Variable sensitivity to diethylene glycol poisoning is related to differences in the uptake transporter for the toxic metabolite diglycolic acid.

INTRODUCTION/CONTEXT: Poisonings with diethylene glycol are characterized by acute kidney injury and peripheral neuropathy. In animal studies on the toxicities of diethylene glycol and its metabolite diglycolic acid, remarkable differences in susceptibility to acute kidney injury were observed in identically-dosed rats. In those studies, only about 60% showed acute kidney injury, yet all rats with acute kidney injury showed marked diglycolic acid accumulation in tissues, while no diglycolic acid accumulated in rats without injury. Diglycolic acid is taken into renal cells via sodium-dependent dicarboxylate transporters. When sodium-dependent dicarboxylate transporter-1 is inhibited or knocked down in human kidney cells, diglycolic acid uptake and toxicity are reduced. We hypothesize that the variation in sensitivity to tissue diglycolic acid retention and to diethylene glycol/diglycolic acid toxicity is explained by differential expression of sodium-dependent dicarboxylate transporter-1 in rat kidneys. METHODS: Using kidney tissue from previous studies, we performed rt-PCR analysis of sodium-dependent dicarboxylate transporter-1 mRNA. In those studies, Wistar-Han rats were either gavage with diethylene glycol 6 g/kg every 12 h for 7 days or with single doses of diglycolic acid 300 mg/kg. Kidney tissue was harvested after euthanasia and preserved in formalin. Tissue slices were homogenized and RNA was isolated using an RNAstorm FFPE RNA Isolation Kit. The expression of sodium-dependent dicarboxylate transporter-1 mRNA was compared between groups that showed diglycolic acid accumulation and acute renal injury with those that showed no diglycolic acid accumulation or toxicity. RESULTS: Significantly higher expression of sodium-dependent dicarboxylate transporter-1 mRNA was present in the kidneys of rats with acute kidney injury and diglycolic acid accumulation compared to those in rats that had no diglycolic acid in their kidneys and no acute kidney injury. DISCUSSION: The likelihood of acute kidney injury after dosing of rats with diethylene glycol or diglycolic acid is linked with an enhanced ability to take up diglycolic acid into renal cells via the sodium-dependent dicarboxylate transporter-1. The variability in diethylene glycol toxicity in humans, as reported in epidemiological studies, may also be linked with differences in tissue uptake of diglycolic acid. CONCLUSIONS: Animals with acute kidney injury after exposure to diethylene glycol or diglycolic acid had higher sodium-dependent dicarboxylate transporter-1 expression and greater diglycolic acid accumulation in renal tissues than animals without acute kidney injury.

Lack of efflux of diglycolic acid from proximal tubule cells leads to its accumulation and to toxicity of diethylene glycol.

Diethylene glycol (DEG) mass poisonings have resulted from ingestion of adulterated pharmaceuticals, leading to proximal tubular necrosis and acute kidney injury. Diglycolic acid (DGA), one of the primary metabolites, accumulates greatly in kidney tissue and its direct administration results in toxicity identical to that in DEG-treated rats. DGA is a dicarboxylic acid, similar in structure to Krebs cycle intermediates such as succinate. Previous studies have shown that DGA is taken into kidney cells via the succinate-related dicarboxylate transporters. These studies have assessed whether the DGA that is taken up by primary cultures of human proximal tubule (HPT) cells is effluxed. In addition, a possible mechanism for efflux, via organic anion transporters (OATs) that exchange external organic anions for dicarboxylates inside the cell, was assessed using transformed cell lines that actively express OAT activities. When HPT cells were cultured on membrane inserts, then loaded with DGA and treated with the OAT4/5 substrate estrone sulfate or the OAT1/3 substrate para-aminohippurate, no DGA efflux was seen. A repeat of this experiment utilizing RPTEC/TERT1 cells with overexpressed OAT1 and OAT3 had similar results. In these cells, but not in HPT cells, co-incubation with succinate increased the uptake of PAH, confirming the presence of OAT activity in the RPTEC/TERT1 cells. Thus, despite OATs stimulation in cells with OAT activity, there was little to no efflux of DGA from the cells. This study concluded that DGA is poorly transported out of cells and that stimulation of OAT transporters is not a viable target for reducing DGA accumulation in cells.

Role of Plasma Membrane Dicarboxylate Transporters in the Uptake and Toxicity of Diglycolic Acid, a Metabolite of Diethylene Glycol, in Human Proximal Tubule Cells.

Diethylene glycol (DEG) mass poisonings have resulted from ingestion of pharmaceuticals mistakenly adulterated with DEG, typically leading to proximal tubular necrosis and acute kidney injury. The metabolite, diglycolic acid (DGA) accumulates greatly in kidney tissue and its direct administration results in toxicity identical to that in DEG-treated rats. DGA is a dicarboxylic acid, similar in structure to metabolites like succinate. These studies have assessed the mechanism for cellular accumulation of DGA, specifically whether DGA is taken into primary cultures of human proximal tubule (HPT) cells via sodium dicarboxylate transporters (NaDC-1 or NaDC-3) like those responsible for succinate uptake. When HPT cells were cultured on membrane inserts, sodium-dependent succinate uptake was observed from both apical and basolateral directions. Pretreatment with the NaDC-1 inhibitor N-(p-amylcinnamoyl)anthranilic acid (ACA) markedly reduced apical uptakes of both succinate and DGA. Basolateral uptake of both succinate and DGA were decreased similarly following combined treatment with ACA and the NaDC-3 inhibitor 2,3-dimethylsuccinate. When the cells were pretreated with siRNA to knockdown NaDC-1 function, apical uptake of succinate and toxicity of apically applied DGA were reduced, while the reduction in basolateral succinate uptake and basolateral DGA toxicity was marginal with NaDC-3 knockdown. DGA reduced apical uptake of succinate but not basolateral uptake. This study confirmed that primary HPT cells retain sodium dicarboxylate transport functionality and that DGA was taken up by these transporters. This study identified NaDC-1 as a likely and NaDC-3 as a possible molecular target to reduce uptake of this toxic metabolite by the kidney.

Histopathological evidence that diethylene glycol produces kidney and nervous system damage in rats.

Diethylene glycol (DEG) is an organic compound that has been found as an adulterant in consumer products as a counterfeit glycerin. Diethylene glycol is metabolized to two primary metabolites: 2-hydroxyethoxyacetic acid (2-HEAA) and diglycolic acid (DGA), the latter shown to accumulate in the kidney and cause dose-dependent cell necrosis. DEG poisonings are characterized predominately by acute kidney injury (AKI) but have also produced delayed neurological sequelae such as sensorimotor neuropathy. To better understand these effects, Wistar-Han rats were orally administered a water control or doses of 4 g/kg-6 g/kg DEG every 12 or 24 h for 7 days, with kidney, brain, and spinal cord tissue collected for histopathological analysis. This dosing paradigm resulted in approximately 25 % of the DEG-treated animals developing AKI and also neurotoxicity (sensorimotor dysfunction and elevated cerebrospinal fluid (CSF) protein). Kidney pathology included a severe, diffuse acute kidney tubular necrosis predominantly affecting proximal convoluted tubules. Scattered birefringent crystals consistent with calcium oxalate monohydrate were also found in the proximal tubule of animals with AKI. Demyelination in the dorsal and lateral white matter regions of the cervical, thoracic, and lumbar areas of the spinal cord of a DEG-treated animal with AKI was documented, establishing the neuropathology in DEG-treated animals that developed neurotoxicity. There were significant changes in amino acid concentrations in the CSF that may reflect the neurotoxicity of DEG, specifically glutamate and glutamine, but with no ammonia change. These studies characterized the pathologic aspects of the neurotoxicity in a DEG repeat-dose model.

The serum glycolate concentration: its prognostic value and its correlation to surrogate markers in ethylene glycol exposures.

CONTEXT: Ethylene glycol poisoning manifests as metabolic acidemia, acute kidney injury and death. The diagnosis and treatment depend on history and biochemical tests. Glycolate is a key toxic metabolite that impacts prognosis, but assay results are not widely available in a clinically useful timeframe. We quantitated the impact of serum glycolate concentration for prognostication and evaluated whether more readily available biochemical tests are acceptable surrogates for the glycolate concentration. OBJECTIVES: The objectives of this study are to 1) assess the prognostic value of the initial glycolate concentration on the occurrence of AKI or mortality in patients with ethylene glycol exposure (prognostic study); 2) identify surrogate markers that correlate best with glycolate concentrations (surrogate study). METHODS: A systematic review of the literature was performed using Medline/PubMed, EMBASE, Cochrane library, conference proceedings and reference lists. Human studies reporting measured glycolate concentrations were eligible. Glycolate concentrations were related to categorical clinical outcomes (acute kidney injury, mortality), and correlated with continuous surrogate biochemical measurements (anion gap, base excess, bicarbonate concentration and pH). Receiver operating characteristic curves were constructed to calculate the positive predictive values and the negative predictive values of the threshold glycolate concentrations that predict acute kidney injury and mortality. Further, glycolate concentrations corresponding to 100% negative predictive value for mortality and 95% negative predictive value for acute kidney injury were determined. RESULTS: Of 1,531 articles identified, 655 were potentially eligible and 32 were included, reflecting 137 cases from 133 patients for the prognostic study and 154 cases from 150 patients for the surrogate study. The median glycolate concentration was 11.2 mmol/L (85.1 mg/dL, range 0-38.0 mmol/L, 0-288.8 mg/dL), 93% of patients were treated with antidotes, 80% received extracorporeal treatments, 49% developed acute kidney injury and 13% died. The glycolate concentration best predicting acute kidney injury was 12.9 mmol/L (98.0 mg/dL, sensitivity 78.5%, specificity 88.1%, positive predictive value 86.4%, negative predictive value 80.9%). The glycolate concentration threshold for a 95% negative predictive value for acute kidney injury was 6.6 mmol/L (50.2 mg/dL, sensitivity 96.9%, specificity 62.7%). The glycolate concentration best predicting mortality was 19.6 mmol/L (149.0 mg/dL, sensitivity 61.1%, specificity 81.4%, positive predictive value 33.3%, negative predictive value 93.2%). The glycolate concentration threshold for a 100% negative predictive value for mortality was 8.3 mmol/L (63.1 mg/dL, sensitivity 100.0%, specificity 35.6%). The glycolate concentration correlated best with the anion gap (R2 = 0.73), followed by bicarbonate (R2 = 0.57), pH (R2 = 0.50) and then base excess (R2 = 0.25), while there was no correlation between the glycolate and ethylene glycol concentration (R2 = 0.00). These data can assist clinicians in planning treatments such as extracorporeal treatments and prognostication. Potentially, they may also provide some reassurance regarding when extracorporeal treatments can be delayed while awaiting the results of further testing in patients in whom ethylene glycol poisoning is suspected but not yet confirmed. CONCLUSIONS: This systematic review demonstrates that the glycolate concentration predicts mortality (unlikely if <8 mmol/L [61 mg/dL]). The anion gap is a reasonable surrogate measurement for glycolate concentration in the context of ethylene glycol poisoning. The findings are mainly based on published retrospective data which have various limitations. Further prospective validation studies are of interest.

Analysis of Fomepizole Elimination in Methanol- and Ethylene Glycol-Poisoned Patients.

INTRODUCTION: Fomepizole is an anti-metabolite therapy that is used to diminish the toxicity from methanol or ethylene glycol. Although its elimination kinetics have been well described in healthy human subjects, the elimination in poisoned patients have only been described in a few isolated cases. This study was designed to relate the elimination of fomepizole in a series of poisoned patients to that in healthy humans. METHODS: Plasma samples from 26 patients in the clinical trials of the use of fomepizole for methanol and ethylene glycol poisoning were analyzed for fomepizole concentrations. The elimination of fomepizole was assessed after individual doses, both during and without intermittent hemodialysis. RESULTS: In methanol- and ethylene glycol-poisoned patients, fomepizole had a volume of distribution of 0.66-0.68 L/kg. After repeated doses of fomepizole, the minimum trough concentration averaged 86-109 µmol/L, which is 10 times higher than the minimum therapeutic concentration. In healthy human subjects, fomepizole elimination follows Michaelis-Menten kinetics and has been calculated as zero-order elimination rates. Zero-order elimination rates averaged 13 and 17 µmol/L/h in methanol and ethylene glycol patients, respectively, compared to 6-19 µmol/L/h in healthy subjects. Elimination during intermittent hemodialysis followed first-order kinetics, with a half-life of 3 h. CONCLUSIONS: Plasma concentrations during the repeated dosing confirmed that the recommended dosing schedule, with and without intermittent hemodialysis, maintained therapeutic concentrations throughout the treatments. Fomepizole elimination in poisoned patients at therapeutic plasma concentrations appears be similar to that reported previously in healthy human subjects.

Fomepizole dosing during continuous renal replacement therapy - an observational study.

BACKGROUND: Fomepizole is the preferred antidote for treatment of methanol and ethylene glycol poisoning, acting by inhibiting the formation of the toxic metabolites. Although very effective, the price is high and the availability is limited. Its availability is further challenged in situations with mass poisonings. Therefore, a 50% reduced maintenance dose for fomepizole during continuous renal replacement therapy (CRRT) was suggested in 2016, based on pharmacokinetic data only. Our aim was to study whether this new dosing for fomepizole during CRRT gave plasma concentrations above the required 10 µmol/L. Secondly, we wanted to study the elimination kinetics of fomepizole during CRRT, which has never been studied before. METHODS: Prospective observational study of adult patients treated with fomepizole and CRRT. We collected samples from arterial line (pre-filter) = plasma concentration, post-filter and dialysate for fomepizole measurements. Fomepizole was measured using high-pressure liquid chromatography with a reverse phase column. RESULTS: Ten patients were included in the study. Seven were treated with continuous veno-venous hemodialysis (CVVHD) and three with continuous veno-venous hemodiafiltration (CVVHDF). Ninety-eight percent of the plasma samples were above the minimum plasma concentration of 10 µmol/L. Fomepizole was removed during CRRT with a median saturation/sieving coefficient of 0.85 and dialysis clearance of 28 mL/min. CONCLUSION: Fomepizole was eliminated during CCRT. The new dosing recommendations for fomepizole and CRRT appeared safe, by maintaining the plasma concentration above the minimum value of 10 µmol/L. Based on these data, the fomepizole maintenance dose during CRRT could be reduced to half as compared to intermittent hemodialysis.

Diethylene glycol produces nephrotoxic and neurotoxic effects in female rats.

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.

Neurotoxic effects of nephrotoxic compound diethylene glycol.

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.

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

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.

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

Background: 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.Objective: 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.Methods: 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.Results: 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.Conclusion: 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.

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

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.

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

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.

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

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.

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

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.

Antidotes for poisoning by alcohols that form toxic metabolites.

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.

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

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.

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

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.

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

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.

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

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.