Extension of a PBPK model for ethylene glycol and glycolic acid to include the competitive formation and clearance of metabolites associated with kidney toxicity in rats and humans.

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.

The role of calcium oxalate crystal deposition in cerebral vessels during ethylene glycol poisoning.

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.

Folate binding and transport by rat kidney brush-border membrane vesicles.

[3H]Pteroylglutamic acid (PteGlu) uptake was studied using brush-border membrane vesicles isolated from rat kidney. Results on the uptake of [3H]PteGlu by brush-border membrane vesicles incubated in media of increasing osmolarities demonstrated that uptake was contributed by two components, intravesicular transport and membrane binding. Both the components of the uptake exhibited similar pH dependence, with maxima at pH 5.6, and were found to be saturable mechanisms with Km values of 6.7.10(-7) and 11.2.10(-7) M, respectively. These studies show that PteGlu is transported by isolated rat kidney brush-border membrane vesicles in a manner consistent with a saturable system and that a binding component may be functionally associated with this.

Incorporation of therapeutic interventions in physiologically based pharmacokinetic modeling of human clinical case reports of accidental or intentional overdosing with ethylene glycol.

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.

Methanol poisoning in human subjects. Role for formic acid accumulation in the metabolic acidosis.

Whereas a great deal of information is available on the etiology of methanol poisoning in the monkey, very little study has been made in human subjects. The role of formic acid in methanol toxicity in human subjects has not been established. Two patients have been studied who have presented with the characteristics of methanol poisoning--metabolic acidosis and ocular toxicity. This has made possible a confirmation of the role of formate in the toxic syndrome. Acidosis was very severe in both cases with arterial pH values of about 6.9 and plasma bicarbonate concentrations of 3 meq/liter. A sensitive and specific assay was used to measure formic acid levels in blood and other fluids. Formate accumulation was marked with initial blood levels ranging from 11.1 to 26.0 meq/liter. Decreases in blood bicarbonate concentrations of similar magnitude coincided with the increase in formate. Thus, accumulation of formic acid plays a major part in the acidosis observed in human subjects poisoned with methanol, as has been demonstrated in monkeys. Treatment involving bicarbonate administration, ethanol infusion and hemodialysis, rapidly decreased formate levels in the blood to control values. Methanol concentrations were reduced but to lesser extent than that of formate. Despite the reduction in formate and methanol concentrations in both cases, the treatment was successful in only one of the two patients.

Ethylene glycol intoxication: evaluation of kinetics and crystalluria.

Ethylene glycol and glycolate kinetics were studied in two cases of ethylene glycol intoxication with maximal ethylene glycol/glycolate concentrations of 40.9/26.8 and 56.4/22.4 mmol/liter, respectively. Both patients survived, but with prolonged renal failure, upon treatment with bicarbonate, ethanol, and hemodialysis. Glycolic acid was the major cause of the metabolic acidosis in both cases; lactate levels were only slightly elevated. Kinetic calculations showed that both ethylene glycol and glycolate were distributed in total body water with plasma half-lives of 8.4 and 7.0 hours, respectively. The half-life of ethylene glycol was increased more than 10-fold by ethanol treatment alone. Calcium oxalate monohydrate crystalluria was dominant in both cases, but in one was preceded by a short period with mainly dihydrate excretion; crystalluria was not present upon admission. Repetitive urine microscopy in search of needle- or envelope-shaped crystals should be performed when ethylene glycol intoxication is suspected.

Role of ethanol metabolism in the alcohol-induced increase in urinary folate excretion in rats.

Chronic ethanol use can lead to folic acid deficiency in humans. In rats, acute doses of ethanol produce a marked increase in the urinary excretion of folate which is followed by a decrease in plasma folate levels. To assess the respective roles of ethanol and its metabolism in these effects, five groups of male Sprague-Dawley rats were treated orally as follows: (1) ethanol in four doses of 1 g/kg each at 0, 1, 2 and 3 hr; (2) ethanol as above plus the alcohol dehydrogenase inhibitor 4-methylpyrazole (4-MP) at 50 mg/kg, i.p., 15 min prior to 0 hr; (3) glucose in four isocaloric doses; (4) glucose plus 4-MP as above; and (5) methanol in four doses of 1 g/kg. Total folate levels in the urine peaked in both ethanol- and methanol-treated rats at the same time as the urine alcohol levels (after 6-8 hr) and then declined over the same time course as the alcohol levels. Concurrent administration of 4-MP inhibited the metabolism of ethanol and maintained the increase in urinary folate excretion throughout 24 hr. Ethanol administration produced minor changes in the relative distribution of folate derivatives in the urine, and these changes were not prevented by 4-MP treatment. The urinary levels of formic acid, which is metabolized by folate-dependent processes, were increased by ethanol administration; this increase was prevented by 4-MP. These results suggest that ethanol is not unique among alcohols in increasing urinary folate excretion and that ethanol metabolism plays no role in the increased urinary folate excretion. However, ethanol metabolism contributes to a second effect of ethanol on the folate system, which leads to increased urinary levels of formic acid.

Methyl alcohol poisoning III. Ocular toxicity.

The ocular toxicity of methyl alcohol has been investigated in six rhesus monkeys. All the animals developed fundus changes within 43 to 171 hours after its ingestion. The only fundus lesion seen was optic disc edema and associated changes, usually of a marked degree. Fluorescein fundus angiography confirmed the findings. The retinal and choroidal circulations, including the retinal capillary bed, were normal. Ophthalmoscopically and angiographically, optic disc edema in methyl alcohol poisoning was indistinguishable from that seen in raised intracranial pressure, except that no increased intracranial pressure was observed. It is postulated that optic disc edema in methyl alcohol poisoning is due to an axoplasmic flow stasis.

Methyl alcohol poisoning. IV. Alterations of the morphological findings of the retina and optic nerve.

The ocular morphological findings of three methyl alcohol-intoxicated rhesus monkeys with optic disc swelling was investigated with light and electron microscopy in conjunction with intravascular horse radish peroxidase. Alterations observed in the optic nerve head were confined to the axons and consisted of swelling and clustering of the mitochondria, disruption of the neurotubules, the formation of vesicles, and enlargement of the axon segments in the prelaminar region. Swelling of the oligodendroglial cytoplasm in contact with the axons and of the astrocytes was seen in the retrolaminar optic nerve and the intraorbital optic nerve. Alterations were not observed in the retina. It is hypothesized that the alterations in the axons are the result of disrupted axoplasmic flow. Possible mechanisms relating methyl alcohol intoxication to disruption of axoplasmic flow are discussed.

Methyl alcohol poisoning. II. Development of a model for ocular toxicity in methyl alcohol poisoning using the rhesus monkey.

Rhesus monkeys were intoxicated with methyl alcohol, using an initial dose of 2 gm/kg and subsequent doses were administered in order to maintain an attenuated and prolonged state of intoxication. Arterial blood samples were drawn for methyl alcohol, formate, PO2, PCO2, and pH, which were monitored periodically throughout the course of the experiment. With the use of these procedures monkeys developed metabolic acidosis with the accumulation of formic acid in the blood and a corresponding decrease in blood bicarbonate. These animals served as models, which allowed for ocular evaluation for early signs related to methyl alcohol poisoning. A mechanism to explain toxicity is proposed and discussed.

Propylene glycol-mediated cell injury in a primary culture of human proximal tubule cells.

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.

Determination of folate transport pathways in cultured rat proximal tubule cells.

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.

Transplacental pharmacokinetics of cocaine and benzoylecgonine in plasma and hair of rhesus monkeys.

There is large variability in the rate and extent of fetal damage from cocaine in humans; however, the sources of such variability are not presently known. In order to study the relationship between maternal cocaine pharmacokinetics at the end of pregnancy and maternal or infant cocaine and benzoylecgonine hair concentrations at birth, ten rhesus monkeys were administered cocaine intramuscularly throughout pregnancy. Cocaine and benzoylecgonine hair concentrations were determined at birth and correlated with maternal pharmacokinetics during pregnancy. There were no correlations between either maternal cocaine Cmax or AUC0-infinity and maternal and infant hair cocaine or benzoylecgonine concentrations. There were no significant correlations between maternal hair benzoylecgonine concentrations and either maternal benzoylecgonine AUC0-120 (r = 0.60; P = 0.07) or benzoylecgonine Cmax (r = 0.60; P = 0.07). No correlations existed between infant hair benzoylecgonine concentrations and either maternal benzoylecgonine AUC0-120 (r = 0.30; P = 0.40) or benzoylecgonine Cmax (r = 0.30; P = 0.40). Also, no correlation was found between maternal cocaine dose and maternal or infant cocaine and benzoylecgonine hair concentrations. In comparison to toxicants such as nicotine and carbon monoxide for which there is a good correlation between maternal systemic exposure and neonatal concentrations, the lack of a similar relationship for cocaine is consistent with the role of the placenta in contributing to the variability in the amounts of cocaine reaching the fetus and hence, potentially to the risk of adverse fetal outcome.

Relationship of alcohol metabolism to folate deficiency produced by ethanol in the rat.

Chronic ethanol use can lead to folic acid deficiency in humans. In rats, acute doses of ethanol produce a marked increase in urinary folate excretion, which precedes a decrease in plasma folate levels. To assess the role of ethanol and its metabolism in these effects, five groups of male Sprague-Dawley rats were treated orally as follows: (1) ethanol in 4 doses of 1 g/kg each at 0, 1, 2 and 3 hr; (2) ethanol, as above, plus the alcohol dehydrogenase inhibitor 4-methylpyrazole (4-MP) at 50 mg/kg IP 15 min prior to 0 hr; (3) glucose in 4 isocaloric doses; (4) glucose plus 4-MP as above; (5) methanol in 4 doses of 1 g/kg. Urinary folate levels (by Lactobacillus casei assay) peaked in both ethanol- and methanol-treated rats at the same time as the urine alcohol levels (6-8 hr) and then declined with a similar time course. Urinary levels of formic acid, which is eliminated by oxidation by a folate-dependent pathway, were significantly increased by ethanol administration, thus indicating another ethanol-folate interaction. Concurrent administration of 4-MP suppressed the increased excretion of formate but had no effect on the increased excretion of folate in ethanol-treated rats. These studies suggest that ethanol has two distinct effects on folate metabolism, one dependent and one independent of ethanol metabolism.

Enzymatic isolation and serum-free culture of human renal cells : retaining properties of proximal tubule cells.

Procedures for the culture of the human renal proximal tubule (HPT) cellutilizing explanted tissue have been previously reported by this laboratory (1). Several other investigators have also reported the isolation and culture of human renal tubule cells (2, and references therein). Although explantation of tissue fragments remains an effective way to initiate cell cultures, cell outgrowth and the attainment of confluent cultures may take several weeks. The cell-culture methodology described in this chapter results in a high yield of confluent primary cultures in 7-10 d. The technique involves the digestion of minced cortical tissue with collagenase, followed by a filtering step to remove tissue fragments. The filtrate is centrifuged, and the cell pellet is resuspended in serum-free growth medium and dispensed onto prepared growth surfaces.

The effect of chronic cocaine exposure throughout pregnancy on maternal and infant outcomes in the rhesus monkey.

To explore the effects of gestational cocaine exposure in a nonhuman primate model, pregnant rhesus monkeys were either treated (N = 10) with escalating doses of cocaine up to 7.5 mg/kg (IM), three times per day, 5 consecutive days per week, prior to conception and throughout gestation, or were not treated (N = 10) with cocaine at all. Substantial levels of both cocaine and its major metabolite, benzoylecgonine, were observed in samples of hair taken at birth from mothers and infants of the cocaine-treated group. Despite these differences in cocaine exposure, the experimental groups did not differ significantly with respect to maternal outcome, as measured by body weight again during pregnancy and length of pregnancy. On the other hand, the experimental groups did differ significantly with respect to infant outcome, as measured at birth by body weight, overall length, and crown circumference, all of which were decreased in the cocaine-treated group. A variety of reflexes tested at birth were normal in the cocaine-treated group. It was concluded that, in a rhesus monkey model, chronic cocaine exposure throughout pregnancy had no significant effect on maternal outcome, but did significantly affect infant outcome as assessed in this investigation.

Normal rat kidney proximal tubule cells in primary and multiple subcultures.

An in vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys and separating the cortex, the cortical tissue is digested with the enzymes DNAse-collagenase (Type I) resulting in a high yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency in a serum-free, hormonally defined medium. The cell yield can be increased by transferring the conditioned medium on Day 1 to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces than on bovine collagen Type I coated surfaces. The culture medium was a 1:1 mixture of Ham's F-12 and Dulbecco's modified Eagle's medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal growth factor, and glutamine. The RPT cells became confluent in 7-10 d, at which point they could be subcultured by trypsinizing and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The cells were histochemically positive for alkaline phosphatase and gamma-glutamyl transpeptidase activity and synthesized the intermediate filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence, the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture model will be a valuable tool for substrate uptake and nephrotoxicity studies.

Transient alterations in cellular permeability in cultured human proximal tubule cells: implications for transport studies.

Primary cultures of human proximal tubule (HPT) cells possess the characteristics of a tight epithelium and retain the characteristics of in vivo renal function. HPT cells form confluent monolayers when grown on collagen-coated polycarbonate inserts in a hormonally defined serum-free medium. However, initial studies of transepithelial transport observed large bidirectional fluxes of the paracellular probe inulin. The present studies were designed to assess the transformation of HPT cell tight junctions to a "leaky" state and subsequent recovery. The apparent transepithelial electrical resistance of HPT cells at confluence was 952.0 +/- 70.0 ohms*cm2, suggesting a well-developed tight junction-mediated paracellular pathway in this epithelium. However, replacement of the growth media produced an immediate 90% drop in the initial resistance, which was paralleled by an increased flux of inulin and of phenol red. This transient abolition of barrier function spontaneously reestablished over 1-2 h by a process that was dependent on the ionic composition of the added media. Complete recovery of cellular resistance was paralleled by markedly decreased fluxes of inulin and of phenol red. The recovery of cellular barrier function was inhibited by cytochalasin B suggesting an intracellular action, not a physical disruption of the monolayer. These results suggest that the tight junctions in these cells appear to transiently produce a leaky state during removal of the media, but rearrange to a "tight conformation" when incubated in the appropriate media.

Effect of ethanol on folate binding by isolated rat renal brush border membranes.

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.

Acute ethanol ingestion by humans and subacute treatment of rats increase urinary folate excretion.

Previous studies with rats showed that acute treatment with ethanol (4 g/kg) produce a marked increase in urinary folate levels, followed by a decrease in plasma folate levels. Analogous studies with human volunteer subjects using a lower dose of ethanol showed that there were small, but statistically significant increases in urinary folate levels after four hours. The initial ethanol dose was 1.0 g/kg with a single supplement of 0.1-0.2 g/kg to maintain ethanol blood levels at about 100 mg/dl for six hours. Further studies with rats were designed to test the cumulative effects of repeated daily doses of ethanol. Male Sprague-Dawley rats were treated for 1, 2, 3, or 4 days either with ethanol orally in 4 doses of 1 g/kg each at 0, 1, 2, and 3 hours or with glucose orally in 4 isocaloric doses. Urine was collected at timed intervals up to 12 hours after each daily dose. The pattern of the increase in urinary folate levels was similar in all groups, whether treated for 1, 2, 3 or 4 days. These results suggest that repeated ethanol treatment can lead to a marked cumulative folate loss via increased urinary excretion and that increased urinary folate excretion may contribute to the development of folate deficiency in humans.