RESUMO
CONTEXT: Diethylene glycol (DEG) mass poisoning is a persistent public health problem. Unfortunately, there are no human biological data on DEG and its suspected metabolites in poisoning. If present and associated with poisoning, the evidence for use of traditional therapies such as fomepizole and/or hemodialysis would be much stronger. OBJECTIVE: To characterize DEG and its metabolites in stored serum, urine, and cerebrospinal fluid (CSF) specimens obtained from human DEG poisoning victims enrolled in a 2006 case-control study. METHODS: In the 2006 study, biological samples from persons enrolled in a case-control study (42 cases with new-onset, unexplained AKI and 140 age-, sex-, and admission date-matched controls without AKI) were collected and shipped to the Centers for Disease Control and Prevention (CDC) in Atlanta for various analyses and were then frozen in storage. For this study, when sufficient volume of the original specimen remained, the following analytes were quantitatively measured in serum, urine, and CSF: DEG, 2-hydroxyethoxyacetic acid (HEAA), diglycolic acid, ethylene glycol, glycolic acid, and oxalic acid. Analytes were measured using low resolution GC/MS, descriptive statistics calculated and case results compared with controls when appropriate. Specimens were de-identified so previously collected demographic, exposure, and health data were not available. The Wilcoxon Rank Sum test (with exact p-values) and bivariable exact logistic regression were used in SAS v9.2 for data analysis. RESULTS: The following samples were analyzed: serum, 20 case, and 20 controls; urine, 11 case and 22 controls; and CSF, 11 samples from 10 cases and no controls. Diglycolic acid was detected in all case serum samples (median, 40.7 mcg/mL; range, 22.6-75.2) and no controls, and in all case urine samples (median, 28.7 mcg/mL; range, 14-118.4) and only five (23%) controls (median, < Lower Limit of Quantitation (LLQ); range, < LLQ-43.3 mcg/mL). Significant differences and associations were identified between case status and the following: 1) serum oxalic acid and serum HEAA (both OR = 14.6; 95% C I = 2.8-100.9); 2) serum diglycolic acid and urine diglycolic acid (both OR > 999; exact p < 0.0001); and 3) urinary glycolic acid (OR = 0.057; 95% C I = 0.001-0.55). Two CSF sample results were excluded and two from the same case were averaged, yielding eight samples from eight cases. Diglycolic acid was detected in seven (88%) of case CSF samples (median, 2.03 mcg/mL; range, < LLQ, 7.47). DISCUSSION: Significantly elevated HEAA (serum) and diglycolic acid (serum and urine) concentrations were identified among cases, which is consistent with animal data. Low urinary glycolic acid concentrations in cases may have been due to concurrent AKI. Although serum glycolic concentrations among cases may have initially increased, further metabolism to oxalic acid may have occurred thereby explaining the similar glycolic acid concentrations in cases and controls. The increased serum oxalic acid concentration results in cases versus controls are consistent with this hypothesis. CONCLUSION: Diglycolic acid is associated with human DEG poisoning and may be a biomarker for poisoning. These findings add to animal data suggesting a possible role for traditional antidotal therapies. The detection of HEAA and diglycolic acid in the CSF of cases suggests a possible association with signs and symptoms of DEG-associated neurotoxicity. Further work characterizing the pathophysiology of DEG-associated neurotoxicity and the role of traditional toxic alcohol therapies such as fomepizole and hemodialysis is needed.
Assuntos
Etilenoglicóis/sangue , Etilenoglicóis/líquido cefalorraquidiano , Etilenoglicóis/intoxicação , Etilenoglicóis/urina , Intoxicação/diagnóstico , Acetatos/líquido cefalorraquidiano , Acetatos/intoxicação , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/urina , Estudos de Casos e Controles , Centers for Disease Control and Prevention, U.S. , Feminino , Fomepizol , Cromatografia Gasosa-Espectrometria de Massas , Glicolatos/sangue , Glicolatos/líquido cefalorraquidiano , Glicolatos/intoxicação , Glicolatos/urina , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Modelos Logísticos , Masculino , Síndromes Neurotóxicas/tratamento farmacológico , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/fisiopatologia , Panamá , Intoxicação/tratamento farmacológico , Intoxicação/etiologia , Pirazóis/uso terapêutico , Diálise Renal , Manejo de Espécimes , Estados UnidosRESUMO
A previously developed PBPK model for ethylene glycol and glycolic acid was extended to include glyoxylic acid, oxalic acid, and the precipitation of calcium oxalate that is associated with kidney toxicity in rats and humans. The development and evaluation of the PBPK model was based upon previously published pharmacokinetic studies coupled with measured blood and tissue partition coefficients and rates of in vitro metabolism of glyoxylic acid to oxalic acid, glycine and other metabolites using primary hepatocytes isolated from male Wistar rats and humans. Precipitation of oxalic acid with calcium in the kidneys was assumed to occur only at concentrations exceeding the thermodynamic solubility product for calcium oxalate. This solubility product can be affected by local concentrations of calcium and other ions that are expressed in the model using an ion activity product estimated from toxicity studies such that calcium oxalate precipitation would be minimal at dietary exposures below the NOAEL for kidney toxicity in the sensitive male Wistar rat. The resulting integrated PBPK predicts that bolus oral or dietary exposures to ethylene glycol would result in typically 1.4-1.6-fold higher peak oxalate levels and 1.6-2-fold higher AUC's for calcium oxalate in kidneys of humans as compared with comparably exposed male Wistar rats over a dose range of 1-1000 mg/kg. The converse (male Wistar rats predicted to have greater oxalate levels in the kidneys than humans) was found for inhalation exposures although no accumulation of calcium oxalate is predicted to occur until exposures are well in excess of the theoretical saturated vapor concentration of 200mg/m(3). While the current model is capable of such cross-species, dose, and route-of-exposure comparisons, it also highlights several areas of potential research that will improve confidence in such predictions, especially at low doses relevant for most human exposures.
Assuntos
Etilenoglicol/farmacocinética , Glicolatos/farmacocinética , Nefropatias/induzido quimicamente , Ácido Oxálico/metabolismo , Animais , Oxalato de Cálcio/metabolismo , Relação Dose-Resposta a Droga , Vias de Administração de Medicamentos , Etilenoglicol/toxicidade , Feminino , Glicolatos/toxicidade , Glioxilatos/metabolismo , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Modelos Biológicos , Ratos , Ratos WistarRESUMO
Translational toxicology can be defined as the movement of potential antidotes for the treatment of poisonings from basic mechanistic research to the marketplace. Because poisonings are infrequent, the clinical development of antidotes is fraught with trials and tribulations. Academic scientists often conduct basic mechanistic work with antidotes but are infrequently involved in further drug development. This article presents the development of 4-methylpyrazole (4MP) (fomepizole) as an antidote against toxic alcohol poisonings, particularly by methanol and ethylene glycol (EG).
Assuntos
Antídotos/farmacologia , Desenho de Fármacos , Pesquisa Translacional Biomédica/métodos , Animais , Antídotos/uso terapêutico , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Etilenoglicol/intoxicação , Fomepizol , Humanos , Metanol/intoxicação , Pirazóis/farmacologia , Pirazóis/uso terapêuticoRESUMO
Male Wistar rats have been shown to be the most sensitive sex, strain and species to ethylene glycol-induced nephrotoxicity in subchronic studies. A chronic toxicity and dosimetry study was therefore conducted in male Wistar rats administered ethylene glycol via the diet at 0, 50, 150, 300, or 400 mg/kg/day for up to twelve months. Subgroups of animals were included for metabolite analysis and renal clearance studies to provide a quantitative basis for extrapolating dose-response relationships from this sensitive animal model in human health risk assessments. Mortality occurred in 5 of 20 rats at 300 mg/kg/day (days 111-221) and 4 of 20 rats at 400 mg/kg/day (days 43-193), with remaining rats at this dose euthanized early (day 203) due to excessive weight loss. Increased water consumption and urine volume with decreased specific gravity occurred at 300 mg/kg/day presumably due to osmotic diuresis. Calculi (calcium oxalate crystals) occurred in the bladder or renal pelvis at > or =300 mg/kg/day. Rats dying early at > or =300 mg/kg/day had transitional cell hyperplasia with inflammation and hemorrhage of the bladder wall. Crystal nephropathy (basophilic foci, tubule or pelvic dilatation, birefringent crystals in the pelvic fornix, or transitional cell hyperplasia) affected most rats at 300 mg/kg/day, all at 400 mg/kg/day, but none at < or =150 mg/kg/day. No significant differences in kidney oxalate levels, the metabolite responsible for renal toxicity, were observed among control, 50 and 150 mg/kg/day groups. At 300 and 400 mg/kg/day, oxalate levels increased proportionally with the nephrotoxicity score supporting the oxalate crystal-induced nephrotoxicity mode of action. No treatment-related effects on the renal clearance of intravenously infused (3)H-inulin, a marker for glomerular filtration, and (14)C-oxalic acid were observed in rats surviving 12 months of exposure to ethylene glycol up to 300 mg/kg/day. In studies with naïve male Wistar and F344 rats (a less sensitive strain), a significant difference was observed in oxalate clearances between young rats (i.e. Wistar clearance < F344) but not in age-matched old rats. Regardless, the ratios of oxalate:inulin clearances in these two strains of rats, including those exposed to ethylene glycol, were all < 1, suggesting that a fraction of the filtered oxalate is reabsorbed. Other species, including humans, typically have clearance ratios >1 and are more effective at clearing oxalic acid by both glomerular filtration and active secretion. Thus, the lower renal clearance and kidney accumulation of oxalates in male Wistar rats enhances their sensitivity, which will be a factor in human risk assessments. The benchmark dose values (BMD05, BMDL05) were 170 mg/kg/day and 150 mg/kg/day for nephropathy, and 170 mg/kg/day and 160 mg/kg/day for birefringent crystals, using incidence times severity data in each case. The NOAEL of 150 mg/kg/day is the same as that reported after 16-week exposure and appears to be a threshold dose below which no renal toxicity occurs, regardless of exposure duration.
Assuntos
Etilenoglicol/toxicidade , Cálculos Renais/induzido quimicamente , Rim/efeitos dos fármacos , Administração Oral , Animais , Oxalato de Cálcio/urina , Diurese/efeitos dos fármacos , Relação Dose-Resposta a Droga , Etilenoglicol/administração & dosagem , Humanos , Rim/patologia , Rim/fisiopatologia , Cálculos Renais/patologia , Cálculos Renais/urina , Masculino , Nível de Efeito Adverso não Observado , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Fatores de Tempo , Testes de Toxicidade Crônica/métodos , Redução de PesoRESUMO
Ethylene glycol (EG) poisoning can lead to serious morbidity or death, which occurs following conversion of ethylene glycol to toxic metabolites. These metabolites affect multiple organ/systems leading to metabolic acidosis, cardiopulmonary depression, acute renal failure and central nervous system deficits. Treatment consists of correcting metabolic acidosis with bicarbonate administration, dialysis to remove toxic metabolites and administration of fomepizole or ethanol to prevent conversion of EG to toxic intermediates. Occasionally in the literature, fatal cases of EG poisoning have been described in which calcium oxalate crystal deposition has occurred in the walls of CNS vessels, sometimes with associated neuropathy. We describe a case of fatal EG poisoning in which the development of rapid cerebral edema was documented by CT scan and was accompanied by definitive evidence of birefringent crystals within walls of CNS blood vessels, with associated inflammation and edema. This case and others in the literature suggest that cerebral edema, and perhaps injury to other organs, could result from oxalate crystal deposition in small blood vessels in the brain and other organs.
Assuntos
Edema Encefálico , Oxalato de Cálcio/metabolismo , Etilenoglicol/intoxicação , Adulto , Vasos Sanguíneos/metabolismo , Edema Encefálico/diagnóstico por imagem , Edema Encefálico/etiologia , Edema Encefálico/metabolismo , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/metabolismo , Etilenoglicol/farmacocinética , Evolução Fatal , Humanos , Inativação Metabólica , Masculino , Intoxicação/complicações , Intoxicação/metabolismo , RadiografiaRESUMO
Although occupational uses of the high production volume (HPV) chemical ethylene glycol (EG) have not been associated with adverse effects, there are case reports where humans have either intentionally or accidentally ingested large quantities of EG, primarily from antifreeze. The acute toxicity of EG can proceed through three stages, each associated with a different metabolite: central nervous system depression (ethylene glycol), cardiopulmonary effects associated with metabolic acidosis (glycolic acid), and ultimately renal toxicity (oxalic acid), depending on the total amounts consumed and the effectiveness of therapeutic interventions. A physiologically based pharmacokinetic (PBPK) model developed in a companion paper (Corley et al., 2005). Development of a physiologically based pharmacokinetic model for ethylene glycol and its metabolite, glycolic acid, in rats and humans. Toxicol. Sci., in press 2005) was refined in this study to include clinically relevant treatment regimens for EG poisoning such as hemodialysis or metabolic inhibition with either ethanol or fomepizole. Such modifications enabled the model to describe data from several human case reports, confirming the ability of the previous model to describe the pharmacokinetics of EG and its metabolite, glycolic acid, in humans across a broad range of doses and multiple exposure routes. By integrating the case report data sets with controlled studies in this PBPK model, it was demonstrated that fomepizole, if administered early enough in a clinical situation, can be more effective than ethanol or hemodialysis in preventing the metabolism of EG to more toxic metabolites. Hemodialysis remains an important option, however, if treatment is instituted after a significant amount of EG is metabolized or if renal toxicity has occurred.
Assuntos
Antídotos/uso terapêutico , Exposição Ambiental/efeitos adversos , Etilenoglicol , Modelos Biológicos , Etilenoglicol/efeitos adversos , Etilenoglicol/farmacocinética , Etilenoglicol/intoxicação , Medicina Baseada em Evidências , Humanos , Inativação Metabólica , Absorção Intestinal , Intoxicação/terapia , Diálise Renal , Medição de RiscoRESUMO
CASE REPORT: We report a case of methanol poisoning exhibiting complete recovery from severe visual impairment following treatment with ethanol, fomepizole, and hemodialysis. An adult male presented with central blindness after ingesting methanol. Initial visual acuity was <20/800 (finger counting at 1-2 feet) with retinal edema on fundoscopy, arterial pH 7.19, methanol 97 mg/dL (30 mmol/L), formate 14.3 mmol/L, and ethanol undetectable. The patient was treated with ethanol, then fomepizole intravenously (15, 10, then 5 mg/kg), and hemodialysis. Methanol metabolism was effectively blocked by fomepizole even after ethanol had been eliminated, and the patient recovered 20/20 vision by day 14 with normal fundoscopy. This case report confirms highly efficient inhibition of alcohol dehydrogenase by fomepizole, as well as demonstrate the safety of fomepizole in a patient already exhibiting end-organ retinal toxicity. The potential for fomepizole to inhibit retinol dehydrogenase, an isoenzyme of alcohol dehydrogenase essential to vision, did not appear to be clinically significant in this symptomatic methanol-poisoned patient.
Assuntos
Antídotos/uso terapêutico , Metanol/antagonistas & inibidores , Metanol/intoxicação , Pirazóis/uso terapêutico , Doenças Retinianas/induzido quimicamente , Solventes/intoxicação , Transtornos da Visão/induzido quimicamente , Transtornos da Visão/tratamento farmacológico , Adulto , Antídotos/efeitos adversos , Soluções Tampão , Depressores do Sistema Nervoso Central/sangue , Depressores do Sistema Nervoso Central/uso terapêutico , Etanol/sangue , Etanol/uso terapêutico , Fomepizol , Humanos , Masculino , Metanol/sangue , Pirazóis/efeitos adversos , Pirazóis/sangue , Bicarbonato de Sódio/uso terapêuticoRESUMO
We studied 62 patients aged 48 years as an average and diagnosed with bilateral optical neuropathy during an epidemics in Pinar del Río province. Of these patients, 42 showed the optical form whereas 20 had the mixed form of optical neuropathy. We researched into the levels of formate and folate in serum and cerebrospinal fluid samples and we found a marked deficiency of folates in more than 50% of samples and high formate concentration levels in almost 25% of samples. We concluded that nutritional shortages that lead to a reduction of folates, and the intake of small amounts of methanol in alcoholic drinks could lead to lacking energetic states which would facilitate that the optical nerve be affected and the epidemic optical neuropathy appear.
Assuntos
Surtos de Doenças , Deficiência de Ácido Fólico/complicações , Ácido Fólico/sangue , Ácido Fólico/líquido cefalorraquidiano , Formiatos/sangue , Formiatos/líquido cefalorraquidiano , Doenças do Nervo Óptico/epidemiologia , Consumo de Bebidas Alcoólicas , Feminino , Humanos , Masculino , Metanol/efeitos adversos , Pessoa de Meia-Idade , Doenças do Nervo Óptico/sangue , Doenças do Nervo Óptico/líquido cefalorraquidiano , Doenças do Nervo Óptico/induzido quimicamente , Fatores de Risco , Solventes/efeitos adversosAssuntos
Etilenoglicol/metabolismo , Etilenoglicol/toxicidade , Rim/efeitos dos fármacos , Rim/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Transformada , Células Cultivadas , Etídio/análogos & derivados , Etilenoglicol/intoxicação , Fluorescência , Fomepizol , Glicolatos/sangue , Glicolatos/metabolismo , Glicolatos/toxicidade , Humanos , Rim/patologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Ácido Oxálico/sangue , Ácido Oxálico/metabolismo , Ácido Oxálico/toxicidade , Ácido Oxálico/urina , Pirazóis/uso terapêuticoRESUMO
STUDY OBJECTIVE: The elimination kinetics of ethylene glycol (EG) in human subjects treated with fomepizole (4-methylpyrazole) were analyzed to establish the efficacy of alcohol dehydrogenase (ADH) inhibition and to characterize elimination pathways. METHODS: Drug concentration data from patients enrolled in the EG arm of the Methylpyrazole for Toxic Alcohols trial, a prospective, multicenter, open-label trial of fomepizole, were analyzed and compared with published estimates. RESULTS: In 19 patients analyzed (EG concentrations of 3.5 to 211 mg/dL), elimination was first order during fomepizole monotherapy (half-life of 19.7+/-1.3 hours) and was not affected by the presence of ethanol. The elimination rate was significantly faster (half-life of <8.6+/-1.1 hours, P <.001) in the absence of fomepizole and ethanol. EG elimination by the kidneys was directly proportional to remaining renal function as estimated by creatinine clearance, with a fractional excretion of 25.5%+/-9.4%. Renal elimination and hemodialysis were the only significant routes of EG elimination as long as fomepizole concentrations were maintained well above 10 micromol/L (EG/fomepizole molar ratio, <100:1). All patients with normal serum creatinine concentrations at the initiation of fomepizole treatment had rapid rates of renal elimination (half-life of 16.8+/-0.8 hours). CONCLUSION: At doses used, fomepizole effectively inhibits ADH-mediated metabolism of EG. Serum creatinine concentration at presentation and creatinine clearance can be used to predict EG elimination during fomepizole therapy and can help determine which patients will require hemodialysis to expedite EG elimination. An absolute EG concentration above 50 mg/dL should no longer be used as an independent criterion for hemodialysis in patients treated with fomepizole.
Assuntos
Álcool Desidrogenase/efeitos dos fármacos , Intoxicação Alcoólica/terapia , Antídotos/administração & dosagem , Etilenoglicol/farmacocinética , Etilenoglicol/intoxicação , Pirazóis/administração & dosagem , Adolescente , Adulto , Idoso , Creatina/sangue , Relação Dose-Resposta a Droga , Serviço Hospitalar de Emergência , Feminino , Fomepizol , Humanos , Injeções Intravenosas , Testes de Função Renal , Masculino , Pessoa de Meia-Idade , Probabilidade , Diálise Renal , Resultado do TratamentoRESUMO
We studied 62 patients aged 48 years as an average and diagnosed with bilateral optical neuropathy during an epidemics in Pinar del RÝo province. Of these patients, 42 showed the optical form whereas 20 had the mixed form of optical neuropathy. We researched into the levels of formate and folate in serum and cerebrospinal fluid samples and we found a marked deficiency of folates in more than 50 of samples and high formate concentration levels in almost 25 of samples. We concluded that nutritional shortages that lead to a reduction of folates, and the intake of small amounts of methanol in alcoholic drinks could lead to lacking energetic states which would facilitate that the optical nerve be affected and the epidemic optical neuropathy appear.
Assuntos
Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Ácido Fólico/sangue , Ácido Fólico/líquido cefalorraquidiano , Deficiência de Ácido Fólico/complicações , Surtos de Doenças , Doenças do Nervo Óptico/epidemiologia , Formiatos , Consumo de Bebidas Alcoólicas , Doenças do Nervo Óptico/sangue , Doenças do Nervo Óptico/induzido quimicamente , Doenças do Nervo Óptico/líquido cefalorraquidiano , Metanol , Fatores de Risco , SolventesRESUMO
OBJECTIVE: Toxic manifestations following ethylene glycol exposure are due to accumulation of metabolites, particularly glycolate. We characterized glycolate elimination kinetics and dialysis properties in a series of ethylene glycol poisonings. METHODS: Patients who ingested ethylene glycol and received fomepizole (4-methylpyrazole; 4-MP) +/- hemodialysis were prospectively evaluated. Serial blood samples for ethylene glycol, glycolate, pH, and bicarbonate were drawn to determine glycolate elimination rate, t1/2, and correlations between initial glycolate and initial markers of acidosis. Dialyzer inlet and outlet samples were obtained to measure hemodialysis glycolate clearance. Plasma ethylene glycol and glycolate were determined by gas chromatography. RESULTS: Ten patients, mean age 49 years (range 28-73 years), presented a mean of 10.5 hours (range 3.5-21.5 hours) after ethylene glycol ingestion. Mean initial ethylene glycol was 18.5 mmol/L (range 0.8-62.2 mmol/L) (115 mg/dL; range 5-386 mg/dL) and glycolate was 17.0 mmol/L (range 10.0-23.7 mmol/L). Nine of 10 underwent hemodialysis. Nonhemodialysis (n = 4) elimination rate was 1.08 +/- 0.67 mmol/L/h (mean +/- SD) and t1/2 was 626 +/- 474 minutes. Elimination t1/2 during hemodialysis (n = 8) was 155 +/- 42 minutes. Hemodialysis clearance (n = 5) was 170 +/- 23 mL/min with flow rates 250-400 mL/min. Pearson correlation coefficients were: anion gap vs glycolate r2 = 0.65 (p = 0.005), bicarbonate vs glycolate r2 = 0.10 (NS) and pH vs glycolate r2 = 0.06 (NS). CONCLUSION: Glycolate has a slow elimination rate and long half-life. Hemodialysis effectively clears glycolate. An increased anion gap correlates with the presence of glycolate. Hemodialysis is projected as useful for ethylene glycol-poisoned patients with anion gap acidosis and low ethylene glycol blood levels.
Assuntos
Etilenoglicol/farmacocinética , Etilenoglicol/intoxicação , Glicolatos/sangue , Diálise Renal , Adulto , Idoso , Antídotos/uso terapêutico , Cromatografia Gasosa , Etilenoglicol/sangue , Feminino , Fomepizol , Meia-Vida , Humanos , Masculino , Pessoa de Meia-Idade , Intoxicação/terapia , Estudos Prospectivos , Pirazóis/uso terapêuticoRESUMO
Deficiency of the vitamin folic acid has recently been linked with increased incidence of neural tube defects and of cardiovascular disease, through elevated plasma homocysteine levels. The kidney has an important role in conserving folate to counteract development of deficiency. Urinary folate excretion is regulated by the degree of reabsorption of folate by the proximal tubule cell. To evaluate an in vitro model for studies of the regulation of urinary folate excretion, the present studies examined the transport of 5-methyltetrahydrofolate (5-CH3-H4PteGlu), the primary form of folate in the glomerular filtrate, by normal rat proximal tubule (RPT) cells in confluent monolayer cultures. Specific binding of 5-CH3-H4PteGlu to the apical membrane was saturable (K(D) = 27 nM), but intracellular transport was not saturated up to 100 nM concentrations. 5-CH3-H4PteGlu transport was decreased 50% by concentrations of folic acid that completely blocked 5-CH3-H4PteGlu binding by the apical folate receptor. Probenecid (10 mM), an anion exchange (reduced folate carrier) inhibitor, reduced 5CH3-H4PteGlu transport by 50% without significantly affecting binding. Aspirin (3 mM) did not alter 5-CH3-H4PteGlu transport, but significantly enhanced the inhibition due to probenecid. Similarly, indomethacin (5 microM) potentiated the inhibition of 5-CH3-H4PteGlu transport by probenecid. These data suggest that RPT cells take up 5-CH3-H4PteGlu by both the folate receptor and the reduced folate carrier, implying a role for both pathways in regulating urinary folate excretion.
Assuntos
Proteínas de Transporte/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas de Membrana , Proteínas de Membrana Transportadoras , Receptores de Superfície Celular , Tetra-Hidrofolatos/metabolismo , Animais , Proteínas de Transporte de Ânions , Aspirina/farmacologia , Transporte Biológico , Proteínas de Transporte/urina , Células Cultivadas , Receptores de Folato com Âncoras de GPI , Ácido Fólico/análogos & derivados , Ácido Fólico/metabolismo , Taxa de Filtração Glomerular , Indometacina/farmacologia , Masculino , Probenecid/farmacologia , Ratos , Ratos Sprague-Dawley , Proteína Carregadora de Folato Reduzido , Fármacos Renais/farmacologia , Tetra-Hidrofolatos/urinaRESUMO
Propylene glycol (propane-1,2-diol; PD) is a widely used solvent for intravenous drugs. Clinical studies have reported serious side effects, including the development of renal insufficiency in patients receiving PD as drug vehicle. Despite such clinical reports, the data on the toxicity of PD in isolated renal cells are limited. Using primary cultured human proximal tubule (HPT) cells as an in vitro model, we have previously shown the acute toxic effects of PD in HPT cells (Morshed et al., Fundam. Appl. Toxicol. 23, 38-43, 1994). Since most cases of clinical toxicity are noted after prolonged administration of PD, the current studies were designed to investigate the toxicity of repeated exposure of PD in HPT cells. The onset of toxicity was determined using 10-50 mM racemic, sinister, and rectus PD (rac-, S-, and R-PD, respectively) for periods up to 6 days. Cytotoxicity was noted by decreases in thymidine incorporation, in mitochondrial metabolic activity, and in lysosomal accumulation of neutral red. Exposure of HPT cells to 50 mM PD produced toxic responses, while at 10 mM, responses were not significantly greater than those of osmotic controls. The toxicity was caused by a PD-specific mechanism and by a secondary mechanism without any enantiomeric specificity. The HPT cell toxicity was associated with a 35% increase in cellular thiobarbituric acid-reactive substances and a 20% decrease in glutathione. These findings suggest the development of a mild, subacute toxicity in normally proliferating HPT cells at concentrations that could be achieved in human plasma when PD is used as a drug vehicle.
Assuntos
Túbulos Renais/efeitos dos fármacos , Mitocôndrias/metabolismo , Vermelho Neutro/farmacocinética , Propilenoglicol/toxicidade , Timidina/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Glutationa/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Estereoisomerismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Fatores de TempoRESUMO
Although reabsorption across the apical (AP) membrane of the renal proximal tubule cell plays a vital role in the conservation of plasma 5-methyltetrahydrofolate, basolateral (BL) membrane-directed secretory pathways may also be important in regulating the urinary excretion of folate. Folate transport proteins, folate receptor and the reduced folate carrier have been implicated in the renal conservation of folate across the AP membrane, but their role in BL membrane-directed folate transport has not been studied. 5-Methyltetrahydrofolate transport across the AP and BL membranes of human proximal tubule cells was studied in cells grown on membrane inserts to allow optimum differentiation of AP and BL domains. Colchicine, an inhibitor of vesicular-mediated endocytosis, inhibited AP binding and AP-directed transport without affecting BL transport. Probenecid, an inhibitor of anion exchange, did not affect binding, but inhibited both AP and BL-directed transport with a greater effect on BL transport. Folic acid abolished AP binding of 5-methyltetrahydrofolate, but diminished AP-mediated transport by only 50%. These data suggest that both the folate receptor and the reduced folate carrier participate in AP uptake of folates by human kidney cells, but that BL-mediated uptake occurs primarily by the reduced folate carrier. Folate transport from the secretory direction occurred as readily as that from the reabsorptive direction, indicating that altered secretion could mediate excess urinary folate excretion.
Assuntos
Ácido Fólico/metabolismo , Túbulos Renais Proximais/metabolismo , Receptores de Superfície Celular , Tetra-Hidrofolatos/farmacocinética , Antídotos/metabolismo , Membrana Basilar/fisiologia , Transporte Biológico , Proteínas de Transporte/fisiologia , Células Cultivadas , Receptores de Folato com Âncoras de GPI , Ácido Fólico/urina , Humanos , Leucovorina/metabolismo , Tetra-Hidrofolatos/metabolismoRESUMO
Decreases in plasma folate levels leading to folate deficiency can result from increased urinary loss of folate, due to changes in either the renal reabsorption or secretion of folate. Hence, human proximal tubule (HPT) cells were cultured on microporous membranes to separate apical (AP) and basolateral (BL) domains and to assess the transport of 5-methyltetrahydrofolate from AP-to-BL (i.e., reabsorptive) and BL-to-AP (secretory) directions. Cellular uptake of alpha-methylglucoside occurred specifically from the AP direction, and transport of p-aminohippurate occurred more readily from the BL direction, demonstrating cell polarity similar to that in vivo. Under tight monolayer conditions, binding of folate to the AP membrane occurred more readily from the AP direction, although AP binding also occurred from the BL chamber. Intracellular transport occurred equally from both AP and BL directions. When loaded from either direction, folate was effluxed from HPT cells into both AP and BL chambers. About 20-30% of the internalized substrate was converted to nonfolate catabolites. Thus HPT cells readily take up folate via both the AP and BL membranes, metabolize it intracellularly and secrete the products across both membranes. These studies suggest that renal folate homeostasis is regulated bidirectionally.
Assuntos
Córtex Renal/metabolismo , Túbulos Renais Proximais/metabolismo , Tetra-Hidrofolatos/farmacocinética , Absorção , Transporte Biológico , Membrana Celular/metabolismo , Células Cultivadas , Técnicas de Cultura/métodos , Humanos , Cinética , Metilglucosídeos/farmacocinéticaRESUMO
This study has examined whether elevated glucose can induce lipid peroxidation and contribute to the inhibition of cell growth in human kidney proximal tubule(HPT) cells. HPT cells were cultured in media containing glucose concentrations of 8 mM (control), 25 mM, and 50 mM. Lipid peroxidation was assessed by the thiobarbituric acid reactivity and cell growth was assessed by 3H-thymidine uptake. Results show decreased (59%, p < 0.01) growth of HPT cells cultured in 50 mM glucose. Cells cultured in 50 mM mannitol did not show any growth inhibition, suggesting that the decreased cell growth associated with glucose is not due to osmolarity changes. There was an increase (108%, p < 0.02) in lipid peroxidation in cells cultured with high levels of glucose (50 mM) compared with controls and cells cultured with 50 mM mannitol. To examine if membrane lipid peroxidation or malondialdehyde (MDA, an end product of lipid peroxidation) has any role in the inhibition of cell growth, we examined the effect of tertiary butylhydroperoxide (TBH, known to cause lipid peroxidation and generate MDA) on the growth of HPT cells. TBH decreased cell growth (49, 17 and 3% of controls at 0.1, 0.25, and 0.5 mumole TBH/ml medium). Similarly, a marked reduction in the growth was observed with exogenous MDA (72, 69 and 34% of controls at 0.1, 0.25, and 0.5 mumole MDA/ml medium). This suggests that elevated glucose can induce membrane lipid peroxidation and accumulation of MDA, which in turn can inhibit cellular growth and contribute to the altered structure and function of HPT cells in diabetes.
Assuntos
Glucose/farmacologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Peroxidação de Lipídeos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Malondialdeído/metabolismo , Malondialdeído/farmacologia , Peróxidos/farmacologia , Espécies Reativas de Oxigênio , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , terc-Butil HidroperóxidoRESUMO
Deficiency of folic acid, an essential vitamin involved in critical metabolic pathways, occurs in several conditions, including alcoholism. In humans and animal models, chronic ethanol consumption leads to decreased plasma levels and increased urinary levels of folate. An isolated perfused rat kidney model has shown that ethanol produces increased urinary excretion of folate, suggesting a direct effect of ethanol on the kidney. Because the folate binding protein, located in the brush border membrane (BBM) of proximal tubule cells, is thought to be involved in renal folate reabsorption, the effects of ethanol on BBM binding of folate were assessed. Binding studies were conducted using isolated rat kidney cortex BBM preparations, incubated with 3H-labeled 5-methyltetrahydrofolate (5-CH3H4PteGlu) at varying concentrations (0.1-100 nM). Ethanol at 500 mg/dl did not significantly affect [3H]5-CH3H4PteGlu binding in BBM. The structural analogue, folic acid, decreased [3H]5-CH3H4PteGlu binding under similar conditions. Because of the lack of effect of ethanol on binding to isolated BBM, the effects of ethanol probably occur at other steps in the renal uptake and metabolism of folate.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Ácido Fólico/metabolismo , Hematínicos/metabolismo , Rim/metabolismo , Animais , Ácido Fólico/farmacologia , Hematínicos/farmacologia , Técnicas In Vitro , Rim/efeitos dos fármacos , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Masculino , Membranas/efeitos dos fármacos , Membranas/metabolismo , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Ratos , Ratos Sprague-Dawley , Tetra-Hidrofolatos/metabolismoRESUMO
4-Methylpyrazole (4-MP), a potent inhibitor of alcohol dehydrogenase activity, is a candidate to replace ethanol as the antidote for methanol and ethylene glycol intoxications, because it has a longer duration of action and apparently fewer adverse effects. To study a probable mutual inhibitory effect between ethanol and 4-MP on their elimination, two studies were performed in healthy human volunteers using double-blind crossover designs. In study A1 4-MP in the presumed therapeutic dose range of 10 to 20 mg/kg caused a 40% reduction in the rate of elimination of ethanol in 12 subjects given 0.5 to 0.7 g/kg of ethanol. These data suggest that such doses of 4-MP inhibit alcohol dehydrogenase activity in humans in vivo and would be effective at blocking methanol or ethylene glycol metabolism. In study B, ethanol (0.6 g/kg followed by 0.2 g/kg twice) significantly decreased the rate of elimination of 4-MP (5 mg/kg, given intravenously to four subjects). These moderate doses of ethanol also inhibited the rate of urinary excretion of 4-carboxypyrazole, the primary metabolite of 4-MP in humans. Data suggest that ethanol inhibits 4-MP metabolism, thereby increasing the duration of therapeutic blood levels of 4-MP in the body. This mutual interaction may have clinical implications, because most self-poisoned patients have also ingested ethanol. Theoretically, methanol and ethylene glycol might also show such interactions with 4-MP.