Publications by authors named "Kenneth McMartin"

40 Publications

Diethylene glycol produces nephrotoxic and neurotoxic effects in female rats.

Clin Toxicol (Phila) 2021 Jul 19:1-8. Epub 2021 Jul 19.

Department of Pharmacology, Toxicology and Neuroscience, LSU Health Sciences Center, Shreveport, LA, USA.

Context: Diethylene glycol (DEG) is an organic compound found in household products but also as a counterfeit solvent in medicines. DEG poisonings are characterized by acute kidney injury (AKI) and by neurological sequelae such as decreased reflexes or face and limb weakness. Previous studies in male rats have demonstrated that neurotoxic effects develop only with the establishment of AKI, but the dose sensitivity of females to DEG toxicity is unknown.

Objectives: Assessing whether subacute administration of DEG in female rats would delineate any sex-differences in neuropathy or in kidney injury.

Methods: Female Wistar-Han rats were orally administered doses of 4 - 6 g/kg DEG every 12 h and monitored for 7 days. Urine was collected every 12 h and endpoint blood and cerebrospinal fluid (CSF) were collected for renal plasma parameters and total protein estimation, respectively. Motor function tests were conducted before and after treatment. Kidney and brain tissue were analyzed for metabolite content.

Results: Of 12 animals treated with DEG, 3 developed AKI as confirmed by increased BUN and creatinine concentrations. Renal and brain DGA contents were increased in animals that developed AKI compared to animals without AKI. Total CSF protein content in animals with AKI was markedly elevated compared to control and to treated animals without AKI. Decreases in forelimb grip strength and in locomotor and rearing activity were observed in animals with AKI compared to control and to animals without AKI.

Discussion: Repeated dosing with DEG in a female model produced nephrotoxic effects at a dose similar to that in males. The decrease in motor function and increase in CSF protein were only present in females that developed AKI. However, kidney and neurologic effects were assessed only at the end of the treatments, thus limiting determination of which effect occurs first. Limb function and coordination were measured globally and more sensitive tests such as nerve conduction studies might offer a detailed neurotoxicity assessment of the effects of DEG.

Conclusions: These studies show that DEG toxicity does not appear to be sex-specific and that, in males and females, neurological symptoms are present only when DGA accumulation and kidney injury also occur.
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http://dx.doi.org/10.1080/15563650.2021.1953049DOI Listing
July 2021

Neurotoxic effects of nephrotoxic compound diethylene glycol.

Clin Toxicol (Phila) 2021 Jan 21:1-17. Epub 2021 Jan 21.

Department of Pharmacology, Toxicology and Neuroscience, LSU Health Sciences Center, Shreveport, LA, USA.

Context: Diethylene glycol (DEG) is an organic compound found in household products but also as an adulterant in medicines by acting as a counterfeit solvent. DEG poisonings have been characterized predominately by acute kidney injury (AKI), but also by delayed neurological sequelae such as decreased reflexes or face and limb weakness.

Objectives: Characterizing the neurological symptoms of DEG poisoning in a subacute animal model would create a clearer picture of overall toxicity and possibly make mechanistic connections between kidney injury and neuropathy.

Methods: Male Wistar-Han rats were orally administered doses of 4 - 6 g/kg DEG every 12 or 24 h and monitored for 7 days. Urine was collected every 12 h and endpoint blood and cerebrospinal fluid (CSF) were collected for a renal plasma panel and total protein estimation, respectively. Motor function tests were conducted before and after treatment. Kidney and brain tissue was harvested for metabolic analysis.

Results: Of the 43 animals treated with DEG, 11 developed AKI as confirmed by increased BUN and creatinine levels. Renal and brain DGA accumulation was markedly increased in animals that developed AKI compared to animals without AKI. The total protein content in CSF in animals with kidney injury was markedly elevated compared to control and to treated animals without AKI. Significant decreases in forelimb grip strength and decreases in locomotor and rearing activity were observed in animals with AKI compared to control and to animals without AKI.

Discussion: Repeated dosing with DEG in an animal model produced nephrotoxic effects like those in studies with acute DEG administration. The decrease in motor function and increase in CSF protein were only present in animals that developed AKI.

Conclusions: These studies show development of neurotoxicity in this DEG animal model and suggest that neurological symptoms are observed only when DGA accumulation and kidney injury also occur.
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http://dx.doi.org/10.1080/15563650.2021.1874403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292454PMC
January 2021

Calcium Oxalate Monohydrate is Associated with Endothelial Cell Toxicity But Not with Reactive Oxygen Species Accumulation.

Cardiovasc Toxicol 2020 12;20(6):593-603

Department of Pharmacology, Toxicology & Neuroscience, Louisiana State University Health Sciences Center, Shreveport, LA, 71130-3932, USA.

One characteristic of ethylene glycol overdose is a cardiopulmonary syndrome including hypertension and pulmonary edema with pathology indicating damage to the endothelium of heart, lung and brain vessels. The mechanism of the cardiopulmonary toxicity is unknown, but has been linked with accumulation of the metabolite calcium oxalate monohydrate (COM) in the endothelium. These studies have evaluated the hypothesis that COM or the oxalate ion produces endothelial damage in vitro and that damage is linked with induction of reactive oxygen species (ROS). In cultured human umbilical vein endothelial cells (HUVEC), COM, but not the oxalate ion, produced cytotoxicity in a dose- and time-dependent manner. Using three ROS-sensitive dyes, HUVEC exposed to COM did not significantly increase ROS production. Additionally, co-treatment with three antioxidants that operate by different mechanisms did not reduce COM cytotoxicity. As such, an increase in ROS production does not explain cell death in endothelial cells. Aluminum citrate, uniquely among citrate compounds, significantly reduced COM cytotoxicity to endothelial cells and thus may act as an adjunct therapy for ethylene glycol poisoning to reduce endothelial damage. These results imply that accumulation of COM in endothelial cells is an important aspect of the cardiopulmonary toxicity from ethylene glycol.
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http://dx.doi.org/10.1007/s12012-020-09584-4DOI Listing
December 2020

Consensus statements on the approach to patients in a methanol poisoning outbreak.

Clin Toxicol (Phila) 2019 12 22;57(12):1129-1136. Epub 2019 Jul 22.

The Norwegian CBRNE Centre of Medicine, Department of Acute Medicine, Oslo University Hospital, Oslo, Norway.

Methanol poisoning is an important cause of mortality and morbidity worldwide. Although it often occurs as smaller sporadic events, epidemic outbreaks are not uncommon due to the illicit manufacture and sale of alcoholic beverages. We aimed to define methanol poisoning outbreak (MPO), outline an approach to triaging an MPO, and define criteria for prioritizing antidotes, extracorporeal elimination treatments (i.e., dialysis), and indications for transferring patients in the context of an MPO. We convened a group of experts from across the world to explore geographical, socio-cultural and clinical considerations in the management of an MPO. The experts answered specific open-ended questions based on themes aligned to the goals of this project. This project used a modified Delphi process. The discussion continued until there was condensation of themes. We defined MPO as a sudden increase in the number of cases of methanol poisoning during a short period of time above what is normally expected in the population in that specific geographic area. Prompt initiation of an antidote is necessary in MPOs. Scarce hemodialysis resources require triage to identify patients most likely to benefit from this treatment. The sickest patients should not be transferred unless the time for transfer is very short. Transporting extracorporeal treatment equipment and antidotes may be more efficient. We have developed consensus statements on the response to a methanol poisoning outbreak. These can be used in any country and will be most effective when they are discussed by health authorities and clinicians prior to an outbreak.
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http://dx.doi.org/10.1080/15563650.2019.1636992DOI Listing
December 2019

Curcumin gum formulation for prevention of oral cavity head and neck squamous cell carcinoma.

Laryngoscope 2019 07 13;129(7):1597-1603. Epub 2018 Nov 13.

Department of Otolaryngology-Head and Neck Surgery, Louisiana State University- Health Sciences Center, Shreveport, Louisiana, U.S.A.

Objectives/hypothesis: Head and neck squamous cell carcinoma represents the sixth most common cancer. As a result of field cancerization, second primaries and recurrences are high. Hence, research has focused on chemoprevention. Curcumin, a polyphenol compound with anticarcinogenic properties, is one such promising nutraceutical. As poor bioavailability limits curcumin's use, a novel gum formulation was tested allowing for direct mucosal absorption into the bloodstream. This preliminary study validates curcumin gum efficacy by assessing release and transmucosal absorption, along with measuring its effects on serum cytokine levels.

Study Design: Clinical trial.

Methods: Protocols consisting of initial chew (chewing gum for 30 minutes) and revised chew (alternating chewing and parking gum against buccal mucosa for 30 minutes) were tested in healthy volunteers. High-performance liquid chromatography measured remnant curcumin in chewed gum, serum, and saliva. Serum levels were assayed for 15 proinflammatory cytokines via multiplex analysis.

Results: Revised chew samples demonstrated significantly higher curcumin release and absorption (P = .0078). Curcumin serum levels were significantly higher at 4 hours in samples > 2.0 g of curcumin release (P = .01). As saliva levels decreased, a concurrent increase in serum levels was observed, with no significance in the inverse relationship (P = .1423). When evaluating differences between gender, race, and age, the Asian population showed significantly lower curcumin release and serum levels (P = .009). CXCL1 (GRO-α) and TNF-α were significantly decreased in serum after chewing the gum (P = .036, P < .001, respectively).

Conclusions: Enhanced mucosal contact appears critical in improving curcumin release and absorption. CXCL1 and TNF-α both represent potential biomarkers for the future study of curcumin chemoprevention.

Level Of Evidence: 2b Laryngoscope, 129:1597-1603, 2019.
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http://dx.doi.org/10.1002/lary.27542DOI Listing
July 2019

Human health assessment for long-term oral ingestion of diethylene glycol.

Regul Toxicol Pharmacol 2017 Jun 29;87 Suppl 2:S1-S20. Epub 2017 Apr 29.

Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, MI 48640 USA. Electronic address:

Diethylene glycol (DEG) is an organic chemical that is used mostly as a chemical intermediate and has minor uses as a solvent or antifreeze in consumer products; these minor uses could result in potential human exposure. Potential short and long-term human exposures also occur from misuses. The considerable reporting of DEG misuses as a substitute for other solvents in drug manufacturing and summaries of important events in the history of DEG poisonings are reviewed. Given the potential for human exposure, the disposition and toxicity of DEG were examined, and a health assessment was performed. Toxicokinetics and metabolism studies are evaluated, along with a discussion on the renal toxicity mode of action in the rat. Additionally, in-depth assessments of the key animal research studies on the toxic effects of DEG from oral ingestion for various exposure time periods are presented with determination of NOAELs and LOAELs from the long-term exposure animal studies. These are applied in the derivation of a reference dose for a non-cancer endpoint from chronic exposure, resulting in a value of 0.3 mg DEG/kg bw.
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http://dx.doi.org/10.1016/j.yrtph.2017.03.027DOI Listing
June 2017

In-vivo evidence of nephrotoxicity and altered hepatic function in rats following administration of diglycolic acid, a metabolite of diethylene glycol.

Clin Toxicol (Phila) 2017 Mar 11;55(3):196-205. Epub 2017 Jan 11.

a Department of Pharmacology, Toxicology and Neuroscience , Louisiana State University Health Sciences Center - Shreveport , Shreveport , LA , USA.

Context: Diglycolic acid (DGA) is one of the two primary metabolites of diethylene glycol (DEG). DEG is an industrial solvent that has been implicated in mass poisonings resulting from product misuse in the United States and worldwide, with the hallmark toxicity being acute kidney injury, hepatotoxicity, encephalopathy and peripheral neuropathy. Our laboratory has generated in-vitro evidence suggesting that DGA is the metabolite responsible for the proximal tubule necrosis and decreased kidney function observed following DEG ingestion. Furthermore, we have shown that DGA specifically accumulates in kidney tissues (100× higher than peak blood concentrations) following DEG administration.

Objective: To examine renal and hepatic accumulation and dysfunction following direct administration of DGA in-vivo. We hypothesize that administration of DGA will result in renal and hepatic DGA accumulation, as well as proximal tubular necrosis and liver injury.

Materials And Methods: Adult male Wistar rats were divided into three groups dosed with 0, 100 or 300 mg/kg DGA via single oral gavage. Urine was collected every 6-12 h and blood, kidneys and liver were removed upon sacrifice at 48 h post-dosing for analysis.

Results: DGA accumulated significantly in both kidney and liver tissue only at 300 mg DGA/kg. DGA concentrations in the kidneys and liver correlated with renal and hepatic injury, respectively. Histopathological and clinical chemistry analysis revealed that DGA-treated animals exhibited moderate liver fatty accumulation and marked renal injury, again only at 300 mg/kg.

Discussion: DGA-induced kidney injury demonstrated a steep dose response threshold, where severe damage occurred only in animals given 300 mg/kg DGA, while no toxicity was observed at 100 mg/kg.

Conclusion: These results provide evidence for in-vivo toxicity following direct administration of DGA, a metabolite of DEG. The steep dose-response threshold for toxicity suggests mechanistically that there is likely a saturable step that results in DGA accumulation in target organs.
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http://dx.doi.org/10.1080/15563650.2016.1271128DOI Listing
March 2017

Diglycolic acid, the toxic metabolite of diethylene glycol, chelates calcium and produces renal mitochondrial dysfunction in vitro.

Clin Toxicol (Phila) 2016 Jul 22;54(6):501-11. Epub 2016 Mar 22.

b Department of Pharmacology, Toxicology, and Neuroscience , Louisiana State University Health Sciences Center , Shreveport , LA , USA ;

Context: Diethylene glycol (DEG) has caused many cases of acute kidney injury and deaths worldwide. Diglycolic acid (DGA) is the metabolite responsible for the renal toxicity, but its toxic mechanism remains unclear.

Objective: To characterize the mitochondrial dysfunction produced from DGA by examining several mitochondrial processes potentially contributing to renal cell toxicity.

Materials And Methods: The effect of DGA on mitochondrial membrane potential was examined in normal human proximal tubule (HPT) cells. Isolated rat kidney mitochondria were used to assess the effects of DGA on mitochondrial function, including respiratory parameters (States 3 and 4), electron transport chain complex activities and calcium-induced opening of the mitochondrial permeability transition pore. DGA was compared with ethylene glycol tetraacetic acid (EGTA) to determine calcium chelating ability. DGA cytotoxicity was assessed using lactate dehydrogenase leakage from cultured proximal tubule cells.

Results: DGA decreased the mitochondrial membrane potential in HPT cells. In rat kidney mitochondria, DGA decreased State 3 respiration, but did not affect State 4 respiration or the ADP/O ratio. DGA reduced glutamate/malate respiration at lower DGA concentrations (0.5 mmol/L) than succinate respiration (100 mmol/L). DGA inhibited Complex II activity without altering Complex I, III or IV activities. DGA blocked calcium-induced mitochondrial swelling, indicating inhibition of the calcium-dependent mitochondrial permeability transition. DGA and EGTA reduced the free calcium concentration in solution in an equimolar manner. DGA toxicity and mitochondrial dysfunction occurred as similar concentrations.

Discussion: DGA inhibited mitochondrial respiration, but without uncoupling oxidative phosphorylation. The more potent effect of DGA on glutamate/malate respiration and the inhibition of mitochondrial swelling was likely due to its chelation of calcium.

Conclusion: These results indicate that DGA produces mitochondrial dysfunction by chelating calcium to decrease the availability of substrates and of reducing equivalents to access Complex I and by inhibiting Complex II activity at higher concentrations.
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http://dx.doi.org/10.3109/15563650.2016.1162312DOI Listing
July 2016

Enhanced Systemic Bioavailability of Curcumin Through Transmucosal Administration of a Novel Microgranular Formulation.

Anticancer Res 2015 Dec;35(12):6411-8

Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA, U.S.A. Department of Otolaryngology-Head and Neck Surgery, Louisiana State University Health Sciences Center, Shreveport, LA, U.S.A.

Background/aim: Curcumin is a promising nutraceutical for chemoprevention of head and neck squamous cell carcinoma (HNSCC). Capsular formulations of curcumin demonstrate low systemic bioavailability. We aimed to determine if curcumin levels were higher in healthy volunteers and cancer patients with microgranular curcumin that allows for transmucosal absorption and identify a consistent biomarker.

Patients And Methods: Eight healthy volunteers and 15 HNSCC patients completed the trials. Serum levels of curcumin were measured by HPLC. Biological activity of curcumin was assessed with Multiplex Immunoassay and immunohistochemistry.

Results: We achieved higher serum levels of curcumin compared to trials using capsular formulation. In cancer patients a significant decrease in expression of fibroblast growth factor-2 (FGF-2) in post-biopsy samples and decreased serum levels of FGF-2, granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-17 (IL-17) (p<0.05) was observed.

Conclusion: Transmucosal administration of microgranular curcumin leads to enhanced curcumin bioavailability that is associated with significant biological effects.
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December 2015

Antidotes for poisoning by alcohols that form toxic metabolites.

Br J Clin Pharmacol 2016 Mar 4;81(3):505-15. Epub 2016 Jan 4.

The Norwegian CBRNe Centre of Medicine, Department of Acute Medicine, Division of Medicine, Oslo University Hospital, NO-0424, Oslo, Norway.

The alcohols, methanol, ethylene glycol and diethylene glycol, have many features in common, the most important of which is the fact that the compounds themselves are relatively non-toxic but are metabolized, initially by alcohol dehydrogenase, to various toxic intermediates. These compounds are readily available worldwide in commercial products as well as in homemade alcoholic beverages, both of which lead to most of the poisoning cases, from either unintentional or intentional ingestion. Although relatively infrequent in overall occurrence, poisonings by metabolically-toxic alcohols do unfortunately occur in outbreaks and can result in severe morbidity and mortality. These poisonings have traditionally been treated with ethanol since it competes for the active site of alcohol dehydrogenase and decreases the formation of toxic metabolites. Although ethanol can be effective in these poisonings, there are substantial practical problems with its use and so fomepizole, a potent competitive inhibitor of alcohol dehydrogenase, was developed for a hopefully better treatment for metabolically-toxic alcohol poisonings. Fomepizole has few side effects and is easy to use in practice and it may obviate the need for haemodialysis in some, but not all, patients. Hence, fomepizole has largely replaced ethanol as the toxic alcohol antidote in many countries. Nevertheless, ethanol remains an important alternative because access to fomepizole can be limited, the cost may appear excessive, or the physician may prefer ethanol due to experience.
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http://dx.doi.org/10.1111/bcp.12824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767193PMC
March 2016

Diethylene glycol-induced toxicities show marked threshold dose response in rats.

Toxicol Appl Pharmacol 2015 Feb 26;282(3):244-51. Epub 2014 Dec 26.

Department of Pharmacology, Toxicology, & Neuroscience, Louisiana State University Health Sciences Center, Shreveport, LA, United States. Electronic address:

Diethylene glycol (DEG) exposure poses risks to human health because of widespread industrial use and accidental exposures from contaminated products. To enhance the understanding of the mechanistic role of metabolites in DEG toxicity, this study used a dose response paradigm to determine a rat model that would best mimic DEG exposure in humans. Wistar and Fischer-344 (F-344) rats were treated by oral gavage with 0, 2, 5, or 10g/kg DEG and blood, kidney and liver tissues were collected at 48h. Both rat strains treated with 10g/kg DEG had equivalent degrees of metabolic acidosis, renal toxicity (increased BUN and creatinine and cortical necrosis) and liver toxicity (increased serum enzyme levels, centrilobular necrosis and severe glycogen depletion). There was no liver or kidney toxicity at the lower DEG doses (2 and 5g/kg) regardless of strain, demonstrating a steep threshold dose response. Kidney diglycolic acid (DGA), the presumed nephrotoxic metabolite of DEG, was markedly elevated in both rat strains administered 10g/kg DEG, but no DGA was present at 2 or 5g/kg, asserting its necessary role in DEG-induced toxicity. These results indicate that mechanistically in order to produce toxicity, metabolism to and significant target organ accumulation of DGA are required and that both strains would be useful for DEG risk assessments.
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http://dx.doi.org/10.1016/j.taap.2014.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5237385PMC
February 2015

Quantitation of diethylene glycol and its metabolites by gas chromatography mass spectrometry or ion chromatography mass spectrometry in rat and human biological samples.

J Anal Toxicol 2014 May 25;38(4):184-93. Epub 2014 Mar 25.

1Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, MI, USA.

The misuse of the commonly used chemical diethylene glycol (DEG) has lead to many poisonings worldwide. Methods were developed for analysis of DEG and its potential metabolites; ethylene glycol, glycolic acid, oxalic acid, diglycolic acid and hydroxyethoxy acetic acid in human urine, serum and cerebrospinal fluid samples, collected following a DEG-associated poisoning in the Republic of Panama during 2006. In addition, methods were developed for rat blood, urine, kidney and liver tissue to support toxicokinetic analysis during the conduct of DEG acute toxicity studies in the rat. Sample analysis was conducted using two techniques; ion chromatography with suppressed conductivity and negative ion electrospray ionization with MS detection or with gas chromatography using electron impact ionization or methane negative chemical ionization with MS detection. Stable-isotope-labeled analogs of each analyte were employed as quantitative internal standards in the assays.
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http://dx.doi.org/10.1093/jat/bku018DOI Listing
May 2014

Diglycolic acid inhibits succinate dehydrogenase activity in human proximal tubule cells leading to mitochondrial dysfunction and cell death.

Toxicol Lett 2013 Aug 1;221(3):176-84. Epub 2013 Jul 1.

Department of Pharmacology, Toxicology, and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, LA, USA.

Diethylene glycol (DEG) is a solvent used in consumer products allowing the increased risk for consumer exposure. DEG metabolism produces two primary metabolites, 2-hydroxyethoxyacetic acid (2-HEAA) and diglycolic acid (DGA). DGA has been shown to be the toxic metabolite responsible for the proximal tubule cell necrosis seen in DEG poisoning. The mechanism of DGA toxicity in the proximal tubule cell is not yet known. The chemical structure of DGA is very similar to citric acid cycle intermediates. Studies were designed to assess whether its mechanism of toxicity involves disruption of cellular metabolic pathways resulting in mitochondrial dysfunction. First, DGA preferentially inhibited succinate dehydrogenase, including human kidney cell enzyme, but had no effect on other citric acid cycle enzyme activities. DGA produces a cellular ATP depletion that precedes cell death. Human proximal tubule (HPT) cells, pre-treated with increasing DGA concentrations, showed significantly decreased oxygen consumption. DGA did not increase lactate levels, indicating no effect on glycolytic activity. DGA increased reactive oxygen species (ROS) production in HPT cells in a concentration and time dependent manner. These results indicate that DGA produced proximal tubule cell dysfunction by specific inhibition of succinate dehydrogenase and oxygen consumption. Disruption of these processes results in decreased energy production and proximal tubule cell death.
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http://dx.doi.org/10.1016/j.toxlet.2013.06.231DOI Listing
August 2013

Aluminum citrate blocks toxicity of calcium oxalate crystals by preventing binding with cell membrane phospholipids.

Am J Nephrol 2013 4;37(1):41-9. Epub 2013 Jan 4.

Department of Pharmacology, Toxicology and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA.

Background/aims: Renal damage from ethylene glycol and primary hyperoxaluria is linked to accumulation of calcium oxalate monohydrate (COM) crystals in the renal proximal tubule (PT). In vitro studies have shown that aluminum citrate (AC), uniquely among citrate salts, blocks COM cytotoxicity to tubular cells. These studies were designed to evaluate the interaction of COM with membrane phospholipids and the ability of AC to reduce COM toxicity by interfering with this interaction.

Methods: Interaction of COM with phospholipids was assessed using differential scanning calorimetric analysis of structural changes in specific liposomes. Interaction of COM with cell membranes was studied by measuring binding of radiolabeled crystals by human PT (HPT) cells.

Results: Analysis of liposomes prepared from phosphatidylserine (PS) or phosphatidylcholine (PC) showed that COM interfered with the gel-liquid transition of PS liposomes, but not that of PC liposomes. AC reversed the COM-induced changes in liposomal structure. AC inhibited the binding of [(14)C]-COM by HPT cells in a concentration-dependent manner. AC blocked COM binding by interacting with the crystal surface and not the cell membrane.

Conclusion: These results indicate that AC blocks the binding of COM by PT cells, and consequently its cytotoxicity, by attaching to the surface of the crystal. Thus, AC, or a related compound that works by the same mechanism, could be a useful adjunct therapy to reduce the renal damage produced by severe hyperoxaluria.
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http://dx.doi.org/10.1159/000345985DOI Listing
August 2013

Oral Reference Dose for ethylene glycol based on oxalate crystal-induced renal tubule degeneration as the critical effect.

Regul Toxicol Pharmacol 2013 Mar 22;65(2):229-41. Epub 2012 Dec 22.

Snellings Toxicology Consulting, LLC, 23 Trails End Lane, Ridgefield, CT 06877, USA.

Several risk assessments have been conducted for ethylene glycol (EG). These assessments identified the kidney as the primary target organ for chronic effects. None of these assessments have incorporated the robust database of species-specific toxicokinetic and toxicodynamic studies with EG and its metabolites in defining uncertainty factors used in reference value derivation. Pertinent in vitro and in vivo studies related to one of these metabolites, calcium oxalate, and its role in crystal-induced nephropathy are summarized, and the weight of evidence to establish the mode of action for renal toxicity is reviewed. Previous risk assessments were based on chronic rat studies using a strain of rat that was later determined to be less sensitive to the toxic effects of EG. A recently published 12-month rat study using the more sensitive strain (Wistar) was selected to determine the point of departure for a new risk assessment. This approach incorporated toxicokinetic and toxicodynamic data and used Benchmark Dose methods to calculate a Human Equivalent Dose. Uncertainty factors were chosen, depending on the quality of the studies available, the extent of the database, and scientific judgment. The Reference Dose for long-term repeat oral exposure to EG was determined to be 15 mg/kg bw/d.
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http://dx.doi.org/10.1016/j.yrtph.2012.12.005DOI Listing
March 2013

Aluminum citrate prevents renal injury from calcium oxalate crystal deposition.

J Am Soc Nephrol 2012 Dec 8;23(12):2024-33. Epub 2012 Nov 8.

Department of Pharmacology, Toxicology, and Neuroscience, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932, USA.

Calcium oxalate monohydrate crystals are responsible for the kidney injury associated with exposure to ethylene glycol or severe hyperoxaluria. Current treatment strategies target the formation of calcium oxalate but not its interaction with kidney tissue. Because aluminum citrate blocks calcium oxalate binding and toxicity in human kidney cells, it may provide a different therapeutic approach to calcium oxalate-induced injury. Here, we tested the effects of aluminum citrate and sodium citrate in a Wistar rat model of acute high-dose ethylene glycol exposure. Aluminum citrate, but not sodium citrate, attenuated increases in urea nitrogen, creatinine, and the ratio of kidney to body weight in ethylene glycol-treated rats. Compared with ethylene glycol alone, the addition of aluminum citrate significantly increased the urinary excretion of both crystalline calcium and crystalline oxalate and decreased the deposition of crystals in renal tissue. In vitro, aluminum citrate interacted directly with oxalate crystals to inhibit their uptake by proximal tubule cells. These results suggest that treating with aluminum citrate attenuates renal injury in rats with severe ethylene glycol toxicity, apparently by inhibiting calcium oxalate's interaction with, and retention by, the kidney epithelium.
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http://dx.doi.org/10.1681/ASN.2012040357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507364PMC
December 2012

Kinetics of fomepizole in pregnant rats.

Clin Toxicol (Phila) 2012 Sep 14;50(8):743-8. Epub 2012 Aug 14.

Department of Pharmacology, Toxicology and Neuroscience, Lousiana State University Health Sciences Center, Shreveport, LA 71130, USA.

Context/objectives: Fomepizole has been utilized with remarkable success for ethylene glycol and methanol poisonings in children and adults. However, very little information is available regarding the safe and effective use of fomepizole in pregnancy. The goal of this research was to utilize an animal model to investigate the kinetics of fomepizole in pregnancy.

Materials/methods: Male and pregnant Sprague-Dawley rats, which were obtained at 19 days gestation, were administered fomepizole 15 mg/kg intraperitoneally. The animals were anesthetized and blood, liver, kidney, and fetus samples were collected at 1-24 hours post administration. Tissue samples were homogenized, deproteinized and prepared by solid phase extraction. Fomepizole concentrations from tissue and serum samples were analyzed using high pressure liquid chromatography.

Results: Between males and pregnant females, tissue and serum fomepizole levels were similar. Fomepizole concentrations in whole fetal tissue were similar to those in the maternal liver and kidney tissue. Fetal fomepizole concentrations were fivefold higher than maternal serum concentrations. The zero order elimination rate of fomepizole from maternal serum was 7.6 mol/L/h, which was slightly slower than the elimination rate in male rats (12.9 mol/L/h). Elimination of fomepizole from the fetus followed a similar time course to that in the maternal tissues.

Discussion/conclusion: Elevated concentrations of fomepizole were detected in the fetus following maternal administration. Although the levels of fomepizole in the fetal tissue would imply significant protection against fetal formation of toxic alcohol metabolites, further research is needed to explore the long-term effects of fomepizole on the fetus.
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http://dx.doi.org/10.3109/15563650.2012.716518DOI Listing
September 2012

Kinetics and metabolism of fomepizole in healthy humans.

Clin Toxicol (Phila) 2012 Jun 4;50(5):375-83. Epub 2012 May 4.

Department of Pharmacology, Toxicology and Neuroscience, Lousiana State University Health Sciences Center - Shreveport, Shreveport , LA 71130-3932, USA.

Context/objective: Fomepizole, a potent inhibitor of alcohol dehydrogenase, has replaced ethanol as antidote for methanol and ethylene glycol intoxications because of a longer duration of action and fewer adverse effects. Prior human studies have indicated that single doses of fomepizole are eliminated by Michaelis-Menten kinetics, but two studies in poisoned patients have suggested that first order elimination occurs after multiple doses. Because of the contrast in fomepizole kinetics among existing studies and the lack of information regarding its metabolism in humans, kinetic and metabolic studies were conducted after single doses and after multiple oral doses in healthy human subjects.

Materials/methods: In a single-dose, crossover study, healthy humans received fomepizole IV or orally (7 mg/kg). Also to define the metabolism and kinetics of fomepizole when administered over the presumed antidotal period, subjects were divided into three groups, which were given oral loading doses of 10-15 mg/kg, followed by supplemental doses of 3-10 mg/kg/12 h through 96 hours.

Results: The single dose study confirmed that fomepizole was eliminated by saturable, nonlinear kinetics, primarily by metabolism, and subsequent renal excretion of the metabolite 4-carboxypyrazole (4-CP). In the multi-dose study, the zero order elimination rate of fomepizole increased with increasing duration of treatment (from mean of 3 μmol/L/h after first dose to 14 μmol/L/h after 72 hours). Consistent with the enhanced elimination of fomepizole, the rate of urinary excretion of 4-CP increased with time. After 96 hours, fomepizole elimination apparently changed to first order kinetics with a t(½) of 1.5-2 hours.

Discussion/conclusion: The results suggest that fomepizole induces its own metabolism via cytochrome P-450, leading to enhanced fomepizole elimination and 4-CP excretion. Thus, to maintain relatively constant plasma levels of fomepizole during therapy, increased supplemental doses at about 36-48 hours are needed to overcome the increased elimination of fomepizole. As such, these kinetic evaluations in healthy humans support the current dosing recommendations for fomepizole.
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http://dx.doi.org/10.3109/15563650.2012.683197DOI Listing
June 2012

Diglycolic acid is the nephrotoxic metabolite in diethylene glycol poisoning inducing necrosis in human proximal tubule cells in vitro.

Toxicol Sci 2011 Nov 18;124(1):35-44. Epub 2011 Aug 18.

Department of Pharmacology, Toxicology, and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932, USA.

Diethylene glycol (DEG), a solvent and chemical intermediate, can produce an acute toxic syndrome, the hallmark of which is acute renal failure due to cortical tubular degeneration and proximal tubular necrosis. DEG is metabolized to two primary metabolites, 2-hydroxyethoxyacetic acid (2-HEAA) and diglycolic acid (DGA), which are believed to be the proximate toxicants. The precise mechanism of toxicity has yet to be elucidated, so these studies were designed to determine which metabolite was responsible for the proximal tubule cell death. Human proximal tubule (HPT) cells in culture, obtained from normal cortical tissue and passaged 3-6 times, were incubated with increasing concentrations of DEG, 2-HEAA, or DGA separately and in combination for 48 h at pH 6 or 7.4, and various parameters of necrotic and apoptotic cell death were measured. DEG and 2-HEAA did not produce any cell death. DGA produced dose-dependent necrosis at concentrations above 25 mmol/l. DGA did not affect caspase-3 activity and increased annexin V staining only in propidium iodide-stained cells. Hence, DGA induced necrosis, not apoptosis, as corroborated by severe depletion of cellular adenosine triphosphate levels. DGA is structurally similar to citric acid cycle intermediates that are taken up by specific transporters in kidney cells. HPT cells, incubated with N-(p-amylcinnamoyl)anthranilic acid, a sodium dicarboxylate-1 transporter inhibitor showed significantly decreased cell death compared with DGA alone. These studies demonstrate that DGA is the toxic metabolite responsible for DEG-induced proximal tubular necrosis and suggest a possible transporter-mediated uptake of DGA leading to toxic accumulation and cellular dysfunction.
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http://dx.doi.org/10.1093/toxsci/kfr204DOI Listing
November 2011

Role of tissue metabolite accumulation in the renal toxicity of diethylene glycol.

Toxicol Sci 2011 Oct 29;123(2):374-83. Epub 2011 Jul 29.

Department of Pharmacology, Toxicology and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932, USA.

Misuse of diethylene glycol (DEG) has led to numerous epidemic poisonings worldwide. DEG produces toxicity because of its metabolism, although the mechanism of its toxicity has not been further defined. The purpose of this study was to investigate the accumulation of specific metabolites in blood and target organ tissues and to determine the relationship between tissue accumulation of metabolites and the resulting toxicity. Wistar rats were treated with water, 2 g/kg DEG (low dose), 10 g/kg DEG (high dose), or 10 g/kg DEG + fomepizole (15 mg/kg then 10 mg/kg per 12 h, to inhibit DEG metabolism), and blood and tissue samples were collected up to 48 h. After high doses of DEG, 2-hydroxyethoxyacetic acid (HEAA) was the primary metabolite in the blood (∼4 mmol/l), with only low concentrations of diglycolic acid (DGA) (∼0.04 mmol/l). In contrast, renal and hepatic concentrations of DGA and of HEAA at 48 h were similar (∼4 mmol/l), indicating a 100-fold concentrative uptake of DGA by kidney tissue. Treatment with fomepizole blocked the formation of HEAA and DGA and the kidney toxicity. Both HEAA and DGA concentrations in the kidney correlated strongly with the degree of kidney damage. Accumulation of HEAA in blood correlated with increased anion gap and decreased blood bicarbonate so appeared responsible for the DEG-induced acidosis. Although these studies suggest that either metabolite may be involved in producing kidney toxicity, the unexpected renal accumulation of DGA at toxic doses of DEG suggests that it must also be considered a possible toxic metabolite of DEG.
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http://dx.doi.org/10.1093/toxsci/kfr197DOI Listing
October 2011

Involvement of urinary proteins in the rat strain difference in sensitivity to ethylene glycol-induced renal toxicity.

Am J Physiol Renal Physiol 2010 Sep 9;299(3):F605-15. Epub 2010 Jun 9.

Department of Pharmacology, Toxicology, and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, USA.

Ethylene glycol (EG) exposure is a common model for kidney stones, because animals accumulate calcium oxalate monohydrate (COM) in kidneys. Wistar rats are more sensitive to EG than Fischer 344 (F344) rats, with greater COM deposition in kidneys. The mechanisms by which COM accumulates differently among strains are poorly understood. Urinary proteins inhibit COM adhesion to renal cells, which could alter COM deposition in kidneys. We hypothesize that COM accumulates more in Wistar rat kidneys because of lower levels of inhibitory proteins in urine. Wistar and F344 rats were treated with 0.75% EG in drinking water for 8 wk. Twenty-four-hour urine was collected every 2 wk for analysis of urinary proteins. Similar studies were conducted for 2 wk using 2% hydroxyproline (HP) as an alternative oxalate source. Total urinary protein was higher in F344 than Wistar rats at all times. Tamm-Horsfall protein was not different between strains. Osteopontin (OPN) levels in Wistar urine and kidney tissue were higher and were further increased by EG treatment. This increase in OPN occurred before renal COM accumulation. Untreated F344 rats showed greater CD45 and ED-1 staining in kidneys than untreated Wistars; in contrast, EG treatment increased CD45 and ED-1 staining in Wistars more than in F344 rats, indicating macrophage infiltration. This increase occurred in parallel with the increase in OPN and before COM accumulation. Like EG, HP induced markedly greater oxalate concentrations in the plasma and urine of Wistar rats compared with F344 rats. These results suggest that OPN upregulation and macrophage infiltration do not completely protect against COM accumulation and may be a response to crystal retention. Because the two oxalate precursors, EG and HP, produced similar elevations of oxalate, the strain difference in COM accumulation may result more so from metabolic differences between strains than from differences in urinary proteins or inflammatory responses.
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http://dx.doi.org/10.1152/ajprenal.00419.2009DOI Listing
September 2010

Inhibition of metabolism of diethylene glycol prevents target organ toxicity in rats.

Toxicol Sci 2010 Sep 7;117(1):25-35. Epub 2010 Jun 7.

Department of Pharmacology, Toxicology and Neuroscience, Louisiana State University Health Sciences Center-Shreveport, Shreveport, Louisiana 71130-3932, USA.

Diethylene glycol (DEG) is an industrial chemical, the misuse of which has led to numerous epidemic poisonings worldwide. The mechanism of its toxicity has not been defined as to the precise relationship between the metabolism of DEG and target organ toxicity. The purpose of this study was to investigate the mechanism for the acute toxicity of DEG, and the effect of the alcohol dehydrogenase inhibitor 4-methylpyrazole (fomepizole), by determining the relationship between accumulation of DEG or its metabolites and the resulting kidney and liver toxicity. Rats were treated by oral gavage with water, 2 g/kg DEG (low dose), 10 g/kg DEG (high dose), or 10 g/kg DEG + fomepizole, and blood and urine were collected over 48 h. Rats treated with high-dose DEG had metabolic acidosis, increased BUN and creatinine, and marked kidney necrosis, noted by histopathology. A minor degree of liver damage was noted at the high dose. After low and high doses of DEG, 2-hydroxyethoxyacetic acid (HEAA) was the primary metabolite in the urine, with only minor amounts of urinary diglycolic acid (DGA). Small amounts of ethylene glycol (EG), but not oxalate or glycolate, were observed in the urine. Treatment with fomepizole blocked the formation of HEAA and DGA and the development of metabolic acidosis and the kidney and liver toxicity. These results indicate that the mechanism for the target organ toxicity results from metabolites of DEG, and not DEG itself nor formation of EG from DEG, and that fomepizole may be a useful antidote for treating DEG poisoning.
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http://dx.doi.org/10.1093/toxsci/kfq167DOI Listing
September 2010

Renal toxicity of ethylene glycol results from internalization of calcium oxalate crystals by proximal tubule cells.

Toxicol Lett 2010 Feb 18;192(3):365-72. Epub 2009 Nov 18.

Department of Pharmacology, Toxicology & Neuroscience, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA.

Ethylene glycol exposure can lead to the development of renal failure due to the metabolic formation of calcium oxalate monohydrate (COM) crystals. The renal damage is closely linked to the degree of COM accumulation in the kidney and most likely results from a COM-induced injury to proximal tubule (PT) cells. The present studies have measured the binding and internalization of COM by primary cultures of normal PT cells from humans and from Wistar and Fischer-344 rats in order to examine the roles of these uptake processes in the resulting cytotoxicity. Internalization was determined by incubation of cells with [(14)C]-COM at 37 degrees C, removal of bound COM with an EDTA incubation, followed by solubilization of cells, as well as by transmission electron microscopy of COM-exposed cells. COM crystals were internalized by PT cells in time- and concentration-dependent manners. COM crystals were bound to and internalized by rat cells about five times more than by human cells. Binding and internalization values were similar between PT cells from Wistar and Fischer-344 rats, indicating that a differential uptake of COM does not explain the known strain difference in sensitivity to ethylene glycol renal toxicity. Internalization of COM correlated highly with the degree of cell death, which is greater in rat cells than in human cells. Thus, surface binding and internalization of COM by cells play critical roles in cytotoxicity and explain why rat cells are more sensitive to COM crystals. At the same level of COM accumulation after ethylene glycol exposure or hyperoxaluria in vivo, rats would be more susceptible than humans to COM-induced damage.
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http://dx.doi.org/10.1016/j.toxlet.2009.11.013DOI Listing
February 2010

Are calcium oxalate crystals involved in the mechanism of acute renal failure in ethylene glycol poisoning?

Authors:
Kenneth McMartin

Clin Toxicol (Phila) 2009 Nov;47(9):859-69

Department of Pharmacology, Toxicology & Neuroscience, Louisiana State University Health Sciences Center, Shreveport, LA, USA.

Introduction: Ethylene glycol (EG) poisoning often results in acute renal failure, particularly if treatment with fomepizole or ethanol is delayed because of late presentation or diagnosis. The mechanism has not been established but is thought to result from the production of a toxic metabolite.

Methods: A literature review utilizing PubMed identified papers dealing with renal toxicity and EG or oxalate. The list of papers was culled to those relevant to the mechanism and treatment of the renal toxicity associated with either compound. ROLE OF METABOLITES: Although the "aldehyde" metabolites of EG, glycolaldehyde, and glyoxalate, have been suggested as the metabolites responsible, recent studies have shown definitively that the accumulation of calcium oxalate monohydrate (COM) crystals in kidney tissue produces renal tubular necrosis that leads to kidney failure. In vivo studies in EG-dosed rats have correlated the severity of renal damage with the total accumulation of COM crystals in kidney tissue. Studies in cultured kidney cells, including human proximal tubule (HPT) cells, have demonstrated that only COM crystals, not the oxalate ion, glycolaldehyde, or glyoxylate, produce a necrotic cell death at toxicologically relevant concentrations. COM CRYSTAL ACCUMULATION: In EG poisoning, COM crystals accumulate to high concentrations in the kidney through a process involving adherence to tubular cell membranes, followed by internalization of the crystals. MECHANISM OF TOXICITY: COM crystals have been shown to alter membrane structure and function, to increase reactive oxygen species and to produce mitochondrial dysfunction. These processes are likely to be involved in the mechanism of cell death.

Conclusions: Accumulation of COM crystals in the kidney is responsible for producing the renal toxicity associated with EG poisoning. The development of a pharmacological approach to reduce COM crystal adherence to tubular cells and its cellular interactions would be valuable as this would decrease the renal toxicity not only in late treated cases of EG poisoning, but also in other hyperoxaluric diseases such as primary hyperoxaluria and kidney stone formation.
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http://dx.doi.org/10.3109/15563650903344793DOI Listing
November 2009

Relationship between serum glycolate and falsely elevated lactate in severe ethylene glycol poisoning.

J Anal Toxicol 2009 Apr;33(3):174-6

New York City Poison Control Center, Department of Health and Mental Hygiene, New York, New York, USA.

In the setting of ethylene glycol (EG) poisoning, a falsely elevated serum lactate concentration is suggested to be an assay cross-reaction with glycolate, but a concentration-dependent relationship has never been identified. We correlate serum lactate and glycolate concentrations in a case of severe EG poisoning. Serial EG [by gas chromatography (GC)], glycolate (derivatized to methyl glycolate, analysis by GC), and lactate (both enzymatic spectrophotometry and GC) concentrations were correlated at five time points. False-positive lactate was confirmed by absence of lactate on GC analysis. The correlation coefficient (Pearson's r) between lactate (by enzymatic spectrophotometry) and glycolate was 0.984 and was statistically significant (p < 0.01). The mean lactate/glycolate conversion factor was 2.58 +/- 0.95. We demonstrate the linear correlation between falsely elevated serum lactate and glycolate concentrations in a case of severe EG poisoning. Our data provide further support to the belief that the lactate assay may cross-react with glycolate in EG poisoning.
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http://dx.doi.org/10.1093/jat/33.3.174DOI Listing
April 2009

Strain differences in urinary factors that promote calcium oxalate crystal formation in the kidneys of ethylene glycol-treated rats.

Am J Physiol Renal Physiol 2009 May 25;296(5):F1080-7. Epub 2009 Feb 25.

Department of Pharmacology, Toxicology and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USA.

Ethylene glycol (EG)-induced hyperoxaluria is the most commonly employed experimental regimen as an animal model of calcium oxalate (CaOx) stone formation. The variant sensitivity to CaOx among different rat strains has not been fully explored, although the Wistar rat is known to accumulate more CaOx in kidney tissue after low-dose EG exposure than in the Fischer 344 (F344) rats. Supersaturation of CaOx in tubular fluid contributes to the amount of CaOx crystal formation in the kidney. We hypothesized that the urinary supersaturation of CaOx in Wistar rats is higher than that of F344 rats, thereby allowing for greater CaOx crystal deposition in the Wistar rat. Age-matched male Wistar and F344 rats were treated with 0.75% EG or drinking water for 8 wk. Twenty-four-hour urine was collected at 0, 2, 4, 6, and 8 wk for analysis of key electrolytes to calculate the CaOx supersaturation. Plasma oxalate level was also measured. Our data confirmed the different sensitivity to renal toxicity from EG between the two rat strains (Wistar > F344). After EG treatment, the plasma oxalate level and urine oxalate excretion were markedly greater in the Wistar rats than in the F344 rats, while urine calcium was slightly decreased in Wistars. Thus, the CaOx supersaturation in urine of Wistar rats was higher, which led to a greater crystal deposition in kidney in Wistar rats. These studies suggest that during EG treatment, changes in urine electrolytes and in CaOx supersaturation occur to a greater extent in the Wistar rat, in agreement with its greater sensitivity to EG toxicity.
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http://dx.doi.org/10.1152/ajprenal.90727.2008DOI Listing
May 2009

Methods to evaluate the effect of ethanol on the folate analogue: fluorescein methotrexate uptake in human proximal tubular cells.

Adv Pharmacol Sci 2009 29;2009:291349. Epub 2009 Sep 29.

Department of Pharmacology, LSU Health Sciences Center, Shreveport, LA 71118, USA.

Ethanol-induced folate deficiency is due to effects of ethanol on folate metabolism and absorption. We have already shown by using different methods that ethanol interferes with reabsorption of folate from the proximal tubule. In this study, we have used the folate analogue, the fluorescein methotrexate (FL-MTX), in order to evaluate effects of ethanol on FL-MTX uptake by the human proximal tubular (HPT) cells by using a confocal microscope and fluoroskan microplate reader. Since endothelins (ETs) play a major role in a number of diseases and also in the damage induced by a variety of chemicals, we have used endothelin-B (ET-B) and protein kinase-C (PKC) inhibitors to evaluate the role of endothelin in ethanol-mediated FL-MTX uptake by using fluoroskan microplate reader. Confocal microscope and fluoroskan studies reveal that cellular absorption of FL-MTX is concentration-dependent. Moreover, ethanol concentration has an impact on FL-MTX uptake. Fluoroskan studies reveal that the ethanol-induced decrease in FL-MTX uptake is reversed by adding the ET-B receptor antagonist (RES-701-1) or PKC-selective inhibitor (BIM). Thus, we can conclude that ethanol may act via ET and ET in turn may act via ET-B receptor and the PKC signaling pathway to impair FL-MTX transport.
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http://dx.doi.org/10.1155/2009/291349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2990105PMC
July 2011

Inhibition of NADPH oxidase activity promotes differentiation of B16 melanoma cells.

Oncol Rep 2008 May;19(5):1225-30

Department of Pharmacology, Toxicology and Neuroscience, LSU Health Sciences Center-Shreveport, Shreveport, LA 71130, USA.

The activity of NADPH oxidase is increased in malignant skin keratinocytes. We demonstrated that inhibition of NADPH oxidase activity by diphenyleneiodonium (DPI) suppressed free radical production, inhibited cell growth and promoted cell differentiation of B16 melanoma cells, as indicated by cell morphology, increased production of melanin, and increased expression of microphthalmia-associated transcription factor (MITF). siRNA to NADPH oxidase subunit Rac1 or p47 induced the expression of MITF, verifying that the pro-differentiation effects are due to the inhibition of NADPH oxidase. Biochemical studies suggest that ERK plays a positive role whereas PKCalpha plays a negative role during this differentiation event. In addition, the protein levels of the tumor suppressor p53 were suppressed by DPI, suggesting that p53 is activated by oxidative stress and may negatively regulate differentiation in melanoma cells. Taken together, these results suggest that inhibiting NADPH oxidase activity promotes cell differentiation of B16 melanoma cells.
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May 2008

Oral administration of fomepizole produces similar blood levels as identical intravenous dose.

Clin Toxicol (Phila) 2008 Mar;46(3):181-6

Department of Emergency Medicine, SUNY Upstate Medical University, Syracuse, New York 13210, USA.

Introduction: Fomepizole is available intravenously (i.v.) for the treatment of methanol and ethylene glycol poisoning. Few studies demonstrate that fomepizole achieves effective serum concentrations after i.v. or oral (p.o.) use. The objective was to describe the comparative pharmacokinetics of fomepizole after a single p.o. and i.v. dose.

Methods: This was a prospective, randomized, crossover trial in 10 healthy volunteers. Each received 15 mg/kg fomepizole, p.o. and by 30 minute i.v. infusion. Serum was collected at 0, 0.25, 0.5, 1, 2, 4, 7, 12, 24, 36, and 48 hours (h) and stored at -70 degrees C. Candidate models were fit to the i.v. and p.o. data, simultaneously, using iterative 2-stage analysis weighted by the estimated inverse observation variance. Time above the MEC (T>MEC) was determined by numeric integration of the fitted functions using 10 micromoles/L as the minimum effective concentration (MEC).

Results: Seven females and 3 males were enrolled. Sole complaints included headache and dizziness in 3 subjects and 10/10 reported an unpleasant taste. The final PK model was 2-compartment with 0-order i.v. and 1(st)-order p.o. input (following a fitted TLag) and Michaelis-Menten elimination. p.o. fomepizole was rapidly absorbed with a bioavailability of approximately 100%. The Km was 0.935+/-0.98 micromoles/L and the Vmax was 18.57+/-9.58 micromoles/L/h. T>MEC was 32 h with agreement between p.o. and i.v. dosing.

Conclusions: This is the first study that effectively determines a human Vmax and Km for p.o. and i.v. fomepizole. p.o. and i.v. administration of fomepizole result in similar pharmacokinetic parameters.
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http://dx.doi.org/10.1080/15563650701373796DOI Listing
March 2008

Calcium oxalate, and not other metabolites, is responsible for the renal toxicity of ethylene glycol.

Toxicol Lett 2007 Aug 20;173(1):8-16. Epub 2007 Jun 20.

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 (147-735microg/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.
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http://dx.doi.org/10.1016/j.toxlet.2007.06.010DOI Listing
August 2007
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