ameliorate hyperoxaluria, and therefore should be evaluated further as a potential treatment of PH1.

Male Wistar rats have been shown to be the most sensitive sex, strain and species to ethylene glycol-induced nephrotoxicity in subchronic studies. A chronic toxicity and dosimetry study was therefore conducted in male Wistar rats administered ethylene glycol via the diet at 0, 50, 150, 300, or 400 mg/kg/day for up to twelve months. Subgroups of animals were included for metabolite analysis and renal clearance studies to provide a quantitative basis for extrapolating dose-response relationships from this sensitive animal model in human health risk assessments. Mortality occurred in 5 of 20 rats at 300 mg/kg/day (days 111-221) and 4 of 20 rats at 400 mg/kg/day (days 43-193), with remaining rats at this dose euthanized early (day 203) due to excessive weight loss. Increased water consumption and urine volume with decreased specific gravity occurred at 300 mg/kg/day presumably due to osmotic diuresis. Calculi (calcium oxalate crystals) occurred in the bladder or renal pelvis at > or =300 mg/kg/day. Rats dying early at > or =300 mg/kg/day had transitional cell hyperplasia with inflammation and hemorrhage of the bladder wall. Crystal nephropathy (basophilic foci, tubule or pelvic dilatation, birefringent crystals in the pelvic fornix, or transitional cell hyperplasia) affected most rats at 300 mg/kg/day, all at 400 mg/kg/day, but none at < or =150 mg/kg/day. No significant differences in kidney oxalate levels, the metabolite responsible for renal toxicity, were observed among control, 50 and 150 mg/kg/day groups. At 300 and 400 mg/kg/day, oxalate levels increased proportionally with the nephrotoxicity score supporting the oxalate crystal-induced nephrotoxicity mode of action. No treatment-related effects on the renal clearance of intravenously infused (3)H-inulin, a marker for glomerular filtration, and (14)C-oxalic acid were observed in rats surviving 12 months of exposure to ethylene glycol up to 300 mg/kg/day. In studies with naïve male Wistar and F344 rats (a less sensitive strain), a significant difference was observed in oxalate clearances between young rats (i.e. Wistar clearance < F344) but not in age-matched old rats. Regardless, the ratios of oxalate:inulin clearances in these two strains of rats, including those exposed to ethylene glycol, were all < 1, suggesting that a fraction of the filtered oxalate is reabsorbed. Other species, including humans, typically have clearance ratios >1 and are more effective at clearing oxalic acid by both glomerular filtration and active secretion. Thus, the lower renal clearance and kidney accumulation of oxalates in male Wistar rats enhances their sensitivity, which will be a factor in human risk assessments. The benchmark dose values (BMD05, BMDL05) were 170 mg/kg/day and 150 mg/kg/day for nephropathy, and 170 mg/kg/day and 160 mg/kg/day for birefringent crystals, using incidence times severity data in each case. The NOAEL of 150 mg/kg/day is the same as that reported after 16-week exposure and appears to be a threshold dose below which no renal toxicity occurs, regardless of exposure duration.

Objective: To study the expression of hyaluronan (HA) and CD44, a transmembrane glucoprotein and cell surface receptor of HA, in impaired renal tubular epithelial cells and relation thereof with the retention of calcium oxalate (CaOx) crystals in the renal tubule., Methods: Forty-eight SD rats were randomly divided into four equal groups, ethylene glycol (EG) 1-day group, treated with drinking water containing 0.75% EG for 1 day, EG 5-day group, treated with drinking water containing 0.75% EG for 5 days, general drinking water 1-day group, treated with tap water for 1 day, and general water 5-day group, treated with tap water for 5 days. The rats were killed 1 day or 5 days later respectively with their kidneys taken out. Immunohistochemistry was used to detect the expression of HA and CD44 in the renal tissues. Polarized light microscopy was used to detect the CaOx crystals in the renal tubules., Results: The expression rates of HA and CD44, and proportion of crystal retention of the tap water 1-day group were 25.0%, 25.0%, and 16.7% respectively, all not significantly different from those of the EG 1-day group (8.3%, 16.7%, and 25.0% respectively, all P > 0.05). The expression rates of HA and CD44, and proportion of crystal retention of the tap water 5-day group were 33.3%, 25.0%, and 16.7% respectively, all significantly lower than those of the EG 5-day group (83.3%, 75.0%, and 66.7% respectively, all P < 0.05). Microscopy showed that the CaOx crystals adhered only to the injured tubular epithelial cells expressing HA and CD44 at their luminal membrane., Conclusion: The expression of HA and CD44 by injured tubular cells seems to play a role in the retention of CaOx crystals in the kidney.

The study investigated the effects of internal (DMSO, 1,2-propanediol, glycerol, ethylene glycol, methanol, N,N-dimethylacetamide) and external cryoprotectants (glucose, sucrose) on the viability and on morphometric parameters of zebrafish embryos. From the tested internal cryoprotectants, DMSO had the lowest toxicity, followed by 1,2-propanediol and glycerol. The external cryoprotectants were less toxic then the internal ones. Early ontogenetic stages were more sensible to cryoprotectant exposure than advanced stages. Two-step incubation procedures in increasing concentrations of internal and external cryoprotectants were superior to multiple-step exposure procedures. All tested vitrification solutions exceeded the tolerance limit of embryos. The tolerance of zebrafish embryos to cryoprotectants was highly variable in a concentration range causing approximately 50% embryo mortality. The width of the perivitelline space showed significant morphometrical changes due to cryoprotectant exposure. In the germinative tissue non-significant changes occurred. The yolk did not change morphometrically after exposure to internal cryoprotectants and showed no sign of dehydration after exposure to external cryoprotectants. Based on these results the study comes to the following conclusions: as yolk dehydration was impossible and as vitrification solutions were over the tolerance limit it seems unlikely that successful vitrification of zebrafish embryos can be achieved. Under these considerations slow freezing methods would be a better option as lower cryoprotectant concentrations can be used and embryos can be dehydrated during freezing.

Intracellular concentrations of potassium and sodium in two-cell mouse embryos in G1/S phase after exposure to vitrification solutions containing vitrificant agents (VFAs) ethane-1,2-diol (ethylene glycol, EG), propane-1,2-diol (propylene glycol, PG), or dimethyl sulfoxide (DMSO), and sucrose (S) after incubation in Dulbecco solution were measured by electron probe microanalysis (EPMA) as described earlier (CryoLetters, 2006, 27: 87-98). The 4-step protocol was as followed: 10% VFA for 10 min => 30% VFA + 0.7 M S for 1.5 min ==> 0.5 M S for 10 min ==> 10 min pure Dulbecco's. The cytoplasmic concentration of potassium and sodium in immediately flashed out from the oviduct embryos was in range of 120 +/- 2 mM, with good concordance with the previous data (CryoLetters, 2006, 27:87-98). Exposure in Dulbecco's for 30 min did not alter elemental composition, neither did exposure in PG or DMSO for 1.5 min. In contrast, exposure for 1.5 min in EG dropped the level of potassium to 96 +/- 2 mM while elevating level of cytoplasmic sodium to 136 +/- 3 mM. Further exposure to 30%-EG for 3 min led to a two-fold decrease of both elements (60 +/- 3 mM and 66 +/- 2 mM for K and Na, respectively).

Vitrification of laser treated human blastocysts using reduced concentrations of permeable cryoprotectants was carried out by submerging cut standard straws (CSS) into liquid nitrogen. The CSS were made by cutting a standard 0.25 ml straw at an angle of approximately 45 degrees . After laser assisted hatching, 6 day blastocysts (n=250) were loaded into droplets of approximately 0.75 microl in the CSS and were either plunged directly into liquid nitrogen or first encased in a standard 0.5 ml straw (aseptic technique) before being vitrified. Permeable cryoprotectants (ethylene glycol+Me(2)SO) at concentrations of 15% and 20% v:v were tested for their effect on post warming re-expansion and post transfer pregnancy rates. Our results indicate that the use of reduced concentrations of cryoprotectants and aseptic packaging of blastocysts did not have any statistically significant impact on the study outcomes.

Background: A patient presented with severe acidosis and a point-of-care lactate measurement of 42 mmol/L. Mesenteric ischemia was suspected, with a potential need for laparotomy; however, plasma lactate measurements were below 4 mmol/L. Ethylene glycol ingestion was subsequently diagnosed. We therefore wished to determine why discrepancies in lactate measurements occur and whether this "lactate gap" could be clinically useful., Methods: We phlebotomized blood, added various concentrations of metabolites of ethylene glycol, and tested the resulting samples with the 5 most common lactate analyzers., Results: With the Radiometer 700 point-of-care analyzer, glycolate addition resulted in an artifactual, massive lactate elevation, even at low glycolate concentrations. Another major ethylene glycol metabolite, glyoxylate (but not oxalate or formate), caused similar elevations. The i-STAT and Bayer point-of-care analyzers and the Beckman and Vitros laboratory analyzers reported minimal lactate elevations. Lactate gap was determined by comparing the Radiometer result with the corresponding result from any of the other analyzers., Interpretation: We demonstrated how inappropriate laparotomy or delayed therapy might occur if clinicians are unaware of this phenomenon or have access to only a single analyzer. We also showed that lactate gap can be exploited to expedite treatment, diagnose late ethylene-glycol ingestion and terminate dialysis. By comparing lactate results from the iSTAT or Bayer devices with that from the Radiometer, ethylene-glycol ingestion can be diagnosed at the point of care. This can expedite diagnosis and treatment by hours, compared with waiting for laboratory results for plasma ethylene glycol.

Objective: To evaluate a vitrification solution using a mixture of two cryprotectant agents, dimethyl sulfoxide and ethylyne glycol plus sucrose, on the survival of human oocytes., Design: Clinical study of cryopreservation of human metaphase II (MII) oocytes by vitrification., Setting: University-affiliated IVF center., Patient(s): Infertile couples who agreed to have their surplus oocytes vitrified during the fresh IVF cycle., Intervention(s): Vitrification of surplus oocytes subsequently used in the next cycle and assisted fertilization by intracytoplasmic sperm injection., Main Outcome Measure(s): Morphologic survival and normal fertilization, embryo development, and clinical outcome., Result(s): A total of 53 surplus MII oocytes from 6 patients were vitrified, of which 24 were thawed, resulting in 18 which survived morphologically (75%). Following insemination, 14 of the 18 surviving eggs were fertilized (77.7%). All zygotes developed into viable embryos that were replaced into each patient's uterus, resulting in two healthy pregnancies: one singleton and one twin. The pregnancies were ongoing., Conclusion(s): Cryopreservation of human MII oocytes by vitrification appears to be a promising procedure, though to assure optimal effectiveness of this protocol further studies should be undertaken.

We developed a simple vitrification technique for bovine embryos that could permit direct transfer. Embryos were produced in-vitro by standard procedures. The base medium for cryopreservation was a chemically defined medium similar to SOF + 25 mM Hepes and 0.25% fatty acid free bovine serum albumin (FAF-BSA) (HCDM2). In experiment 1, embryos were first exposed to 3.5M ethylene glycol (V1) for 1, 2 or 3 min at room temperature (20-24 degrees C), and then moved to 7 M ethylene glycol (V2) at 4 or 20-24 degrees C and loaded in 0.25-mL straws. After 45 s in 7 M ethylene glycol, straws were placed in liquid nitrogen. Embryos that were loaded at 20-24 degrees C had higher survival rates than those loaded at 4 degrees C (P<0.05). Exposure for 1 min was best for morulae, while 3 min was best for blastocysts. In experiment 2, blastocysts were handled at 24 degrees C and exposed to two concentrations of ethylene glycol in V1 (3.5 or 5 M) followed by V2 as in experiment 1, two warming temperatures (20 or 37 degrees C) and two post-warming holding times until culture (5 or 15 min). Exposure to 5 M ethylene glycol and warming at 37 degrees C was the optimal combination of procedures, and embryos survived well after 15 min in straws if warmed at 37 degrees C. In experiment 3, ethylene glycol concentration (3, 4 or 5 M) and exposure time (0.5 or 1 min) during two-step addition of cryoprotectant were studied for bovine morulae. In experiment 4, morulae were exposed to V2 for 30 or 45 s in HCDM2 or Vigro holding medium and then held in 22-24 degrees C air or 37 degrees C water post-warming. Experiment 5 was like experiment 4 except blastocysts were used. Overall survival rates of blastocysts in experiment 5 averaged 80% of non-vitrified controls after 48 h culture. The survival rates with in vitro-produced morulae in experiments 1, 3 and 4 were unacceptable. Vitrification solutions based on Vigro tended to result in higher survival than HCDM2 for blastocysts, but not morulae. In experiment 6, the survival rate in vitro of in vivo-produced morulae and blastocysts after two-step vitrification was nearly 100%. Our vitrification technique was very effective for in vitro produced blastocysts, but not for in vitro-produced morulae.

Vitrification using open pulled straw (OPS) has provided encouraging results with embryos from other species. The aim of this study was to compare the survival of 6.5- and 6.75-day-old equine embryos after OPS vitrification and slow-cooling. Eighteen embryos were frozen using a slow-cooling method. Embryos were placed in modified PBS with increasing glycerol concentration (2.5%, 5%, 7.5% and 10% (v/v) 5 min each). Embryos were loaded into 0.25 ml straws then placed in a programmable freezer and subsequently plunged into liquid nitrogen. After thawing, cryoprotectant was removed by five steps with decreasing glycerol and sucrose concentrations. Twenty embryos were vitrified using the OPS method. Embryos were exposed to 7.5% dimethyl-sulfoxide (DMSO)+7.5% ethylene glycol (EG) for 3 min and in 18% DMSO+18% EG+0.4M sucrose for 1 min, loaded in OPS and plunged into liquid nitrogen. After warming, embryos were placed in decreasing sucrose concentrations. All embryos were cultured in synthetic oviduct fluid (SOF) medium for 3h and evaluated using 4',6-diamidino-2-phenylindole (DAPI) staining. The percentage of cells entering in S-phase (%SC) was evaluated by incorporation of BrdU. No significant differences were observed for mean diameter, morphological grade and percentage of degenerate embryos after 3h of culture for slow-cooling and OPS methods. The percentage of dead cells per embryo was similar for the two procedures (42+/-6 versus 46+/-9). The percentage of cells entering in S-phase did not differ significantly between the two procedures (27+/-5 versus 26+/-6). OPS vitrification may be as efficient as slow-cooling for the cryopreservation of equine embryos. However, these results should be confirmed by the transfer of OPS vitrified embryos to recipient mares.

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.

In 2002, poison centers in the US reported 5816 human exposures to ethylene glycol. A guideline that effectively determines the threshold dose for emergency department referral and need for pre-hospital decontamination could potentially avoid unnecessary emergency department visits, reduce health care costs, optimize patient outcome, and reduce life disruption for patients and caregivers. An evidence-based expert consensus process was used to create this guideline. Relevant articles were abstracted by a trained physician researcher. The first draft of the guideline was created by the primary author. The entire panel discussed and refined the guideline before distribution to secondary reviewers for comment. The panel then made changes based on the secondary review comments. The objective of this guideline is to assist poison center personnel in the out-of-hospital triage and initial management of patients with a suspected exposure to ethylene glycol by (1) describing the process by which the exposure might be evaluated, (2) identifying the key decision elements in managing the case, (3) providing clear and practical recommendations that reflect the current state of knowledge, and (4) identifying needs for research. This guideline is based on an assessment of current scientific and clinical information. The panel recognizes that specific patient care decisions may be at variance with this guideline and are the prerogative of the patient and health professionals providing care, considering all of the circumstances involved. Recommendations are in chronological order of likely clinical use. The grade of recommendation is in parentheses. (1) A patient with exposure due to suspected self-harm, misuse, or potentially malicious administration should be referred to an emergency department immediately regardless of the dose reported (Grade D). (2) Patients with inhalation exposures will not develop systemic toxicity and can be managed out-of-hospital if asymptomatic (Grade B). Patients with clinically significant mucous membrane irritation should be referred for evaluation (Grade D). (3) Decontamination of dermal exposures should include routine cleansing with mild soap and water. Removal of contact lenses and immediate irrigation with room temperature tap water is recommended for ocular exposures. All patients with symptoms of eye injury should be referred for an ophthalmologic exam (Grade D). (4) Patients with symptoms of ethylene glycol poisoning should be referred immediately for evaluation regardless of the reported dose (Grade C). (5) The absence of symptoms shortly after ingestion does not exclude a potentially toxic dose and should not be used as a triage criterion (Grade C). (6) Adults who ingest a "swallow" (10-30 mL), children who ingest more than a witnessed taste or lick, or if the amount is unknown of most ethylene glycol products should be referred immediately for evaluation. The potential toxic volume of dilute solutions (e.g., concentration <20%) is larger and can be estimated by a formula in the text (Grade C). (7) A witnessed taste or lick only by a child, or an adult who unintentionally drinks and then expectorates the product without swallowing, does not need referral (Grade C). (8) Referral is not needed if it has been >24 hours since a potentially toxic unintentional exposure, the patient has been asymptomatic, and no alcohol was co-ingested (Grade D). (9) Gastrointestinal decontamination with ipecac syrup, gastric lavage or activated charcoal is not recommended. Transportation to an emergency department should not be delayed for any decontamination procedures (Grade D). (10) Patients meeting referral criteria should be evaluated at a hospital emergency department rather than a clinic. A facility that can quickly obtain an ethylene glycol serum concentration and has alcohol or fomepizole therapy available is preferred. This referral should be guided by local poison center procedures and community resources (Grade D). (11) The administration of alcohol, fomepizole, thiamine, or pyridoxine is not recommended in the out-of-hospital setting (Grade D).

Experience with dose response and mechanisms of toxicity has shown that multiple mechanisms may exist for a single agent along the continuum of the full dose-response curve. It is highly likely that critical, limiting steps in any given mechanistic pathway may become overwhelmed with increasing exposures, signaling the emergence of new modalities of toxic tissue injury at these higher doses. Therefore, dose-dependent transitions in principal mechanisms of toxicity may occur, and could have significant impact on the interpretation of reference data sets for risk assessment. To illustrate the existence of dose-dependent transitions in mechanisms of toxicity, a group of academic, government, and industry scientists, formed under the leadership of the ILSI Health and Environmental Sciences Institute (HESI), developed a series of case studies. These case studies included acetaminophen, butadiene, ethylene glycol, formaldehyde, manganese, methylene chloride, peroxisome proliferator-activated receptor (PPAR), progesterone/hydroxyflutamide, propylene oxide, vinyl acetate, vinyl chloride, vinylidene chloride, and zinc. The case studies formed the basis for technical discourse at two scientific workshops in 2003.

Experiments were conducted to study the effect of cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propanediol (PROH), and glycerol at different concentrations (3.5, 4, 5, 6, and 7 M each with 0.5 M sucrose and 0.4% BSA in DPBS) on survival, in vitro maturation, in vitro fertilization, and post-fertilization development of vitrified-thawed immature buffalo oocytes. The COCs were harvested from the ovaries by aspirating the visible follicles. The recovery of post-thaw morphologically normal oocytes was lower in 3.5 and 4 M DMSO, EG, and PROH compared to 5, 6, and 7 M. In all the concentrations of glycerol, an overall lower numbers of oocytes recovered were normal compared to other cryoprotectants. Less number of oocytes reached metaphase-II (M-II) stage from the oocytes cryopreserved in any of the concentrations of DMSO, EG, PROH, and glycerol compared to fresh oocytes. Among the vitrified groups, highest maturation was obtained in 7 M solutions of all the cryoprotectants. The cleavage rates of oocytes vitrified in different concentrations of DMSO, EG, PROH, and glycerol were lower than that of the fresh oocytes. The cleavage rates were higher in oocytes cryopreserved in 6 and 7 M DMSO, EG, PROH, and glycerol compared with oocytes cryopreserved in other concentrations. However, the percentage of morula and blastocyst formation from the cleaved embryos did not vary in fresh oocytes and vitrified oocytes. In conclusion, this report describes the first successful production of buffalo blastocysts from immature oocytes cryopreserved by vitrification.

Cryopreservation of primordial follicles represents an opportunity to preserve female gametes, and consequently to protect the reproductive capacity of humans and animals, as well as to safeguard genetic material from endangered animal species or rare breeds. The aim of this work was to assess the toxicity of different concentrations of ethylene glycol (EG) to primordial follicles, and verify the viability of these follicles after the freezing-thawing procedure. Primordial follicles were isolated from ovine ovaries and exposed to different EG concentrations to evaluate the cryoprotectant (CPA) toxicity before and after cryopreservation. After isolation of primordial follicle (control), the number (mean+/-S.E.M.) of viable primordial follicles/ml was 3764+/-795.21. The number of viable follicles in the toxicity test using EG at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 M was 1096+/-447.9, 960+/-446.67, 948+/-366.14, 832+/-313.59, 856+/-280.67, and 700+/-255.02, respectively. The number of viable follicles at concentrations of 2.5 M was less than for controls. After cryopreservation, the numbers decreased to 0+/-0, 148+/-85.46, 764+/-246.69, 824+/-291.9, 844+/-296.27, and 588+/-200.65, respectively for 0, 0.5, 1.0, 1.5, 2.0, and 2.5 M EG. The number of viable follicles at 0, 0.5, and 2.5 M was less than for controls. In conclusion, after the freezing and thawing procedure, concentrations of 1.0, 1.5, and 2.0 M EG can be successfully used for the cryopreservation of isolated follicles in sheep.

Hypercalciuria and hyperoxaluria are important risk factors in the pathogenesis of kidney stones. Urinary glycolate has also been reported to be elevated in patients with renal stones. 1,25-Dihydroxyvitamin D(3), the active metabolite of vitamin D, has been reported to induce hyperoxaluria after either oral or intravenous administration. 1-alpha-D(3), a synthetic derivative of vitamin D, together with ethylene glycol, has been reported to induce renal stones in experimental rats. We have examined the effect of 1-alpha-vitamin D(3) on urinary oxalate and glycolate excretion. Our results indicate that 1-alpha-D(3), together with ethylene glycol, caused a significant increase in urinary glycolate, without a parallel rise in urinary oxalate excretion, in ethylene glycol-fed rats. This increase in urinary glycolate was due to the synergistic effect of both drugs.

Background: The effects of exposure time (20 versus 45 s) to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) and freezing rates (1200-10 300 degrees C/min) during rapid freezing of mouse pronuclear stage embryos on survival and development to blastocysts were investigated. Different freezing rates were achieved by directly plunging the straws (rapid freezing) and open pulled straws (OPS) in liquid nitrogen (OPS freezing) and by plunging the straws (super rapid) and OPS (super OPS) in a super cooled liquid nitrogen chamber (at -212 degrees C) before storage in liquid nitrogen., Methods: Morphologically intact mouse zygotes (n = 891) pre-equilibrated in 1.5 mol/l ethylene glycol for 5 min were either loaded in 0.25 ml straws containing cryoprotectant or loaded in OPS with 2 microl cryoprotectant. After 20 or 45 s of loading the straws or mixing in cryoprotectant and loading in OPS, they were plunged either directly in to liquid nitrogen or were plunged first in to liquid nitrogen in a super cooled chamber and then stored in liquid nitrogen. Zygotes were thawed and intact embryos cultured in vitro., Results: The rate of survival was higher (91%, P < 0.01) when zygotes were frozen with rapid freezing compared with super rapid, OPS and super OPS freezing rates with an exposure time of 20 s (70, 65, and 76% respectively). When zygotes were exposed to cryoprotectant for 45 s and frozen with rapid freezing rates, the survival was higher (86%, P < 0.01) compared with those frozen with OPS (62%) but was not different from those frozen with super rapid and super OPS freezing rates (81 and 75%). A higher rate of survival was observed when zygotes were exposed to cryoprotectant for 45 s and frozen with super OPS than with OPS freezing (75 versus 62%; P < 0.05). The rate of cleavage and development of intact zygotes to blastocysts was not different among the different groups., Conclusion: Exposure of zygotes to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) for 20 or 45 s did not influence their survival and development and increasing the freezing rate from 1200-10 300 degrees C/min was of no advantage when using a rapid freezing procedure for freezing mouse pronuclear stage embryos.

The kinetics of orally administered ethylene glycol (EG) and its major metabolites, glycolic acid (GA) and oxalic acid (OX), in pregnant (P; gestation day 10 at dosing, GD 10) rats were compared across doses, and between pregnant and nonpregnant (NP) rats. Groups of 4 jugular vein-cannulated female rats were administered 10 (P and NP), 150 (P), 500 (P), 1000 (P), or 2500 (P and NP) mg (13)C-labelled EG/kg body weight. Serial blood samples and urine were collected over 24-hr postdosing, and analyzed for EG, GA, and OX using GC/MS techniques. Pharmacokinetic parameters including Cmax, Tmax, AUC, and betat((1/2)) were determined for EG and GA. Pregnancy status (GD 10-11) had no impact on the pharmacokinetic parameters investigated. Blood levels of GA were roughly dose-proportional from 10 to 150 mg EG/kg, but increased disproportionately from 500 to 1000 mg EG/kg. EG and GA exhibited dose-dependent urinary elimination at doses > or = 500 mg EG/kg, probably due to saturation of metabolic conversion of EG to GA, and of GA to downstream metabolites. The shift to nonlinear kinetics encompassed the NOEL (500 mg EG/kg) and LOEL (1000 mg EG/kg) for developmental toxicity of EG in rats, providing additional evidence for the role of GA in EG developmental toxicity. The peak maternal blood concentration of GA associated with the LOEL for developmental toxicity in the rat was quite high (363 microg/g or 4.8 mM blood). OX was a very minor metabolite in both blood and urine at all dose levels, suggesting that OX is not important for EG developmental toxicity.