2,2,2-Trichloroethanol lengthens the circadian period of Bmal1-driven circadian bioluminescence rhythms in U2OS cells.

2,2,2-Trichloroethanol (TCOH) is responsible for the pharmacological actions of chloral hydrate (CH), and is a major metabolite of trichloroethylene. Human exposure to TCOH is known to be increasing. Recently, it was reported that TCOH causes a significant phase delay of Per2 expression in mouse liver when injected daily over the course of several days. However, it is not clear whether TCOH directly modulates the molecular clock. In the present study we used a cell-based assay system to test this possibility. We found that the daily oscillation period of Bmal1 was lengthened to 3 h following treatment with 1.5 mM TCOH, and increased to 5 h with 3 mM TCOH treatment. However, low concentrations of TCOH had no noticeable effects. The effect of TCOH on Per2 oscillation was marginal. Interestingly, serum from rats anesthetized with CH also modulated Bmal1 period, suggesting that exposure to anesthesia should be taken into consideration for circadian rhythm studies. In summary, our study reveals a direct regulation of TCOH on molecular clock.

Tween-80 and impurity induce anaphylactoid reaction in zebrafish.

A number of recent reports suspected that Tween-80 in injectable medicines, including traditional Chinese medicine injections could cause life-threatening anaphylactoid reaction, but no sound conclusion was drawn. A drug-induced anaphylactoid reaction is hard to be assayed in vitro and in conventional animal models. In this study, we developed a microplate-based quantitative in vivo zebrafish assay for assessing anaphylactoid reaction and live whole zebrafish mast cell tryptase activity was quantitatively measured at a wavelength of 405 nm using N-benzoyl-dl-arginine p-nitroanilide as a substrate. We assessed 10 batches of Tween-80 solutions from various national and international suppliers and three Tween-80 impurities (ethylene glycol, 2-chloroethanol and hydrogen peroxide) in this model and found that three batches of Tween-80 (nos 2, 20080709 and 20080616) and one Tween-80 impurity, hydrogen peroxide (H2 O2 ), induced anaphylactoid reactions in zebrafish. Furthermore, we found that H2 O2 residue and peroxide value were much higher in Tween-80 samples 2, 20080709 and 20080616. These findings suggest that H2 O2 residue in combination with oxidized fatty acid residues (measured as peroxide value) or more likely the oxidized fatty acid residues in Tween-80 samples, but not Tween-80 itself, may induce anaphylactoid reaction. High-throughput zebrafish tryptase assay developed in this report could be used for assessing safety of Tween-80-containing injectable medicines and potentially for screening novel mast cell-modulating drugs.

Trichloroethylene and trichloroethanol induce skin sensitization with focal hepatic necrosis in guinea pigs.

OBJECTIVES: Occupational exposure to trichloroethylene (TCE) induces trichloroethylene hypersensitivity syndrome (TCEHS), which causes hypersensitivity dermatitis and hepatitis. However, whether TCE itself or its two metabolites, trichloroethanol (TCEOH) and trichloroacetic acid (TCA), are involved in TCEHS remains unclear. Therefore, in this study we explored the allergens causing TCEHS and characterized TCEHS-related liver injury in guinea pigs. METHOD: The guinea pig maximization test was performed using TCE, TCEOH, and TCA as candidate allergens. Skin inflammation was scored, and liver function and histopathological changes were evaluated by biochemical tests and hematoxylin and eosin staining, respectively. RESULTS: The sensitization rates for TCE, TCEOH, and TCA were 90.0%, 50.0%, and 0.0%, respectively. In the TCE and TCEOH experimental groups, the skin showed varying degrees of erythema with eosinophil granulocyte infiltration in the dermis. Additionally, serum alanine aminotransferase and γ-glutamyl transpeptidase levels increased significantly, and histological analysis revealed focal hepatocellular necrosis with inflammatory cell infiltration in the liver. CONCLUSIONS: TCE is the main cause of allergy and TCEOH is a secondary factor for allergy in guinea pigs. TCE and TCEOH can cause immune-mediated skin sensitization complicated by focal hepatic necrosis.

Upregulation of CYP2E1 expression causes oxidative damage induced by 2-chloroethanol in primary cultured rat astrocytes.

Brain edema caused by subacute poisoning with 1,2-dichloroethane (1,2-DCE) has gained much attention during recent years, but its underlying mechanism is poorly understood. As an intermediate metabolite of 1,2-DCE in vivo, 2-chloroethanol (2-CE) can be transformed into chloroacetaldehyde and reactive oxygen species (ROS) through cytochrome P450 2E1 (CYP2E1) mediated metabolism. In previous studies, it was found that CYP2E1 expression is enhanced in the brain of mice treated with 1,2-DCE. This study was designed to verify the roles of CYP2E1 overexpression in 2-CE induced cytotoxicity in rat astrocytes, and the contribution of specific signaling molecules to the upregulation of CYP2E1 expression caused by 2-CE. The results of this study demonstrate that treatment with 2-CE can enhance CYP2E1 protein and mRNA levels, cause an increase in ROS and MDA levels, and higher percentages of apoptotic cells in rat astrocytes. Pretreatment with either diallyl sulfide or vitamin C, the inhibitor of CYP2E1 or scavenger of ROS, respectively, can suppress the levels of CYP2E1 expression, ROS and MDA, ameliorate cell apoptosis, and attenuate phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in these cells. Additionally, pretreatment with the inhibitor of either ERK1/2 or transcriptional factor specificity protein 1 (SP1) can suppress the CYP2E1 expression, and alleviate the oxidative damage caused to these cells. In conclusion, our findings demonstrate that CYP2E1 overexpression plays a crucial role in 2-CE induced oxidative damage of rat astrocytes, and that CYP2E1 expression is upregulated partially through the activation of the ERK1/2 and SP1 signaling pathways by ROS generated during CYP2E1-mediated 2-CE metabolism. This study provides novel information that can be used in elucidating the mechanism by which 1,2-DCE induces brain edema.

Application of cryopreserved human hepatocytes in trichloroethylene risk assessment: relative disposition of chloral hydrate to trichloroacetate and trichloroethanol.

BACKGROUND: Trichloroethylene (TCE) is a suspected human carcinogen and a common groundwater contaminant. Chloral hydrate (CH) is the major metabolite of TCE formed in the liver by cytochrome P450 2E1. CH is metabolized to the hepatocarcinogen trichloroacetate (TCA) by aldehyde dehydrogenase (ALDH) and to the noncarcinogenic metabolite trichloroethanol (TCOH) by alcohol dehydrogenase (ADH). ALDH and ADH are polymorphic in humans, and these polymorphisms are known to affect the elimination of ethanol. It is therefore possible that polymorphisms in CH metabolism will yield subpopulations with greater than expected TCA formation with associated enhanced risk of liver tumors after TCE exposure. METHODS: The present studies were undertaken to determine the feasibility of using commercially available, cryogenically preserved human hepatocytes to determine simultaneously the kinetics of CH metabolism and ALDH/ADH genotype. Thirteen human hepatocyte samples were examined. Linear reciprocal plots were obtained for 11 ADH and 12 ALDH determinations. RESULTS: There was large interindividual variation in the Vmax values for both TCOH and TCA formation. Within this limited sample size, no correlation with ADH/ALDH genotype was apparent. Despite the large variation in Vmax values among individuals, disposition of CH into the two competing pathways was relatively constant. CONCLUSIONS: These data support the use of cryopreserved human hepatocytes as an experimental system to generate metabolic and genomic information for incorporation into TCE cancer risk assessment models. The data are discussed with regard to cellular factors, other than genotype, that may contribute to the observed variability in metabolism of CH in human liver.

Tumorigenicity of chloral hydrate, trichloroacetic acid, trichloroethanol, malondialdehyde, 4-hydroxy-2-nonenal, crotonaldehyde, and acrolein in the B6C3F(1) neonatal mouse.

The tumorigenicity of chloral hydrate (CH), trichloroacetic acid (TCA), trichloroethanol (TCE), malondialdehyde (MDA), crotonaldehyde, acrolein, and 4-hydroxy-2-nonenal (HNE) was tested in the B6C3F(1) neonatal mouse. Mice were administered i.p. injections of CH (1000, 2000, 2500, and 5000 nmol per animal), TCA (1000 and 2000 nmol), TCE (1000 and 2000 nmol), MDA (1500 and 3000 nmol), crotonaldehyde (1500 and 3000 nmol), acrolein (75 and 150 nmol), and HNE (750 and 1500 nmol) at 8 and 15 days of age. At 12 months, only male mice treated with the positive control chemicals, 4-aminobiphenyl (500 and 1000 nmol) and benzo[a]pyrene (150 and 300 nmol), had incidences of tumors in the liver significantly higher than the solvent control. Additional male mice were dosed as described above and their livers were excised at 24, 48 h, and 7 days after the final dose. Liver DNA was isolated and analyzed by 32P-postlabeling/high-performance liquid chromatography (HPLC) and HPLC/electrochemical detection for MDA-derived adduct (M(1)G) and 8-oxo-2'-deoxyguanosine (8-OHdG) formation, respectively. At 24 and 48 h after the final dose, CH- and TCA-treated mice exhibited significantly higher M(1)G levels than the controls. 8-OHdG formation was also induced by CH, TCA, and MDA. These results suggest that under these experimental conditions the B6C3F(1) neonatal mouse is not sensitive to carcinogens that induce an increase in endogenous DNA adduct formation through lipid peroxidation or oxidative stress.

Exposure to trichloroethylene and its metabolites causes impairment of sperm fertilizing ability in mice.

Trichloroethylene (TCE) is a prevalent occupational and environmental contaminant that has been reported to cause a variety of toxic effects. Here, we have undertaken studies to test the hypothesis that TCE exposure adversely affects sperm function and fertilization. Sperm retrieved from mice exposed to TCE (1000 ppm) by inhalation for 1 to 6 weeks were incubated in vitro with eggs isolated from superovulated female mice. The number of sperm bound per egg was significantly decreased when mice were exposed to TCE for 2 and 6 weeks but not at exposures of 1 and 4 weeks. In vivo fertilization was also determined in superovulated female mice mated with males exposed to TCE for 2 to 6 weeks. The percentages of eggs fertilized, as assessed by the presence of two pronuclei, were significantly decreased after 2 and 6 weeks of TCE exposure. A slight but insignificant decrease was observed after 4 weeks of TCE exposure. The direct effects of TCE and its metabolites, chloral hydrate (CH) and trichloroethanol (TCOH), on in vitro sperm-egg binding were also investigated. Sperm-egg binding was significantly decreased when sperm were pretreated with CH (0.1-10 microg/mL). Significantly lower levels of sperm-egg binding were also detected with TCOH (0.1-10 microg/mL), although the decreases were not as pronounced as those for CH. These results showed that TCE exposure leads to impairment of sperm fertilizing ability, which may be attributed to TCE metabolites, CH, and TCOH.

Uncoupling of oxidative phosphorylation in rat liver mitochondria by chloroethanols.

Chloroethanols are toxic chemicals used in industry and also formed as a result of the metabolism of several widely used halogenated hydrocarbons. The effect of 2-chloroethanol (CE), 2,2-dichloroethanol (DCE) and 2,2,2-trichloroethanol (TCE) on rat liver mitochondrial respiration was studied. Rat liver mitochondria were isolated in a medium consisting of 250 mM sucrose, 10mM Tris-HCl and 1 mM EDTA (pH 7.4). Mitochondrial respiration was determined with an oxygen electrode at 30 degrees C and the polarographic buffer consisted of 250 mM mannitol, 10 mM KCl, 10 mM K2HPO4, 5 mM MgCl2, 0.2 mM EDTA and 10 mM Tris-HCl (pH 7.4). With succinate as the respiratory substrate and using chloroethanols (150 mM), CE stimulated respiration by 28.2 +/- 6.5% and DCE by 202.7 +/- 8.2% while TCE inhibited mitochondrial respiration (greater than 95%). The effect of change in the concentration of chloroethanols on mitochondrial respiration was also studied. CE showed maximum stimulation at 600 mM (97.6%), DCE at 150 mM (202.6%) and TCE at 30 mM (313.6%). Respiratory stimulation was independent of mitochondrial protein concentration. Chloroethanols (optimal concentrations for respiratory stimulation with succinate) inhibited mitochondrial respiration when glutamate-malate was used as the respiratory substrate. Estimation of adenosine triphosphate (ATP) showed that chloroethanols inhibited the synthesis of ATP. These results indicate that chloroethanols stimulate mitochondrial respiration by uncoupling oxidative phosphorylation and that the uncoupling potency is proportional to the extent of chlorination at the beta-position of haloethanol.

Inhibition of alcohol dehydrogenase by chloral hydrate and trichloroethanol: possible role in the chloral hydrate-ethanol interaction.

Both chloral hydrate and trichloroethanol inhibited mouse liver alcohol dehydrogenase (LADH) in vitro. The inhibition of LADH by chloral hydrate appears to be non-competitive in nature with an inhibition constant (Ki) of about 2.7 X 10(-4) M. The inhibition of LADH by trichloroethanol was competitive and the (Ki) was about 2.7 X 10(-5) M. The elimination of ethanol from the blood and brain was significantly reduced in chloral hydrate- or trichloroethanol-pretreated mice. Since reduced elimination of ethanol could result in the prolongation of its central depressant activity, we suggest that this should be considered as a factor in the enhanced pharmacological effects of ethanol-chloral hydrate mixtures.

Assessment of cancer risk from ethylene oxide residues in spices imported into New Zealand.

Quantitative estimates of cancer risks from ethylene oxide (ETO) residues were constructed based on 200 retail samples of various spices in New Zealand. Two samples of cinnamon contained detectable ETO. The highest value encountered was 15 ppm. ETO was not detected in the remaining 198 samples. However, 31 samples had detectable levels of ethylene chlorohydrin (ECH) and/or ethylene bromohydrin (EBH). A conservative estimate of ETO intake, based on average spice consumption, was 3.4 x 10(-6) mg/kg/day. Cancer potency factors for ETO ranging from 0.29 to 0.55 (mg/kg/day)(-1) were used to form cancer risk estimates. The resulting estimates of average lifetime excess cancer risk was 0.8 x 10(-6) to 1.7 x 10(-6). The US 97.5 percentile value for spice consumption (2.8 kg spices per year), gave an extreme upper-end estimate of lifetime cancer risk of approximately 1.4 x 10(-5). These risks are practically negligible considering the conservative assumptions used in estimating exposure to ETO. The exposures to ECH and EBH are 200-300-fold higher than to ETO. These compounds are of lesser potency to ETO in terms of mutagenicity or carcinogenicity in studies to date. However, the precise contribution of these compounds to the cancer risk estimate is uncertain due to large toxicological data gaps, including the absence of a 2-year cancer bioassay by the oral route.

Potential halogenated industrial carcinogenic and mutagenic chemicals. III. Alkane halides, alkanols and ethers.

A variety of alkane halides, alkanols and ethers, analogous to the previously considered halogenated unsaturated (Part I) and saturated hydrocarbons (Part II) possess significant utility in a broad spectrum of applications including: solvents, fumigants, propellants and intermediates in the production of other chemicals, textiles, plastics and ion-exchange resins. Ethylene dichloride and dibromide, propylene dichloride; dibromochloropropane; 2-chloroethanol; 1-chloro-2-propanol; 2,3-dibromo-1-propanol; bis(chloromethyl)-, chloromethyl methyl-, bis(2-chloroisopropyl)-, and bis(2-chloroethyl-ethers were reviewed principally in terms of their synthesis, areas of utility, stability, distribution, reactivity, levels of exposure, population at risk, metabolism, carcinogenicity and mutagenicity.

Guinea pig maximization test for trichloroethylene and its metabolites.

OBJECTIVES: To study the contact allergenic activities of trichloroethylene (TCE) and its three metabolites trichloroacetic acid, trichloroethanol and chloral hydrate. METHODS: A modified guinea pig maximization test (GPMT) was adopted. The skin sensitization (edema and erythema) was observed in trichloroethylene, trichloroacetic acid, trichloroethanol, chloral hydrate and 2,4-dinitrochlorobenzene. RESULTS: The allergenic rate of TCE, trichloroacetic acid and 2,4-dinitrochlorobenzene was 71.4%, 58.3% and 100.0% respectively, and that of trichloroethanol and chloral hydrate was 0%. The mean response score of TCE, trichloroacetic acid and 2,4-dinitrochlorobenzene was 2.3, 1.1, 6.0 respectively. The histopathological analysis also showed an induction of allergenic transformation in guinea pig skin by both TCE and trichloroacetic acid. CONCLUSION: TCE appears to be a strong allergen while trichloroacetic acid a moderate one. On the other hand, both trichloroethanol and chloral hydrate are weak sensitization potentials. Immunologic reaction induced by TCE might be postulated as the pathological process of this illness. Consequently, it is suggested that in the mechanism of Occupational Dermatitis Medicamentose-Like (ODML) induced by TCE, the chemical itself might be the main cause of allergy. As one of its metabolic products, trichloroacetic acid might be a subordinate factor.

Trichloroethylene and trichloroethanol-induced formic aciduria and renal injury in male F-344 rats following 12 weeks exposure.

Trichloroethylene (TCE) is widely used as a cleaning and decreasing agent and has been shown to cause liver tumours in rodents and a small incidence of renal tubule tumours in male rats. The basis for the renal tubule injury is believed to be related to metabolism of TCE via glutathione conjugation to yield the cysteine conjugate that can be activated by the enzyme cysteine conjugate ß-lyase in the kidney. More recently TCE and its major metabolite trichloroethanol (TCE-OH) have been shown to cause formic aciduria which can cause renal injury after chronic exposure in rats. In this study we have compared the renal toxicity of TCE and TCE-OH in rats to try and ascertain whether the glutathione pathway or formic aciduria can account for the toxicity. Male rats were given TCE (500mg/kg/day) or TCE-OH at (100mg/kg/day) for 12 weeks and the extent of renal injury measured at several time points using biomarkers of nephrotoxicity and prior to termination assessing renal tubule cell proliferation. The extent of formic aciduria was also determined at several time points, while renal pathology and plasma urea and creatinine were determined at the end of the study. TCE produced a very mild increase in biomarkers of renal injury, total protein, and glucose over the first two weeks of exposure and increased Kim-1 and NAG in urine after 1 and 5 weeks exposure, while TCE-OH did not produce a consistent increase in these biomarkers in urine. However, both chemicals produced a marked and sustained increase in the excretion of formic acid in urine to a very similar extent. The activity of methionine synthase in the liver of TCE and TCE-OH treated rats was inhibited by about 50% indicative of a block in folate synthesis. Both renal pathology and renal tubule cell proliferation were reduced after TCE and TCE-OH treatment compared to controls. Our findings do not clearly identify the pathway which is responsible for the renal toxicity of TCE but do provide some support for metabolism via glutathione conjugation.

Sex differences in metabolism of trichloroethylene and trichloroethanol in guinea pigs.

OBJECTIVES: Trichloroethylene (TRI) has the potential to cause generalized dermatitis complicated with hepatitis. The guinea pig maximization test (GPMT) also suggests that both TRI and its metabolite trichloroethanol (TCE) exhibit immunogenicity and possible sex differences in guinea pigs. However, TRI and TCE metabolisms in guinea pigs have not been elucidated in detail. The first issue to clarify may be the sex differences in relation to the immunogenicity. METHODS: We collected urine from Hartley male and female guinea pigs 24 hours after intracutaneous injection of TRI, TCE or trichloroacetic acid (TCA) during a GPMT and measured the urinary metabolites by gas chromatography-mass spectrometry. RESULTS: After TRI treatment, the amount of TCA was significantly greater in females than males, while there was no sex difference in the total amount (TCA + TCE). TCA was only detected in urine after TCA treatment. Interestingly, not only TCE but also TCA was detected in urine of both sexes after TCE treatment, and the amount of TCA was also greater in females than males. An additional experiment showed that TCE treatment did not result in the detection of urinary TCA in cytochrome P450 (CYP)2E1-null mice TCEbut did in wild-type mice, suggesting the involvement of CYP2E1 in the metabolism from TCE to TCA. The constitutive expression of CYP2E1 in the liver of guinea pigs was greater in females than males. CONCLUSIONS: The sex difference in urinary TCA excretion after TRI and TCE treatments may be due to variation of the constitutive expression of CYP2E1.

Trichloroethylene-induced formic aciduria: effect of dose, sex and strain of rat.

The industrial solvent trichloroethylene (TCE) has been reported to increase the excretion of formic acid in the urine of male Fischer 344 (F-344) rats following large oral doses. We have examined the dose-response relationship for formic aciduria in male and female Fischer 344 rats, the effect of some known metabolites of TCE and examined the response in male Wistar rats to help understand its relevance to renal toxicity. We report that doses of TCE as low as 8 mg/kg for 3 days to both male and female F344 rats produced formic aciduria. The formic aciduria was time-dependent being more marked after 3 doses compared to one dose in male F344 rats and to a lesser extent in female F344 rats. TCE administration to male Wistar rats produced less formic aciduria than in male F344 rats, indicating a strain difference in response. As TCE is primarily metabolised by cytochrome P450 2E1, Wistar rats were administered inducers of cytochrome P450 2E1 followed by TCE, this increased formic acid excretion to a concentration similar to that observed in male F344 rats, indicating a role for P450. Administration of the major metabolites of TCE, trichloroethanol and trichloroacetic acid to male F344 rats also produced a marked and sustained formic aciduria, while the metabolite of TCE formed via glutathione conjugation had no effect on formic acid excretion. The mechanism whereby this response occurs is currently not understood, but the formic acid excreted is not a metabolite of TCE, but appears to be due to interference with the metabolic utilisation of formate by a down stream metabolite of TCE. Over the three days of the studies no histopathological evidence of kidney toxicity was observed in F344 rats given TCE, indicating that the perturbation of formate metabolism does not lead to acute renal injury.

Effects of chloroacetaldehyde in 2-chloroethanol-induced cardiotoxicity.

Cardiovascular effects have often been found in 2-chloroethanol (2-CE) intoxicated patients, but the 2-CE elicits cardiovascular toxicity mechanism is not clear. Recently, we have found that chloroacetaldehyde (CAA) accumulation in 2-CE-intoxicated rat's blood and play an important role in 2-CE intoxication. In this study, we used an isolated rat atrium model to examine the cardiotoxicity of 2-CE and CAA. Results indicated that 2-CE did not cause tension arrest in isolated rat right atria, but CAA did. 2-CE caused tension inhibition in the isolated rat left atria. In addition, CAA caused significant tension inhibition and contracture in the isolated rat left atria. Nifedipine, an L-type calcium channel blocker, decreased CAA-induced tension inhibition and contracture. Meanwhile, atrial nNOS and calmodulin (CaM) had significantly greater expression in the 2-CE group and the CAA group than control group. Nifedipine could decrease CAA-induced nNOS and CaM expression. 2-CE-induced cardiovascular toxicity might be due to its metabolite CAA. CAA-induced cardiovascular toxicity might be mediated by calcium channel and nifedipine protected against nNOS-triggered cardiovascular effects.

Trichloroethanol up-regulates matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in HaCaT cells.

Occupational trichloroethylene (TCE) exposure could induce generalized skin hypersensitivity reactions complicated with severe liver dysfunctions. Active extracellular matrix degradation and remodeling are involved in the skin hypersensitivity reaction induced by chemical exposure. In the present study, we have compared the effects of in vitro exposure to trichloroethanol (TCOH) and trichloroacetic acid (TCA) of a keratinocyte cell line (HaCaT). The modulation of matrix metalloproteinases (MMPs) was selected as marker of sensitization. HaCaT cells were treated with different concentrations of TCOH or TCA up to 6days. The gelatinolyic activities of MMP-2 and MMP-9 were detected by gelatin-zymography. MMP-2, tissue inhibitor of metalloproteinase (TIMP)-2, MMP-9 and TIMP-1 mRNAs were analyzed by real-time PCR and MMP-9 and TIMP-1 proteins were tested by Western blotting. A dose-effect relationship between TCOH treatment and MMP-9 activity, mRNA and protein expression levels was found in HaCaT cells. TCOH also induced up-regulation of TIMP-1 mRNA and protein. We found no such effects in HaCaT cells treated with TCA. Moreover, previously published literatures on patch tests suggested that TCOH could induce moderately positive reactions at low concentrations in hypersensitivity patients caused by occupational TCE exposure. In summary, these observations indicated that TCOH might play an important role in TCE-induced skin hypersensitivity.

Protective effects of fomepizole on 2-chloroethanol toxicity.

2-Chloroethanol (2-CE) is a widely used industrial solvent. In Taiwan, Taiwanese farmers apply 2-CE on grape-vines to accelerate grape growth, a practice that in some cases have caused poisoning in humans. Thus, there is strong interest in identifying antidotes to 2-CE. This study examines the protective role in 2-CE intoxicated rats. Alcohol dehydrogenase and glutathione were hypothesized to be important in the metabolism of 2-CE. This study used fomepizole, an alcohol dehydrogenase inhibitor, and chemicals that affected glutathione metabolism to study 2-CE toxicity. Notably, fomepizole 5 mg/kg significantly increased median lethal dose (LD(50)) of 2-CE from 65.1 to 180 mg/kg and reduced the production of a potential toxic metabolite chloroacetaldehyde (CAA) in animal plasma. In contrast, disulfiram (DSF), an aldehyde dehydrogenase inhibitor, increased the toxicity of 2-CE on the lethality in rats. Additional or pretreatment with N-acetylcysteine (NAC) and fomepizole significantly reduced plasma CAA concentrations. Fomepizole also significantly reduced 2-CEinhibited glutathione activity. Otherwise, pretreatment with NAC for 4 days followed by co-treatment with fomepizole significantly decreased formation of the metabolic CAA. These results indicated that its catalytic enzyme might play a vital role during 2-CE intoxication, and the combination of fomepizole and NAC could be a protective role in cases of acute 2-CE intoxication.