Ethanol - Induced Accumulation of Ethylene Glycol Monoalkyl Ethers in Rats

ABSTRACT

In adult female SPF Sprague-Dawley rats, exposed for 2 hours to 2-methoxy-ethanol (ME, 1600 ppm), 1-acetoxy-2-methoxy-ethane (AME, 800 ppm), 2-ethoxy-ethanol (EE, 420 ppm), or 1-acetoxy-2-ethoxy-ethane (AEE, 170 ppm) the blood level of ME (after ME or AME) or EE (after EE or AEE) was considerably increased after pretreatment with ethanol (20 mmol/kg b.w. i.p.). (ME and EE are metabolites of AME and AEE, respectively.) After i.p. co-administration of ME (10 mmol/kg), EE (10 mmol/kg) or butoxy-ethanol (BE, 2.5 mmol/kg) with ethanol (20 mmol/kg) the blood level of ME, EE, and BE remained nearly constant as long as ethanol levels in blood were above 3 mmol/l. Repeated i.p. dosing (5 times one injection per hour) with EE (4 mmol/kg) or ME (5 mmol/kg) plus ethanol (B or 10 mmol/kg) each resulted in an almost complete accumulation of both ether compounds in the blood. Blood levels of ethanol were increased significantly after EE, but only slightly after ME administration. The prolonged retention of ME, EE, or BE is due to an inhibition of the degradation of these compounds following the competition with ethanol at the alcohol dehydrogenase, the common metabolizing enzyme. This study has demonstrated that glycol ether derivatives are extremely accumulated as long as only very low levels of ethanol are present in blood. Therefore, it is concluded that the elimination of the investigated glycol ethers after occupational exposure can be retarded in alcoholized employees causing an increased health risk of these chemicals following the consumption of alcoholic beverages.     

Disposition of three glycol ethers administered in drinking water to male F344/N rats

The glycol ethers 2-methoxyethanol (ME), 2-ethoxyethanol (EE), and 2-butoxyethanol (BE) are widely used solvents in industrial and consumer applications. The reproductive, teratogenic, and hematotoxic effects of the glycol ethers are due to the alkoxyacetic acid metabolites of these compounds. The effect of alkyl group length on disposition of these three glycol ethers was studied in male F344/N rats allowed access for 24 hr to 2-butoxy[U-14C]ethanol, 2-ethoxy[U-14C]ethanol, or 2-methoxy[U-14C]ethanol in drinking water at three doses (180 to 2590 ppm), resulting in absorbed doses ranging from 100 to 1450 mumols/kg body wt. Elimination of radioactivity was monitored for 72 hr. The majority of the 14C was excreted in urine or exhaled as CO2. Less than 5% of the dose was exhaled as unmetabolized glycol ether. Distinct differences in the metabolism of the glycol ethers as a function of alkyl chain length were noted. For BE 50-60% of the dose was eliminated in the urine as butoxyacetic acid and 8-10% as CO2; for EE 25-40% was eliminated as ethoxyacetic acid and 20% as CO2; for ME 34% was eliminated as methoxyacetic acid and 10-30% as CO2. Ethylene glycol, a previously unreported metabolite of these glycol ethers, was excreted in urine, representing approximately 10, 18, and 21% of the dose for BE, EE, and ME, respectively. Thus, for longer alkyl chain lengths, a smaller fraction of the administered glycol ether was metabolized to ethylene glycol and CO2. Formation of ethylene glycol suggests that dealkylation of the glycol ethers occurs prior to oxidation to alkoxyacetic acid and, as such, represents an alternate pathway in the metabolism of these compounds that does not involve formation of the toxic acid metabolite.     

Hematological effects of four ethylene glycol monoalkyl ethers in short-term repeated exposure in rats

This study was carried out to compare the hematological effects of 2-methoxyethanol (ME), 2-ethoxyethanol (EE), 2-isopropoxyethanol (IPE), and 2-butoxyethanol (BE) in short-term studies in rats. Male rats were subcutaneously treated with ME or EE at a dosage of 0, 1.25, 2.5 and 5.0 mM/kg in saline, 5 days per week, for 4 weeks. Other rats were exposed to IPE or BE at doses of 0, 0.25, 0.5, 0.75 and 1.25 mM/kg in the same manner. Administration of each chemical, except of ME, resulted in a time- and dose-dependent swelling of erythrocytes as evidenced by an increase in mean corpuscular volume (MCV). Subsequently, red blood cells (RBC), packed cell volumes (PCV), hemoglobin concentration (HGB), and mean cell hemoglobin concentration (MCHC) decreased. Furthermore, an increase in mean cell hemoglobin (MCH) and reticulocyte counts was observed. The onset of hemolysis induced by EE, IPE or BE was faster than after ME administration. While in rats exposed to ME hematological changes were strongly pronounced and progressively increased with exposure time beginning from the day 11, those in animals treated with EE were rather persisted at low constant level for all exposure period. In contrast, the rats exposed to IPE and BE demonstrated the dramatic hematological changes more pronounced in case of BE than IPE at the beginning of exposure (on day 4). Despite of exposure duration, these changes were regressed, although the decrease in RBC and MCHC and the increase in MCV and MCH in rats treated with highest doses of both compound (0.5, 0.75, and 1.25 mM/kg) were more persistent, probably due to selective hemolysis of the aged erythrocytes. In addition, significant leukopenia due to reduction of lymphocytes in rats exposed to ME was observed. In summary, this study demonstrated no tolerance to ME- and EE-induced intravascular hemolysis developed under these experimental conditions. On the contrary, tolerance to IPE- and BE-induced hemolysis in rats exposed to these compounds was prompted.     

Effect of Dose on the Disposition of Methoxyethanol, Ethoxyethanol, and Butoxyethanol Administered Dermally to Male F344/N Rats

The glycol ethers methoxyethanol (ME), ethoxyethanol (EE), andbutoxyethanol (BE) are widely used in industrial and householdproducts. Rodent studies indicate the ME and EE are potentiallytoxic compounds causing teratogenic, fetotoxic, hematotoxic,and testicular effects. Exposure of rodents to high concentrationsof BE resulted in anemia due to hemolysis of blood cells, leukopenia,hemoglobinuria, and liver and kidney damage. The purpose ofthis study was to determine the uptake, metabolism, and excretionof dermally administered glycol ethers as a function of theexternally applied dose. Three different amounts of the ¹⁴C-labeledglycol ethers (450-4000 µmole/kg) were applied to same-sizedareas on the clipped backs of F344/ N rats, and nonoccludedpercutaneous absorption was measured. The rates of excretionof the ^l4C-labeled parent compound and metabolites by differentroutes were measured, as well as the amount of ¹⁴C remainingin the carcass. Within the dose range studied, the absorptionand metabolism of these three glycol ethers by F344/N rats waslinearly related to the dermally applied dose. The absorptionof all three glycol ethers was approximately 20–25%, regardlessof the chain length of the alkyl group or the dose administered.The majority of the absorbed dose was excreted in the urine.Feces and exhaled CO₂ represented minor routes of excretion.The alkoxyacetic acid was a major metabolite for all three glycolethers. The formation of small amounts of ethylene glycol indicatedcleavage of the ether bond. Dermally administered glycol etherswere metabolized differently than glycol ethers administeredin drinking water (M. A. Medinsky, G. Singh, W. E. Bechtold,J. A. Bond, P. J. Sabourin, L. S. Birnbaum, and R. F. Henderson,1990, Toxicol. Appl. Pharmacol. 102, 443-455). In general, administrationin drinking water enhanced the production of ethylene glycoland glycol ether-derived CO₂.     

Comparative Immunosuppression of Various Glycol Ethers Orally Administered to Fischer 344 Rats

Oral dosing of adult male F344 rats with the glycol ether 2-methoxyethanol(ME) or its principal metabolite 2-methoxyacetic acid (MAA)results in the suppression of the primary plaque-forming cell(PFC) response to trinitrophenyl-lipopolysaccharide (TNP-LPS).In the present study, the PFC response to TNP-LPS was used toevaluate the immunotoxic potential of ethylene glycol (EG) aswell as the glycol ethers 2-methoxyethyl acetate (MEA), 2-(2-methoxyethoxy)ethanol, bis(2-meth- oxyethyl) ether, 2-ethoxyethanol and itsprincipal metabolite 2-ethoxyacetic acid, 2-ethoxyethyl acetate,and 2-butoxyethanol relative to ME and MAA. Rats were immunizedwith TNP-LPS and then exposed 4 and 28 hr later to 50, 100,200, or 400 mg/kg of glycol ether or EG. Three days followingimmunization, the PFC response to TNP-LPS was determined. Inaddition to ME and MAA, only MEA, which was as effective asME, suppressed the PFC response to TNP-LPS. Concomitant administrationof the alcohol dehydrogenase inhibitor 4-methylpyrazole withME or MEA prevented suppression of the PFC response by theseglycol ethers. These results indicate that of the chemicalstested only ME, MEA, and MAA are immunosup pressive, and thatoxidative metabolism via alcohol dehydrogenase is necessaryfor ME- and MEA-suppression of the response to TNP-LPS.     

Studies on the effects of N,N′-dimethylformamide on ethanol disposition and on monoamine oxidase activity in rats

Pretreatment of male rats with N,N′-dimethylformamide (DMF) by oral or inhalation routes prior to ethanol administration caused marked alterations in blood ethanol and/or acetaldehyde concentrations, depending upon the DMF dose given and on the DMF-ethanol time interval. Rats were given single oral doses of DMF (2 or 20 mmol/kg or disulfiram (2 mmol/kg) and challenged with ethanol (2 g/kg po) 18 hr later. Disulfiram or DMF at equimolar doses produced peak elevations in blood acetaldehyde concentrations which were about five- and fourfold higher, respectively, than those of controls. In contrast, the 20-mmol/kg did not enhance acetaldehyde blood concentrations but resulted in approximately a twofold elevation in the peak blood ethanol concentration. Monoamine oxidase activity in brain or liver was not affected by DMF. No increases in acetaldehyde concentrations occurred when the time interval between DMF and ethanol was reduced to 3 hr. Rats were exposed to DMF by inhalation at 1000 or 6000 ppm for 3 days (4 hr/day) and given ethanol 1 hr after the last exposure. Blood acetaldehyde was increased by 46% in rats exposed to 1000 ppm of DMF when measured 30 min after ethanol. DMF exposure at 6000 ppm decreased blood acetaldehyde concentrations to 50% of control values, but increased blood ethanol concentrations by 55%. When ethanol was given 24 hr after the last DMF exposure period, marked elevations in peak blood acetaldehyde concentrations occurred at both exposure concentrations of DMF. Single oral pretreatment of rats with 2- or 20-mmol/kg doses of N-methylformamide (MF), a metabolite of DMF, 18 hr prior to ethanol also enhanced blood acetaldehyde concentrations. A similar result was obtained when rats were given 2 mmol/kg of MF only 3 hr before ethanol.     

The relationship of embryotoxicity to disposition of 2-methoxyethanol in mice

Paw development of CD-1 mice is uniquely sensitive to 2-methoxyethanol (ME) given by gavage (po) on gestation day (gd) 11 (copulation plug day = gd 0). The relation between induction of paw dysmorphogenesis and disposition of po ME (3.3 or 4.6 mmol/kg) in the maternal and conceptus compartments was investigated. The expression of digit malformations depends on metabolism of ME to methoxyacetic acid (MAA). ME and MAA were equipotent in causing teratogenicity. Alcohol dehydrogenase (ADH) catalyzes the initial rate-limiting oxidation that leads to embryotoxicity. The ADH inhibitor 4-methylpyrazole (0.12 or 1.2 mmol/kg) or ethanol (43.3 mmol/kg, single dose concomitant with ME or additional ethanol 5 and 10 hr later) reduced the incidence of malformations 60-100%, depending on the dosing regimen. Elimination of 14C from 1,2-14C-ME occurred predominantly via urine where 80% of a teratogenic dose was excreted and 6% appeared in CO2. Oxidation of ME to MAA was nearly complete after 1 hr when approximately 90% of 14C in maternal plasma and conceptus coeluted with authentic 14C-MAA upon HPLC. 14C-MAA levels in embryos were 1.2 X those in plasma 1 and 6 hr after dosing, although by 6 hr concentrations had declined to approximately 50% of 1-hr values. Concomitant ethanol did not affect 14C kinetics as measured in maternal blood after oral 14C-ME, but retarded ME conversion to MAA by about 2 hr. Furthermore, embryo 14C-MAA levels then reached only 50% of the peak in embryos from dams dosed with ME alone, an effect that coincided with less 14C incorporation into macromolecules synthesized by the embryo within 6 hr. These data imply that the attenuation of digit malformations by concomitant ethanol may be explained by changes in MAA disposition. However, delayed ethanol (5 and 10 hr after 3.3 mmol ME/kg) reduced teratogenicity by 25%, although MAA was present in the embryo up to 5 hr. Dams given 14C-MAA by iv injection had higher 14C blood levels than after MAA po but their offspring had fewer digit malformations. Peak and steady-state plasma levels of MAA as well as embryo concentrations of the chemical do not appear to determine the embryotoxic outcome whereas further metabolism of MAA does.     

Delayed Ethanol Elimination in Rats after Application of Cefamandole or Cefoperazone

Adult female Sprague-Dawley rats received ethanol (2 g/kg b.w., i.p.) 18 hours after pretreatment with the betalactam-antibiotics cefamandole (CMD) or cefoperazone (CPZ) (1 mmol/kg b.w. each, i.p.). The blood ethanol concentrations, determined repeatedly within 4 hours by head space GC, were increasingly elevated after CMD or CPZ up to twice the respective control values. The possible clinical and forensic significance of these findings for the therapeutic use of CMD or CPZ is pointed out.     

Hematological effects of exposure to mixtures of selected ethylene glycol alkyl ethers in rats

Exposure to various ethylene glycol monoalkyl ethers (EGAEs) is known to result in hemolytic effect caused by their metabolites, appropriate alkoxyacetic acids, generated via both alcohol dehydrogenase and aldehyde dehydrogenase. It has been shown in many studies that administration of single doses of EGAEs to rats lead to dose- and time-dependent hemolytic anemia. The repeated exposure to isopropoxyethanol (IPE), and butoxyethanol (BE), contrary to methoxyethanol (ME) and ethoxyethanol (EE), resulted in significantly less pronounced hematological changes. While the majority of hematological effects were dramatic at the beginning of the exposure, later these changes clearly regressed despite continued weekly exposure to these ethers. The gradual recovery from the hemolytic anemia may be associated with tolerance development to the hemolytic effect of IPE and BE. ME demonstrated high hematotoxicity, which increased progressively and reached a maximum at the end of 4 week exposure, whereas EE revealed moderate hematological effects. It might be suspected that ME and EE may modified of IPE hemolytic activity in rats simultaneously treated with these compounds. In the rats co-exposed to IPE and ME subcutaneously at a relatively low doses of 0.75 mM + 0.75 mM for 4 weeks, a significantly less pronounced hematological changes at the beginning of the exposure in comparison with animals treated with IPE (0.75 mM) alone were observed. At the later period, i.e., at the end of 4 weeks exposure, the hematological alterations in the same animals were markedly pronounced and progressively elevated with exposure time, except for mean corpuscular volume (MCV) values, which were significantly lower in comparison with IPE group. ME at the higher dose of 1.25 mM/kg and EE at both doses of 0.75 and 1.25 mM/kg did not modify the hematotoxicity of IPE (at doses of 0.75 mM and 1.25 mM) at the beginning of the exposure, whereas increased its harmful effects at the end of the treatment. The amelioration in the majority of the hematological parameters at the beginning of the exposure may be caused by inhibitory effect of ME on IPE metabolism. On the contrary, an accumulation of the methoxyacetic acid and ethoxyacetic acid, toxic metabolites of ME and EE, respectively, and no tolerance development to the hemolytic effect of these two chemicals may be responsible for elevated hematological alterations at the end of the exposure.     

Effects of Subchronic Exposure of Rats to 2-Methoxyethanol or 2-Butoxyethanol: Thymic Atrophy and Immunotoxicity

Effects of Subchronic Exposure of Rats to 2-Methoxyethanol and2-Butoxyethanol: Thymic Atrophy and Immunotoxicity. Exon, J.H., Mather, G. G., Bussiere, J. L., Olson, D. P., and Talcott,P. A. (1991) Fundam. Appl Toxicol. 16, 830–840. Male Sprague–Dawleyrats were exposed to either 2000 or 6000 ppm of 2-methoxyethanol(ME) or 2-butoxyethanol (BE) and females were exposed to either1600 or 4800 ppm of these compounds in the drinking water for21 days. Body weights were decreased in male rats exposed tothe high doses of both chemicals, while body weights of femalesexposed to either dose of BE were decreased. Male and femalerats exposed to either concentration of ME had a dose-relatedreduction in thymus weights. Testis weight was significantlylower in male rats exposed to the high dose of ME. Dose-relatedincreases in natural killer (NK) cell cytotoxic activities anddecreases in specific antibody production were observed in allrats treated with ME. Rats exposed to the low dose of BE alsohad enhanced NK cell activity. Splenocyte production of interferon-was decreased in male rats exposed to either dose of ME andin females treated with the high dose of ME. Spleen cell numberswere reduced in males exposed to the high dose of ME and femalesgiven either dose of ME. It appears that the immune system isa sensitive target of ME but not BE. The effects of ME on immunefunction differ depending on the immune parameter assessed.Enhanced NK cell activity may partially explain the observationsof others that certain glycol ethers have antitumor effectsin vivo.     

Comparison of the effects of ethanol and 4-methylpyrazole on the pharmacokinetics and toxicity of ethylene glycol in the dog

The purpose of this investigation was to compare the effects of ethanol and 4-methylpyrazole (4MP) on the toxicity and pharmacokinetics of ethylene glycol (EG) in the dog. All dogs received 173 mmol/kg EG, p.o. Dogs were randomly assigned to 3 groups: EG-treated only, EG + ethanol (19.3 mmol/kg, i.v. 3, 7, 14 and 24 h after EG) and EG + 4MP (0.24 mmol/kg, i.v. 3 h after EG, 0.18 mmol/kg at 24 h and 0.06 mmol/kg at 36 h). EG produced a rapid onset of metabolic acidosis (within 3 h) and acute oliguric renal failure (after 48 h), whereas administration of ethanol or 4MP greatly attenuated acidosis and prevented renal toxicity. The administration of ethanol, however, severely increased the central nervous system (CNS) depression that existed after ingestion of EG. The half-life of FG in serum was 10.8 +/- 0.7 h in the EG-only treatment group, 6.8 +/- 0.7 (P less than 0.05) in the EG + ethanol group and 9.8 +/- 0.9 h in the EG + 4MP group. Approx. 10% and 48% of the dose of EG was excreted unchanged in the urine at the 0-3 and 3-72 h periods, respectively. Treatment with 4MP increased the amount of EG excreted in the urine (71% from 3-72 h), whereas ethanol did not (51%). However, both ethanol and 4MP increased the rate constant of EG excretion into urine approx. 70%. These data demonstrate the utility of 4MP over ethanol for the treatment of EG-induced toxicity in dogs and indicate that ethanol and 4MP cause an increase in the rate constant of EG excretion in the urine and not a prolongation in EG half-life.     

Effect of exposure concentration on the disposition of inhaled butoxyethanol by F344 rats.

The glycol ethers are a class of solvents widely used due to their range of vapor pressures and miscibility in aqueous and organic media. Butoxyethanol (BE) causes anemia and lowered hematocrits in rats due to direct hemolysis of red blood cells. Exposure to BE is most likely to occur by dermal contact or by inhalation. In this paper, we report the uptake, metabolism, and excretion of BE following 6-hr exposure at different inhaled concentrations. The uptake and metabolism of BE were essentially linear up to 438 ppm. The majority of the inhaled butoxy-[14C]ethanol was eliminated in the urine with butoxyacetic acid (BAA) being the major urinary metabolite, accompanied by lesser amounts of ethylene glycol and BE glucuronide. A small proportion (5-8%) of the retained BE was exhaled as 14CO2. Most (greater than 80%) of the [14C]BE-derived material in blood was in the plasma. BAA was the major metabolite of BE in plasma. Ratios of ethylene glycol to BAA in plasma were higher than those in urine. The BE-derived 14C in plasma rapidly became associated with the acid-precipitable (protein) fraction, probably due to binding of metabolites to proteins or incorporation of the BE metabolites into the carbon pool. These results indicate that, in rats, overall metabolism of BE to BAA, the hemolytic metabolite, was linearly related to the exposure concentration up to a concentration that caused severe toxicity (438 ppm). Assuming that the toxicity of inhaled BE is directly proportional to the formation of BAA, the toxicity of inhaled BE can be expected to be linearly related to the exposure concentration up to exposure concentrations that cause mortality.     

Effect of carbon monoxide inhalation exposure in mice on drug metabolism in vivo

Experiments were undertaken to evaluate the action of carbon monoxide (CO) on the mixed-function oxidase (MFO) system in vivo. Mice were exposed to 500 ppm CO for 8 h per day in an inhalation chamber under dynamic airflow conditions. Hexobarbital (150 mg/kg, i.p.), zoxazolamine (150 mg/kg, i.p.) or ethanol (2 mg/g, i.p.) was given to each group of mice during CO exposure and disappearance of the drug from blood or brain was determined while CO exposure continued. The experiments were repeated with different groups of animals which were exposed to CO for 3 or 5 days. Hexobarbital and ethanol metabolism were not affected by CO following either one day exposure or repeated exposure. There was no statistically significant difference in the brain level of zoxazolamine in animals exposed to CO when compared to control. These studies indicate that in vivo metabolism of hexobarbital, zoxazolamine and ethanol in mice is not affected by exposure to 500 ppm CO under the conditions employed in the present study.     

Interactive effect of combined exposure to glycol ethers and alcohols on toxicodynamic and toxicokinetic parameters

 Ethylene glycol monomethyl ether (EGME) exhibits testicular toxicity and ethylene glycol monobutyl ether (EGBE) is a solvent with haemolytic effects in rats. The study of the interaction of two glycol ethers (EGME and EGBE) and three alcohols (ethanol, n-propanol and n-butanol, 10 or 30 mmol/kg), orally co-administered in male rats, was carried out from a toxicodynamic and toxicokinetic point of view. Administered alone, EGME (10 mmol/kg) caused a 30- and 5-fold increase in the urinary creatine/creatinine ratio at 24 and 48 h, respectively, and 24 h urinary excretion of methoxyacetic acid was of 0.71± 0.042 mmol/24 h (mean±SE). The simultaneous administration of one of the three alcohols at either of the doses mentioned above did not significantly modify the urinary creatine/creatinine ratio (24 and 48 h), or the 24 h urinary excretion of methoxyacetic acid. Administered alone, EGBE (5 mmol/kg) caused an average decrease of 26% in the number of circulating red blood cells and a strong (250 times) increase in the level of plasma haemoglobin 4 h after treatment. Urinary excretion of butoxyacetic acid in rats treated with EGBE (1 mmol/kg) was 0.083±0.0039 mmol/24 h (mean±SE). The simultaneous injection of 30 mmol/kg alcohol (ethanol, n-propanol or n-butanol) almost totally inhibits the haemolytic effect of EGBE, and decreases the urinary excretion of butoxyacetic acid by 43–31%. A strong dose of alcohol (30 mmol/kg) decreases the haemolytic effect due to EGBE, and reduces the urinary excretion of butoxyacetic acid. In contrast, the coadministration of alcohol did not modify the testicular toxicity of EGME, or the 24 h urinary excretion of methoxyacetic acid. It is possible that competitive inhibition of alcohol dehydrogenase by alcohols results in the diversion of EGBE metabolism. Received: 3 July 1995/Accepted: 17 November 1995     

Dose and route dependency of metabolism and toxicity of chloroform in ethanol-treated rats

 The effects of a single dose of ethanol on the metabolism and toxicity of chloroform administered to rats per os (p.o.), intraperitoneally (i.p.), or by inhalation (inh) at different doses were investigated. Rats that had been given either ethanol (2 g/kg) or vehicle (water) alone at 4 p.m. on the previous day were challenged with chloroform at 10 a.m. p.o. (0, 0.1, 0.2, or 0.4 g/kg), i.p. (0, 0.1, 0.2, or 0.4 g/kg), or inh (for 6 h each at 0, 50, 100, or 500 ppm). The ethanol treatment, which had no influence on the intake of food and water, increased chloroform metabolism in vitro about 1.5-fold with no significant influence on liver glutathione content. The treatment had a dose-dependent effect on the metabolism and toxicity of chloroform, and the effect differed depending on the route of administration. Compared at the same dose level, the area under the curve (AUC) of blood chloroform concentration was invariably smaller following p.o. than i.p. administration. In accordance with this, chloroform administered p.o. caused more deleterious hepatic damage than the same amount of chloroform administered i.p. Although ethanol treatment had no significant influence on the AUC at any dose by any route of administration, the toxicity of p.o.-administered chloroform was significantly higher in ethanol-treated rats than in control rats at a dose as low as 0.1 g/kg, whereas no significant difference was observed in toxicity between both groups of rats at such a low dose administered i.p. When rats were exposed inh to air containing chloroform vapor, ethanol consumption had no effect on hepatotoxicity until the exposure concentration was raised to 500 ppm, a finding which suggests that a single dose of ethanol (2 g/kg) affects the toxicokinetics of inhaled chloroform in rats only at a concentration as high as 500 ppm. Received: 6 December 1993 / Accepted: 25 May 1994     

The teratogenicity of methoxyacetic acid in the rat

Intraperitoneal (i.p.) administration of methoxyacetic acid (MAA) to rats on Day 8, 10, 12 or 14 of pregnancy was embryolethal and teratogenic. Skeletal anomalies, hydrocephalus and dilatation of the kidney pelvis were the most common malformations. Embryonic response to MAA varied with gestational age and with dosage (0.1 to 2.5 mmol/kg). These actions are similar to those previously reported for 2-methoxyethanol (ME) and dimethoxyethyl phthalate (DMEP). Embryos were also examined on Day 12, 48 h following i.p. administration of 2.5 mmol/kg MAA. Abnormalities were comparable to those previously observed following MAA treatment of rat conceptuses in culture. These data support the conclusion that MAA is the proximal teratogenic metabolite of ME and DMEP.     

Metabolism and disposition of ethylene glycol monobutyl ether (2-butoxyethanol) in rats

Ethylene glycol monobutyl ether (2-butoxyethanol, BE) is a major industrial chemical with multiple and diverse uses that may result in significant risk of human exposure and environmental contamination. The current studies were undertaken to investigate the metabolism and disposition of this chemical in male F344 rats. Data presented in this report showed that BE is rapidly absorbed after gavage administration, metabolized, and eliminated. Tissue distribution of BE revealed that BE is distributed to all tissues with the highest levels (determined 48 hr after dosing) detected in the forestomach followed by the liver, kidney, spleen, and the glandular stomach. However, the increase in the tissue concentration in rats treated with 500 mg/kg (as compared to that in rats treated with 125 mg/kg BE) was not proportional to the increase in BE dose. The major route of BE elimination was in the urine, followed by 14CO2 exhalation. The portion of the BE dose eliminated in urine or as 14CO2 was significantly higher in rats treated with 125 mg/kg than in the rats treated with 500 mg/kg. This may indicate that saturation of BE-metabolizing enzymes occurs at the high dose. A small portion (8%) of the administered dose (500 mg/kg) was excreted in the bile in 8 hr after dosing. Qualitative and quantitative HPLC analysis of the urinary and biliary metabolites of BE revealed that the major urinary metabolite, butoxyacetic acid (BAA), accounted for more than 75% of the radioactivity excreted in the urine. The second major metabolite in urine was the glucuronide conjugate of BE (BEG).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhalation toxicokinetics of butoxyethanol and its metabolite butoxyacetic acid in the male Sprague-Dawley rat

A total of 16 male Sprague-Dawley rats were continuously exposed to 20 ppm or 100 ppm butoxyethanol (BE) vapor for 1, 2, 3, 4, 6, 8, 10, or 12 days. Urine was collected in 24-h intervals and stored at –70°C. At the end of the exposure the animals were euthanized by decapitation and tissue samples of blood, muscle, liver and were rapidly collected and frozen to –70°C. The samples were later derivatized and analyzed for BE and its major metabolite butoxyacetic acid (BAA) by electron capture gas chromatography. BE and BAA were rapidly distributed to the tissues examined. The concentration of BE in blood was slightly higher, and that of BAA markedly higher than in other tissues, indicating weak (BE) and pronounced (BAA) blood protein binding, respectively. BE was efficiently metabolized and the blood clearance averaged 2.6 l/h per kg, corresponding to a hepatic extraction ratio of about 0.75. The renal clearance of BAA (average 0.53 l/h per kg) corresponded to approximately 15% of the renal blood flow. The kinetics of BE and BAA were linear up to 100 ppm. There were no clear indications of changes in the toxicokinetics, such as metabolic induction or inhibition of metabolism or excretion, during the course of the exposure. The recovery of BAA in urine was 64% of the calculated inhaled amount of BE, on an equimolar basis. Received: 15 February 1994/Accepted: 3 May 1994     

Role of glutathione in metabolic degradation of dichloromethane in rats

Dichloromethane (DCM) elimination and carboxyhemoglobin (COHb) generation were examined in adult female SD rats pretreated with a glutathione (GSH) depletor(s). Rats were treated with either buthionine sulfoximine (BSO; 2 mmol/kg, i.p.), diethylmaleate (DEM; 3 mmol/kg, i.p.), phorone (PHO; 1 mmol/kg, i.p.) or BSO plus PHO (BSO; 2 mmol/kg +PHO; 0.5 mmol/kg, i.p.). The hepatic GSH concentration was significantly reduced by each treatment. Decrease in hepatic GSH was maintained at least for 10 h after BSO treatment but recovered rapidly in rats treated with DEM or PHO. The hepatic p-nitrophenol hydroxylase activity was not affected by the GSH depletors at the dose used in this study. Rats were treated with an i.p. injection of DCM (3 mmol/kg) and the concentrations of DCM and the COHb levels in blood were monitored. In rats pretreated with a GSH depletor, the peak COHb level was significantly greater than that of rats treated with DCM only. The peak COHb level attained in each group of rats appeared to be inversely related to the magnitude of reduction in hepatic GSH levels. The half-life of DCM in blood was also increased in rats pretreated with the GSH depletor(s). The results indicate that the GSH-dependent metabolic reaction has an important role in the overall elimination of DCM as well as in the metabolic generation of carbon monoxide (CO) from this solvent.     

Genotoxic and anti-genotoxic properties of Calendula officinalis extracts in rat liver cell cultures treated with diethylnitrosamine.

Calendula officinalis flower extracts are used to cure inflammatory and infectious diseases, for wound healing and even cancer with partial objective evidence of its therapeutic properties or toxic effects, many of which can be attributed to the presence of flavonols. We studied whether C. officinalis extracts induce unscheduled DNA synthesis (UDS) in rat liver cell cultures, and if these extracts can reverse diethylnitrosamine (DEN)-induced UDS. Four different flower extracts were prepared: aqueous (AE), aqueous-ethanol (AEE), ethanol (EE) and chloroform (CE). AE and AEE were evaporated to 6.72 and 4.54 mg of solid material per ml, respectively and final ethanol concentration in AEE was 0.8%. EE and CE were dried and resuspended in dimethyl sulfoxide (DMSO) to 19.2 and 10 mg of solid material per ml. Ethanol residue of EE was 0.34%. In the UDS assay in liver cell cultures, DEN at 1.25 microM produced a maximal increase of 40% (3)H-thymidine ((3)HdTT) incorporation, and both, AE and AEE showed complete reversion of the DEN effect at around 50 ng/ml and between 0.4 to 16 ng/ml, respectively. In the absence of DEN, these two polar extracts induced UDS at concentrations of 25 microg for AE and 3.7 microg/ml for AEE to 100 microg/ml in rat liver cell cultures. Concentrations producing genotoxic damage were three orders of magnitude above concentrations that conferred total protection against the DEN effect. Thus, at the lower end, ng/ml concentrations of the two polar extracts AE and AEE conferred total protection against the DEN effect and at the higher end, g/ml concentrations produced genotoxic effects. These results justify the study of C. officinalis flower extracts to obtain products with biological activity and to define their genotoxic or chemopreventive properties.