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.

Acute Toxicity in the Rat Lung Induced by Intratracheal Instillation of Glycolic Acid.

BACKGROUND/AIM: Inhalation toxicity tests of glycolic acid, which is used in many household products, have been reported, but the pulmonary toxicity of glycolic-acid has not been confirmed. Here, the lung damage caused by glycolic acid was investigated in rats. MATERIALS AND METHODS: An intratracheal instillation test was performed with glycolic acid in male rats. Bronchoalveolar lavage fluid (BALF) and histopathological analysis were conducted to identify the pulmonary toxicities. RESULTS: Intratracheal instillation of glycolic acid caused weight loss in animals and increased the content of lactate dehydrogenase, total protein, polymorphonuclear neutrophils, and inflammatory cytokines in BALF. In addition, pulmonary edema, alveolar/interstitial inflammation, and necrosis and desquamation of bronchial/bronchiolar epithelia were confirmed via histopathological examination. CONCLUSION: Exposure to glycolic acid can be harmful and toxic to the lungs.

Developmental toxicity studies on triclopyr acid, triclopyr butoxyethyl ester and triclopyr triethylamine salt in the rabbit.

Developmental toxicity studies have been conducted in the rabbit on triclopyr acid and its active-ingredient variants, triclopyr triethylamine salt (T-TEA) and triclopyr butoxyethyl ester (T-BEE), which are dissociated or hydrolysed in vivo to triclopyr acid. In this paper, the available developmental toxicity studies on triclopyr acid, T-TEA and T-BEE are summarised and evaluated. For triclopyr acid and T-TEA, there was no evidence of impaired reproductive performance, fetotoxicity, or teratogenicity, even at maternally toxic doses. The no-observed-adverse-effect levels (NOAELs) for developmental toxicity were 75 mg/kg bw per day for triclopyr acid and 100 mg/kg bw per day for T-TEA, equivalent to 72 mg/kg bw per day expressed as triclopyr acid. A study on T-BEE showed increased post-implantation loss and slight increases in skeletal anomalies and variants at the highest dose tested of 100 mg/kg bw per day, a maternally toxic dose. In a follow-up study on T-BEE, focusing on post-implantation loss, no general increase in post-implantation loss was observed, but one animal at 100 mg/kg bw per day with maternal toxicity had complete resorption of implants. The NOAEL for post-implantation loss was 60 mg/kg bw per day, equivalent to 44 mg/kg bw per day expressed as triclopyr acid. It cannot be excluded that T-BEE may be associated with increased post-implantation loss, but it was only seen in association with maternal toxicity. It is concluded that triclopyr acid and its variants are not specifically toxic to the rabbit embryo and fetus, since post-implantation loss only occurred at doses causing maternal toxicity.

Reproductive and developmental evaluations of triclopyr acid, triclopyr butoxyethyl ester and triclopyr triethylamine salt in the rat.

Reproductive and developmental toxicity studies have been conducted in rat and rabbit on triclopyr acid and its active-ingredient variants, triclopyr butoxyethyl ester (T-BEE) and triclopyr triethylamine salt (T-TEA). In this paper the results of a rat two-generation study on triclopyr acid are presented, together with a review of all the reproductive and developmental toxicity data available from the rat studies. In the rat two-generation study, triclopyr acid was administered in the diet, giving doses of 0, 5, 25 or 250 mg/kg bw per day. Parental toxicity, especially maternal toxicity, occurred at 250 mg/kg bw per day with reduced body weight and feed intake, organ weight changes, and kidney toxicity. Slight kidney toxicity was also evident at 25 mg/kg bw per day. Developmental toxicity, in the form of reduced postnatal survival in the F1 and F2 generations and reductions in pre-weaning offspring body weight in both generations, was seen only at a dose causing significant parental toxicity. There were no effects on any other reproductive or developmental parameters at any dose. It is concluded that the developmental toxicity, seen only at the highest dose, was most likely attributable to maternal toxicity. The no-observed-adverse-effect levels were 5 mg/kg bw per day for parental toxicity and 25 mg/kg bw per day for developmental toxicity. From the multigeneration and developmental toxicity studies on triclopyr and its variants, it can also be concluded that triclopyr is not specifically toxic to reproduction and is not selectively toxic to the embryo, fetus or neonate in the rat.

Integrating in vitro chemical transplacental passage into a generic PBK model: A QIVIVE approach.

With the increasing application of cell culture models as primary tools for predicting chemical safety, the quantitative extrapolation of the effective dose from in vitro to in vivo (QIVIVE) is of increasing importance. For developmental toxicity this requires scaling the in vitro observed dose-response characteristics to in vivo fetal exposure, while integrating maternal in vivo kinetics during pregnancy, in particular transplacental transfer. Here the transfer of substances across the placental barrier, has been studied using the in vitro BeWo cell assay and six embryotoxic compounds of different kinetic complexity. The BeWo assay results were incorporated in an existing generic Physiologically Based Kinetic (PBK) model which for this purpose was extended with rat pregnancy. Finally, as a "proof of principle", the BeWo PBK model was used to perform a QIVIVE based on developmental toxicity as observed in various different in vitro toxicity assays. The BeWo results illustrated different transport profiles of the chemicals across the BeWo monolayer, allocating the substances into two distinct groups: the 'quickly-transported' and the 'slowly-transported'. BeWo PBK exposure simulations during gestation were compared to experimentally measured maternal blood and fetal concentrations and a reverse dosimetry approach was applied to translate in vitro observed embryotoxicity into equivalent in vivo dose-response curves. This approach allowed for a direct comparison of the in vitro dose-response characteristics as observed in the Whole Embryo Culture (WEC), and the Embryonic Stem Cell test (cardiac:ESTc and neural:ESTn) with in vivo rat developmental toxicity data. Overall, the in vitro to in vivo comparisons suggest a promising future for the application of such QIVIVE methodologies for screening and prioritization purposes of developmental toxicants. Nevertheless, the clear need for further improvements is acknowledged for a wider application of the approach in chemical safety assessment.

Effects of co-formulants on the absorption and secretion of active substances in plant protection products in vitro.

Currently, the authorisation process for plant protection products (PPPs) relies on the testing of acute and topological toxicity only. Contrastingly, the evaluation of active substances includes a more comprehensive set of toxicity studies. Nevertheless, mixture effects of active ingredients and co-formulants may result in increased toxicity. Therefore, we investigated effects of surface active co-formulants on the toxicity of two PPPs focussing on qualitative and quantitative toxicokinetic effects on absorption and secretion. The respective products are based on the active substances abamectin and fluroxypyr-meptyl and were tested for cytotoxicity in the presence or absence of the corresponding surfactants and co-formulants using Caco-2 cells. In addition, the effect of co-formulants on increased cellular permeation was quantified using LC-MS/MS, while potential kinetic mixture effects were addressed by fluorescence anisotropy measurements and ATPase assays. The results show that surface active co-formulants significantly increase the cytotoxicity of the investigated PPPs, leading to more than additive mixture effects. Moreover, analytical investigations show higher efflux ratios of both active substances and the metabolite fluroxypyr upon combination with certain concentrations of the surfactants. The results further point to a significant and concentration-dependent inhibition of Pgp transporters by most of the surfactants as well as to increased membrane fluidity. Altogether, these findings strongly support the hypothesis that surfactants contribute to increased cytotoxicity of PPPs and do so by increasing the bioavailability of the respective active substances.

Diethylene glycol and its metabolites induce cell death in SH-SY5Y neuronal cells in vitro.

Diethylene glycol (DEG) intoxication results in metabolic acidosis, renal and hepatic dysfunction, and late-stage neurotoxicity. Though the renal and hepatic toxicity of DEG and its metabolites 2-hydroxyethoxyacetic acid (2-HEAA) and diglycolic acid (DGA) have been well characterized, the resultant neurotoxicity has not. SH-SY5Y neuroblastoma cells were incubated with all 3 compounds at increasing concentrations for 24, 48, or 120 h. At all 3 time points, 50 mmol/L DGA and 100 mmol/L DEG showed significant Annexin V and propidium iodide (PI) staining with additional concentrations showing similar staining patterns at 24 h (100 mmol/L DGA) and 48 h (50 mmol/L DEG, 100 mmol/L DGA). Only the 200 mmol/L 2-HEAA concentration induced SH-SY5Y cell death. Interestingly at 24 and 48 h, 100 mmol/L DEG induced significant increases in apoptotic cell death markers, which progressed to necrosis at 120 h. Similar to DEG, 50 mmol/L DGA induced significant increases in SH-SY5Y cell apoptosis and necrosis markers at both 24 and 48 h. As expected, high DGA concentrations (100 mmol/L) at 120 h induced significant SH-SY5Y cell necrosis with no apoptosis detected. However, at 120 h lower DGA concentrations (20 mmol/L) significantly increased oligonucleosome formation alone and in combination with 2-HEAA or DEG. Taken together, these results indicate that DGA and DEG at threshold concentrations induce neurotoxicity in SH-SY5Y cells.

Fluroxypyr-1-methylheptyl ester interferes with the normal embryogenesis of zebrafish by inducing apoptosis, inflammation, and neurovascular toxicity.

Fluroxypyr-1-methylheptyl ester (FPMH) is a synthetic auxin herbicide used to regulate the growth of post-emergence broad-leaved weeds. Although acute exposure to FPMH increases the mortality of several fish species in the juvenile stage, the developmental toxicity of FPMH in aquatic vertebrates has not yet been investigated. In the present study, we assessed the developmental toxicity of FPMH using zebrafish models that offer many advantages for studying toxicology. During embryogenesis, survival rates gradually decreased with increasing FPMH concentrations and exposure times. At 120 h post-fertilization, FPMH-exposed zebrafish larvae showed various abnormalities such as small eye size, heart defects, enlarged yolk sac, and shortened body length. The study results confirmed the induction of apoptosis in the anterior body of zebrafish and upregulation of inflammatory gene expression. Further, defects in vascular networks, especially the loss of central arteries and abnormal aortic arch structures, were seen in the fli1:eGFP transgenic zebrafish model. Neurotoxicity of FPMH was examined using mbp:eGFP zebrafish and which displayed compromised myelination following FPMH administration. Our study has demonstrated the mechanisms underlying FPMH toxicity in developing zebrafish that is a representative model of vertebrates.

Utilization of a model hepatotoxic compound, diglycolic acid, to evaluate liver Organ-Chip performance and in vitro to in vivo concordance.

Microphysiological systems (MPS) are emerging as potentially predictive models for drug safety and toxicity assessment. To assess the utility of these systems, the Food and Drug Administration partnered with Emulate to evaluate the Human Liver Organ-Chip in a regulatory setting. Diglycolic acid (DGA), a known hepatotoxin, was evaluated in the Liver-Chip and compared to a multi-well plate format to assess the Liver-Chip's capabilities, limitations, overall performance, and concordance with other in vivo and in vitro studies. Cryopreserved primary human hepatocytes were exposed to DGA from 1 to 20 mM in Liver-Chips or traditional multi-well plates. We found that 10 mM or 20 mM of DGA was severely cytotoxic in both platforms, while 5 mM was mildly cytotoxic in Liver-Chips. Additionally, some hepatocyte functions were reduced with 5 mM DGA in Liver-Chips and 1 mM in well plates. Individual well effects were greater or occurred sooner than in the Liver-Chips. Examination of the performance of the Liver-Chip showed that variability was low for biochemical endpoints, but higher for imaging endpoints. Sensitivity and specificity were high. Only 3-4 Liver-Chips were necessary to detect an effect depending on the endpoint and effect size. The specifics of the experiment are found herein.

Employment of cytology for in vitro skin irritation test using a reconstructed human epidermis model, Keraskin™.

Skin irritation tests using reconstructed human epidermis (RhE) employ viability as an endpoint, but color interference or borderline results are often problematic. We examined whether the cytology of cells from treated RhE could determine skin irritancy. Six chemicals (three irritants; DnP, 1-B, PH, three non-irritants; DP, APA, HS) were evaluated in a RhE, Keraskin™. DP, HS, and PH were clearly classified with viability, but DnP, 1-B, and APA were often falsely determined, due to borderline values falling near the cutoff, 50%. In histology, the tissues treated with DnP, 1-B, and PH showed erosion of the stratum corneum, vacuolization, and necrosis in the basal layer. DP- and HS-treated tissues showed relatively normal morphology but APA induced necrosis similar to irritants. Cytology revealed that DnP, 1-B or PH depleted cells and induced irregular and abnormal cell shapes. In contrast, relatively regular and normal shapes and clear distinction between the nucleus and cytoplasm was observed for DP, APA and HS. To further confirm it, additional 10 substances, including false positives from OECD TG 439, were tested. Overall (16 substances in total), cytology: total area predicted the skin irritancy of test chemicals with the highest accuracy (87.5%) followed by cytology: cell count (81.3%), histology (75%) and viability (68.8%), confirming the utility of cytology as an alternative endpoint in the skin irritation test using RhE.

Acute and 28-Day Repeated Inhalation Toxicity Study of Glycolic Acid in Male Sprague-Dawley Rats.

BACKGROUND/AIM: The use of glycolic acid is present in a variety of consumer products, including medicines, cleaners, cosmetics, and paint strippers. It has recently led to concerns about toxicity from inhalation exposure. Herein, the pulmonary toxicity of glycolic acid was investigated in rats. MATERIALS AND METHODS: We conducted acute (~458 mg/m3) and sub-acute (~49.5 mg/m3) inhalation tests to identify the potential toxicities of glycolic acid. RESULTS: Inhalation exposure to glycolic acid in the acute and subacute inhalation tests did not cause any specific changes in clinical examinations, including body weight, organ weight, hematology, serum biochemistry, and histopathology. The polymorphonuclear neutrophils (PMNs) and inflammatory cytokines in Bronchoalveolar lavage fluid (BALF) increased in rats exposed to single and repeated inhalations. In the sub-acute test, the changes induced by glycolic acid were minor or returned to normal during the recovery period. CONCLUSION: The No Observed Adverse Effect Concentration (NOAEC) for the nasal and pulmonary toxicity of glycolic acid was determined to be over 50 mg/m3 at the end of a 28-day inhalation test in male rats.

Diglycolic acid induces HepG2/C3A liver cell toxicity in vitro.

Carboxymethyl starches are added to food products for thickening or tablet binding/filling purposes. Although they lack toxicity, their synthesis creates the chemical byproduct diglycolic acid (DGA), which is difficult to eliminate and whose toxicity is in question. A rare case of an accidental direct exposure to extremely high concentrations of DGA in a person revealed that DGA has the potential to be toxic to several organs, with the kidneys and liver being the most affected organs. Given that DGA is present in our food supply as a chemical byproduct of carboxymethyl starch food additives, we sought to perform in vitro testing of its potential hepatotoxicity to help complement a recent in vivo rat acute dose-response study that also tested for the potential hepatotoxic effects of daily DGA ingestion by oral gavage over a period of 28 days. Using the HepG2/C3A cellular in vitro model, we tested how escalating doses of DGA exposure over 24 h could induce hepatotoxicity. Both in vitro and in vivo testing systems revealed that DGA is indeed a hepatotoxin once a certain exposure threshold is reached. The concordance of these models highlights the utility of in vitro testing to support and help predict in vivo findings.

Persistence of auxinic herbicides applied on pasture and toxicity for succeeding crops.

The aim of this work was to determine the persistence of auxinic herbicides applied on tropical pasture and toxicity for succeeding crops. The herbicides were applied in an area of dystrophic red‒yellow latosol with pasture infested of weeds. At 40, 80, and 280 days after application of herbicide, the soil samples were collected at depths of 0 to 20 cm. Soil with residues of 2,4-D, 2,4-D + picloram, triclopyr, and a soil without herbicide application were analyzed with six replicates. Seven crops were cultivated in these soils: cucumber (Cucumis sativus L.), velvet bean [Mucuna pruriens (L.) DC.], pigeon pea [Cajanus cajan (L.) Millsp.], alfalfa (Medicago sativa L.), lablab bean [Lablab purpureus (L.) Sweet], corn (Zea mays L.), and sorghum [Sorghum bicolor (L.) Moench]. The plants of cucumber, pigeon pea, and alfalfa were the most susceptible to the auxinic herbicide residues. However, the lablab bean was the only one among the dicot evaluated that showed tolerance to the 2,4-D + picloram residual when cultivated in soils at 280 days after application of herbicide. Corn and sorghum showed lower chlorophyll content in soils with 2,4-D + picloram residual up to 80 days after application of herbicide.

Editor's Highlight: Ethylene Glycol Teratogenicity: A Role for Embryonic Acidosis?

Ethylene glycol (EG) is a developmental toxicant in pregnant rats and mice. A suggested mechanism for this toxicity is that the EG metabolite, glycolic acid (GA), causes acidosis which may affect the embryonic heart rate (HR). This inhibition would cause periods of embryonic bradycardia and arrhythmia resulting in increased embryonic death and malformation in surviving embryos. This hypothesis was investigated using gestational day (GD) 11 and 13 rat embryos in vitro. Increasing concentrations of GA or lactic acid in the incubation medium caused a decrease in external pH (pHe) and a concentration-dependent decrease in embryonic HR. Increased concentrations of GA or lactic acid with pHe corrected to normal levels did not affect HR. Severely decreased pHe, caused by reduced NaHCO3 in the incubation medium, had little effect on the HR of GD 13 embryos but substantially reduced the HR of GD 11 embryos. These results suggest that increased monocarboxylate concentration (glycolate or lactate) needs to be in combination with increased H+ concentration (low pHe) to influence the embryonic HR. These results implicate the monocarboxylate transporter reported to be present in the early postnatal rat heart, the chick embryonic heart throughout development, and the chorioallantoic placenta. The results showed some evidence that the adverse effect of GA and reduced pHe on the embryonic HR increased with duration of exposure and hence lends support to the suggested mechanism of embryotoxicity for EG.

Effects of chemical management for invasive plants on the performance of Lithobates pipiens tadpoles.

Invasive plants impact amphibians by altering habitat, altering species interactions, and releasing potentially toxic secondary chemicals. Despite being costly and having the potential to affect nontarget wildlife, chemical management is commonly used to control invasive plants. Prior research has indicated that individual effects of invasive plants or herbicides can be harmful to aquatic organisms; however, information is lacking on the combined effect of these factors on amphibians. A laboratory experiment was performed to assess the impact of leachates of the invasive plants Eurasian watermilfoil (Myriophyllum spicatum) and European buckthorn (Rhamnus cathartica), the herbicide Renovate® 3 (triclopyr [3, 5, 6-trichloro-2-pyridinyloxyacetic acid]), and the combined effects of each plant leachate and the herbicide on the growth, morphology, and survival of northern leopard frog (Lithobates pipiens) tadpoles. No effects of treatment on survival were observed. Tadpole exposure to M. spicatum reduced body mass by 17%, exposure to R. cathartica increased body mass by 36%, and exposure to R. cathartica + low herbicide increased body mass by 38% (although only early in the experiment). Exposure to Renovate 3 induced a 16% and 29% decrease in tadpole size in lower (0.22 mg triclopyr active ingredient [a.i.]/L) and higher (0.92 mg triclopyr a.i./L) concentration treatments, respectively. Results from the present study highlight the importance of considering both individual and combined effects of invasive plants and herbicides because they may have different outcomes for tadpole growth and development. Environ Toxicol Chem 2017;36:2958-2964. © 2017 SETAC.

Herbicide Toxicity Testing with Non-Target Boreal Plants: The Sensitivity of Achillea millefolium L. and Chamerion angustifolium L. to Triclopyr and Imazapyr.

Terrestrial plant toxicity tests were conducted to determine the sensitivity of two boreal plants, yarrow (Achillea millefolium L.) and fireweed (Chamerion angustifolium L.), to the herbicides imazapyr and triclopyr. Both plants are common non-target species on northern powerline rights-of-way where the impacts of proposed herbicide applications are of concern. In the vegetative vigour test, triclopyr foliar spray caused extensive damage to A. millefolium at <50% of the maximum field application rate (inhibition concentration (IC)50 = 1443.8 g a.i. ha-1) and was lethal to C. angustifolium at the lowest dose tested (1210.9 g a.i. ha-1). Both species demonstrated extremely high sensitivity to imazapyr foliar spray: IC50s = 8.29 g a.i. ha-1 and 4.82 g a.i. ha-1 (<1.5% of the maximum field rate). The seedling emergence and seedling growth tests were conducted in the organic horizon of five boreal soils. Few differences in herbicide bioavailability between soils were detected. Triclopyr limited growth of A. millefolium, C. angustifolium and standard test species Calamagrostis canadensis at low levels (most IC50 estimates between 2-20 µg g-1). For imazapyr, IC50 estimates could not be calculated as there was >75% inhibition of endpoints at the lowest doses of ~2 µg g-1. A foliar application of triclopyr or imazapyr for woody species control would likely cause significant damage to boreal non-target plants. The high sensitivity of both species to herbicide residues in soil indicates long term impacts are dependent on herbicide degradation rates in northern conditions. A. millefolium performed well and is recommended for use in toxicity testing relevant to boreal regions.

28-day repeated dose response study of diglycolic acid: Renal and hepatic effects.

The acute oral toxicity of diglycolic acid (DGA) was evaluated. Groups of female rats (n = 8 rats/group) received 28 consecutive daily single doses of 0.3, 1.0, 3.0, 10.0, 30.0, 100.0 or 300.0 mg DGA/kg body weight by gastric intubation. One group of animals served as vehicle control. Tissues and blood serum were collected at necropsy on day 29. Select organs were weighed and fixed in formalin for histopathological analysis. Animals from the 300 mg/kg bw dose group were removed from the study after 5 consecutive days of treatment as a consequence of adverse treatment related effects. The animals in the remaining treatment groups survived the exposure period. No adverse clinical signs were observed throughout the exposure period in the surviving animals. No significant differences from controls were observed for feed and fluid consumption or body weight gain in the surviving animals. Lesions were observed in the kidneys, liver, stomach, intestine, thymus, spleen and bone marrow in rats from the 300 mg/kg dose group and signs of renal tubular regeneration were observed only in the 100 mg/kg dose group. These results suggest that high levels of pure DGA would need to be consumed before renal and other forms of organ toxicity are observed.

Effects of the herbicide triclopyr on metamorphic northern red-legged frogs.

Aquatic herbicides are used to manage invasive emergent plants in and around wetlands. Metamorphic frogs that emerge during the aquatic weed management season may be at risk of herbicide exposure. Metamorphic northern red-legged frogs (Rana aurora) were exposed to a triclopyr tank mix labeled for control of broadleaf emergent aquatic weeds such as invasive purple loosestrife (Lythrum salicaria). The tank mix consisted of Renovate® 3 (triclopyr triethylamine salt 44.4%), the modified vegetable oil surfactant Competitor®, and the marker dye Hi-Light® . Metamorphs were exposed to the tank mix and a clean-water control for 96 h, and then reared in clean water for 60 d. Exposure to the tank mix resulted in no treatment-related mortalities, no effects on behavior immediately post exposure, and no effects on body or liver condition indices. Exposure to the tank mix resulted in lethargy during exposure and a 1-d delay in completion of metamorphosis. Deformities present in the rearing population confounded results for some endpoints. Observed effects were minimal, especially compared with the potential for ecological impacts from unmanaged invasive plants. Environ Toxicol Chem 2017;36:2316-2326. © 2017 SETAC.

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.

A conserved phosphatase destroys toxic glycolytic side products in mammals and yeast.

Metabolic enzymes are very specific. However, most of them show weak side activities toward compounds that are structurally related to their physiological substrates, thereby producing side products that may be toxic. In some cases, 'metabolite repair enzymes' eliminating side products have been identified. We show that mammalian glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase, two core glycolytic enzymes, produce 4-phosphoerythronate and 2-phospho-L-lactate, respectively. 4-Phosphoerythronate strongly inhibits an enzyme of the pentose phosphate pathway, whereas 2-phospho-L-lactate inhibits the enzyme producing the glycolytic activator fructose 2,6-bisphosphate. We discovered that a single, widely conserved enzyme, known as phosphoglycolate phosphatase (PGP) in mammals, dephosphorylates both 4-phosphoerythronate and 2-phospho-L-lactate, thereby preventing a block in the pentose phosphate pathway and glycolysis. Its yeast ortholog, Pho13, similarly dephosphorylates 4-phosphoerythronate and 2-phosphoglycolate, a side product of pyruvate kinase. Our work illustrates how metabolite repair enzymes can make up for the limited specificity of metabolic enzymes and permit high flux in central metabolic pathways.