Neuroinflammatory Reactions in the Brain of 1,2-DCE-Intoxicated Mice during Brain Edema.

We previously reported that expression of matrix metalloproteinase-9 (MMP-9) mRNA and protein was upregulated during 1,2-dichloroethane (1,2-DCE) induced brain edema in mice. We also found that the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway resulted in MMP-9 overexpression and nuclear factor-κB (NF-κB) activation in mice treated with 1,2-DCE. In this study, we further hypothesized that inflammatory reactions mediated by the p38 MAPK/ NF-κB signaling pathway might be involved in MMP-9 overexpression, blood-brain barrier (BBB) disruption and edema formation in the brain of 1,2-DCE-intoxicated mice. Our results revealed that subacute poisoning by 1,2-DCE upregulates protein levels of glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba-1), interleukin-1ß (IL-1ß), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS) and p-p65 in mouse brains. Pretreatment with an inhibitor against p38 MAPK attenuates these changes. Moreover, pretreatment with an inhibitor against NF-κB attenuates alterations in brain water content, pathological indications notable in brain edema, as well as mRNA and protein expression on levels of MMP-9, VCAM-1, ICAM-1, iNOS, and IL-1ß, tight junction proteins (TJs), GFAP and Iba-1 in the brain of 1,2-DCE-intoxicated mice. Furthermore, pretreatment with an inhibitor against MMP-9 obstructs the decrease of TJs in the brain of 1,2-DCE-intoxicated mice. Lastly, pretreatment with an antagonist against the IL-1ß receptor also attenuates changes in protein levels of p-p38 MAPK, p-p65, p-IκB, VCAM -1, ICAM-1, IL-1ß, and Iba-1 in the brain of 1,2-DCE-intoxicated-mice. Taken together, findings from the current study indicate that the p38 MAPK/ NF-κB signaling pathway might be involved in the activation of glial cells, and the overproduction of proinflammatory factors, which might induce inflammatory reactions in the brain of 1,2-DCE-intoxicated mice that leads to brain edema.

Lung Tumor Induction by 26-week Dermal Application of 1,2-Dichloroethane in CB6F1-Tg rasH2 Mice.

Short-term alternatives to traditional 2-year carcinogenic studies in rodents are being actively pursued. Recently, a 26-week short-term carcinogenicity study using CB6F1-Tg rasH2@Jcl (rasH2) mice has become a worldwide standard for the evaluation of chemical carcinogenesis. However, an acceptable short-term carcinogenic study model for dermally applied products is still lacking. To investigate the suitability of using the rasH2 mouse to test carcinogenic potential, 1,2-dichloroethane (1,2-DCE) was dermally applied to rasH2 mice: 1,2-DCE is a known carcinogen that causes lung bronchiolo-alveolar adenomas and adenocarcinomas when administered topically, orally, or by inhalation exposure; 1,2-DCE at a dose level of 126 mg/mouse in 200 µl acetone or acetone alone (vehicle control) was applied to the dorsal skin of 10 mice of each sex 3 times a week for 26 weeks. As a positive control, 10 mice of each sex received a single intraperitoneal injection of 75 mg/kg of N-methyl- N-nitrosourea. Bronchiolo-alveolar adenomas and adenocarcinomas were significantly increased in 1,2-DCE-treated rasH2 mice of both sexes, and bronchiolo-alveolar hyperplasias were significantly increased in female mice. Overall, almost all mice of each sex developed adenomas and/or adenocarcinomas with 100% of female rasH2 mice developing bronchiolo-alveolar adenocarcinomas.

Route-to-route extrapolation of 1,2-dichloroethane studies from the oral route to inhalation using physiologically based pharmacokinetic models.

To help develop a comprehensive, quantitative understanding of the hazards of 1,2-dichloroethane (ethylene dichloride, EDC, CAS No. 107-06-2) exposure by the inhalation route, the results of existing subchronic studies and an extended one-generation reproductive toxicity (EOGRT) study recently conducted by the oral route in rats were extrapolated using a physiologically based pharmacokinetic (PBPK) model. The no observed adverse effects levels (NOAELs) for the endpoints of neurotoxicity and reproductive/developmental toxicity were the highest tested doses of 169 and 155 mg/kg-day, respectively. These NOAELs were equivalent to continuous exposure of rats to minimums of 76 ppm and 62 ppm EDC, respectively, using total metabolism of EDC as the dose metric that is equivalent in the oral and inhalation scenarios. In contrast, the subchronic study NOAEL of 37.5 mg/kg-day corresponded to continuous inhalation of 4.4 ppm EDC, based on equivalent extrahepatic metabolism. The selection of the internal metric which serves to establish route-to-route equivalency was found to profoundly influence the NOAEL-equivalent inhalation exposure concentration and thus will be a key determinant of inhalation toxicity reference criteria developed on the basis of EDC studies conducted by the oral route.

Absorption of ethanol, acetone, benzene and 1,2-dichloroethane through human skin in vitro: a test of diffusion model predictions.

The overall goal of this research was to further develop and improve an existing skin diffusion model by experimentally confirming the predicted absorption rates of topically-applied volatile organic compounds (VOCs) based on their physicochemical properties, the skin surface temperature, and the wind velocity. In vitro human skin permeation of two hydrophilic solvents (acetone and ethanol) and two lipophilic solvents (benzene and 1,2-dichloroethane) was studied in Franz cells placed in a fume hood. Four doses of each (14)C-radiolabed compound were tested - 5, 10, 20, and 40µLcm(-2), corresponding to specific doses ranging in mass from 5.0 to 63mgcm(-2). The maximum percentage of radiolabel absorbed into the receptor solutions for all test conditions was 0.3%. Although the absolute absorption of each solvent increased with dose, percentage absorption decreased. This decrease was consistent with the concept of a stratum corneum deposition region, which traps small amounts of solvent in the upper skin layers, decreasing the evaporation rate. The diffusion model satisfactorily described the cumulative absorption of ethanol; however, values for the other VOCs were underpredicted in a manner related to their ability to disrupt or solubilize skin lipids. In order to more closely describe the permeation data, significant increases in the stratum corneum/water partition coefficients, Ksc, and modest changes to the diffusion coefficients, Dsc, were required. The analysis provided strong evidence for both skin swelling and barrier disruption by VOCs, even by the minute amounts absorbed under these in vitro test conditions.

Roles of aquaporins and matrix metalloproteinases in mouse brain edema formation induced by subacute exposure to 1,2-dichloroethane.

The aim of this study was to explore the effects of 1,2-dichloroethane (1,2-DCE) on expression of aquaporins (AQPs) and matrix metalloproteinases (MMPs) in the process of brain edema formation. Two parts were included in this study, establishment of animal model of brain edema, and mechanism of brain edema induced by subacute exposure to 1,2-DCE. In part one, mice were exposed to 0, 1.1, 1.2 or 1.3g/m(3) 1,2-DCE, 3.5h per day for 3days. Pathological analysis and water content detection in the brain were examined. In part two, mice were exposed to 1.2g/m(3) 1,2-DCE, 3.5h per day for 1, 2 or 3days, named group D, E and F, respectively. Expression of AQP4, MMP2 and MMP9 in the brain was determined by immunochemical staining, western blot and real time PCR. According to the results of part one, the 1.2g/m(3) dose was chosen for part two, a follow-up time-course study. In part two, protein expression of MMP2 and MMP9 in group F, and AQP4 in group E and F significantly increased compared to the control. Similarly, mRNA levels of AQP4 in group F, and MMP9 in group E and F significantly increased. Our results suggested that exposure to 1,2-DCE might up-regulate the expression of AQP4 protein and MMP9 mRNA at the early phase of brain edema, and AQP4 may play an important role in the brain edema formation.

Assessment of the developmental toxicity and placental transfer of 1,2-dichloroethane in rats.

This study evaluates the developmental toxicity and placental transfer of 1,2-dichloroethane (DCE) in rats. Sprague-Dawley rats were given 0-2.4 mmol DCE kg-1 day-1 by gavage, or were exposed for 6 hr per day to 0-300 ppm DCE by inhalation, from Day 6 to 20 of gestation. Maternal toxicity was observed after inhalation exposure to 300 ppm DCE and oral administration of 2.0 or 2.4 mmol DCE kg-1. There was no evidence of altered growth nor teratogenic effects after either inhalation or oral administration of DCE at any concentration tested. The time course disposition of 14C was examined over a 48-hr period in 12- and 18-day pregnant rats after a single oral dose of 1.6 mmol [14C]DCE kg-1. Peak concentrations of radiocarbon occurred between 2 and 4 hr postdose. Conceptus (Day 12) and fetal (Day 18) tissues accounted for 0.06 and 0.4% of the administered dose, respectively. Up to 4 hr, levels of radiocarbon in placenta and fetus were slightly less than in maternal plasma of 18-day pregnant rats and were two to five times higher at later periods. At 2 hr, unchanged DCE accounted for most of radioactivity (78-86%) recovered in maternal plasma, placenta, and fetus. Acidic metabolites and radioactivity bound to macromolecules increased up to 24 hr (0.01 mumol-eq DCE g-1) in either placental or fetal tissues. Thereafter, their levels declined more slowly than those in the maternal plasma. Results from this developmental toxicity study in rats confirm embryonic exposure to radiocarbon associated with [14C]DCE and/or its metabolites and has demonstrated the lack of observable teratogenic effects.

Ten and ninety-day toxicity studies of 1,2-dichloroethane in Sprague-Dawley rats.

Male and female Sprague-Dawley rats received 1,2-dichloroethane in corn oil by gavage for 10 or 90 consecutive days. The doses for the 10-day study were 10, 30, 100, and 300 mg/kg; the 90-day study doses were 37.5, 75, and 150 mg/kg. There were ten animals per sex per dose group. In the 10-day study, all female animals died in the high dose group and only 2 of 10 males survived. Final body weights and weight gain along with hematology and clinical chemistry findings were not different from controls. The only relative organ weight which was significantly different was the liver in males exposed to 100 mg/kg. The main histopathological lesion exhibited was multifocal to diffuse inflammation of the mucosal and submucosal layers of the forestomach in the 100 mg/kg dose group. This change was minimal in both males and females. In the 90-day study there were no treatment-related effects pertaining to clinical observations. Body weight gain and total food consumption were significantly decreased in high dose males. There were slight but significant differences in hemoglobin, hematocrit, red blood cell count, platelets, albumin, and alkaline phosphatase values in the 75 and/or 150 mg/kg groups in one or both sexes. In males, relative brain, kidney, and liver weights were significantly increased at 75 and 150 mg/kg. There were also differences in spleen, adrenal, and testes weights (absolute and/or relative). In females, absolute and/or relative kidney and liver weights were significantly increased at 150 mg/kg (liver) and at 75 and 150 mg/kg (kidney). There were no apparent treatment-related effects pertaining to mortality, ophthalmology, gross pathology, or histopathology.

Neoplastic expression in murine cells induced by halogenated hydrocarbons.

The neoplastic expression in mouse embryo fibroblasts exposed to 1,2-dibromoethane and its chloroanalogue, 1,2-dichloroethane in vitro, was examined. Both substances are widely used as fumigants for carpet and upholstery, as gasoline additives, and as organic solvents. Both are known to be highly toxic, mutagenic, and carcinogenic agents. C3H10T1/2 cells treated with these haloalkanes exhibited altered morphology and were selected further by cloning in soft agar. Soft agar clones were found to induce a 100% multitumor occurrence in the nude mouse model. These results suggest that this pair of mutagens have altered the normal phenotype of mouse embryo cells, and these cells have become neoplastic. These neoplastic cell lines will be useful as an in vitro model to study the role of genetic changes in the transformation processes induced by halogenated hydrocarbons.

Effect of inhalation exposure regimen on DNA binding potency of 1,2-dichloroethane in the rat.

1,2-Dichloroethane (DCE) was reported to be carcinogenic in rats in a long-term bioassay using gavage in corn oil (24 and 48 mg/kg/day), but not by inhalation (up to 150-250 ppm, 7 h/day, 5 days/week). The daily dose metabolized was similar in the two experiments. In order to address this discrepancy, the genotoxicity of DCE was investigated in vivo under different exposure conditions. Female F-344 rats (183-188 g) were exposed to [1.2-14C]-DCE in a closed inhalation chamber to either a low, constant concentration (0.3 mg/l = 80 ppm for 4 h) or to a peak concentration (0.3 mg/l = 80 ppm for 4 h) or to a peak concentration (up to 18 mg/l = 4400 ppm) for a few minutes. After 12 h in the chamber, the dose metabolized under the two conditions was 34 mg/kg and 140 mg/kg. DNA was isolated from liver and lung and was purified to constant specific radioactivity. DNA was enzymatically hydrolyzed to the 3'-nucleotides which were separated by reverse phase HPLC. Most radioactivity eluted without detectable or with little optical density, indicating that the major part of the DNA radioactivity was due to covalent binding of the test compound. The level of DNA adducts was expressed in the dose-normalized units of the Covalent Binding Index, CBI = mumol adduct per mol DNA nucleotide/mmol DCE per kg body wt.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative toxicity of ethylene dichloride in F344/N, Sprague-Dawley and Osborne-Mendel rats.

Studies were conducted to compare the toxicity of ethylene dichloride (EDC) in F344/N rats, Sprague-Dawley rats, and Osborne-Mendel rats. Ten rats/sex/group were exposed to EDC in drinking-water at 0, 500, 1000, 2000, 4000 and 8000 ppm for 13 wk. The highest concentration was limited by the maximum solubility of EDC in water (about 9000 ppm). In addition, F344/N rats (10/sex/group) were administered EDC in corn oil by gavage to compare toxicity resulting from bolus administration with that of continuous exposure in drinking-water. Gavage doses of EDC were within the range of total daily doses (in mg/kg body weight/day) resulting from exposure in drinking-water. EDC administered by gavage resulted in greater toxicity to F344/N rats than did administration of similar doses in drinking-water. All males receiving 240 and 480 mg/kg body weight and 9/10 females receiving 300 mg/kg body weight by gavage died before the end of the study. Necrosis of the cerebellum was observed in the brains of 3 males receiving 240 mg/kg body weight and 3 females receiving 300 mg/kg body weight. Hyperplasia and inflammation of the forestomach mucosa were observed in 8 male and 3 female rats that died or were killed in moribund condition. EDC caused minimal toxicity to F344/N, Sprague-Dawley and Osborne-Mendel rats at the drinking-water concentrations used in these studies; only female F344/N rats had EDC-related renal lesions. Based on mortality and EDC-related lesions, the no-effect levels for EDC administered by gavage to F344/N rats were 120 mg/kg body weight for males and 150 mg/kg body weight for females.

Urinary thioether biological monitoring in the interaction between 1,2-dichloroethane and disulfiram in Sprague-Dawley rats.

The interaction between inhaled 1,2-dichloroethane (ethylene dichloride; EDC) and dietary Disulfiram (tetraethylthiuram disulfide; Antabuse; DSF) was investigated for male Sprague-Dawley rats in terms of urinary levels of thio-compounds extractable in ethyl acetate and then hydrolyzed in alkali (the classic urinary thioether assay). The assay was found to be an inadequate biological monitoring indicator for EDC or DSF exposure during the DSF/EDC interaction at exposures of 0, 153, 304 and 455 ppm EDC (7 hr/day, 5 days/week, 30 exposure days) for rats fed with AIN-76 diet fortified with 0.15% DSF. EDC inhibited the excretion of DSF-derived thio-compounds with increasing EDC concentration; the thioether content was dose-related in the absence of DSF. In situations where confounding agents generate neutral S-containing urinary metabolites without involvement of endogenous glutathione, the classic thioether assay requires supplementation by other biochemical monitoring strategies.

Inhalation pharmacokinetics of 1,2-dichloroethane after different dietary pretreatments of male Sprague-Dawley rats.

The effect of the pretreatment of male Sprague-Dawley rats with phenobarbital (PB), butylated hydroxyanisole (BHA) and disulfiram (DSF) on the inhalation kinetics of 1,2-dichloroethane [ethylene dichloride (EDC)] was studied by the gas uptake method. A closed recirculating system was constructed and characterized. The rate curves in all the pretreatment regimens showed saturable dependence on EDC concentration. These saturable dependencies (Michaelis-Menten) appeared to be associated with enzymatic metabolism. In general, a two-compartment, steady-state pharmacokinetic model described the uptake data. Data were transformed by Hanes plots to calculate the inhalational Km, the ambient EDC concentration at which uptake proceeded at half maximum rate, and Vmax, the maximum rate of uptake (i.e., maximum rate of metabolism). Although PB and BHA pretreatments did not affect the Km of EDC, PB pretreatment increased the Vmax while DSF pretreatment decreased both the Km and Vmax.

Interaction between 1,2-dichloroethane and disulfiram. I. Toxicologic effects.

Inhalation and intraperitoneal (ip) studies with 1,2-dichloroethane (1,2-DCE) using Sprague-Dawley rats fed 0.15% disulfiram (tetraethylthiuram disulfide; Antabuse; DSF) in AIN-76 diet [approx. (79 +/- 11) mg DSF/kg body wt/day] resulted in the observation of a toxic interaction relative to either agent alone. The combination treatment caused testicular atrophy and histopathology in the liver and testes at 1,2-DCE inhalation concentrations greater than 300 ppm (an estimated dose of 194 mg/kg body wt/day) administered 5 days/week for 30 days or at ip doses of 150 mg 1,2-DCE/kg body wt/day over 30 days, 7 days/week. DSF lowered the 1,2-DCE dose at which liver enlargement, decreased liver/body weight ratios, and decreased body weight gains appeared relative to exposure to the 1,2-DCE alone. For the ip study, a decrease in spleen weight also occurred. Testicular atrophy and liver pathology had previously been observed in rats exposed to 20 ppm of ethylene dibromide (EDB) and fed 0.05% DSF in rat chow diet; EDB or DSF alone did not elicit testicular atrophy. These toxic effects indicate that 1,2-DCE may interact with DSF as does EDB, but at a much higher dose.

In vivo genotoxicity and acute hepatotoxicity of 1,2-dichloroethane in mice: comparison of oral, intraperitoneal, and inhalation routes of exposure.

The in vivo genotoxicity of 1,2-dichloroethane (DCE) was studied in the liver of male C57BL/6 X C3H F1 (hereafter called B6C3F1) mice after single p.o., i.p., and inhalation exposures. The acute hepatotoxicity of DCE was also examined in order to determine nonnecrogenic exposure levels for each route of administration. Single-strand breaks and/or alkali-labile lesions were demonstrated by alkaline DNA-unwinding/hydroxylapatite chromatography in hepatic DNA at 4 hr after p.o. or i.p. administration of nonnecrogenic doses (100 mg/kg, p.o.; 150 mg/kg, i.p.) of DCE to groups of four to six mice. No evidence of hepatic DNA damage was found immediately following 4-hr inhalation exposures of mice to a nonnecrogenic (150 ppm) or necrogenic (500 ppm) concentration of DCE. Four-hr inhalation exposures of mice to concentrations of DCE causing high mortality within 24 hr (1000 to 2000 ppm) produced evidence of hepatic DNA damage at 4 hr, but the possibility that this damage was due to the acute necrogenic effects of the exposures could not be excluded. A significant fraction of the hepatic DNA damage observed 4 hr after i.p. administration of DCE (200 mg/kg) was still evident after 24 hr, indicating the persistence of unrepaired lesions in the DNA. These findings are consistent with the seemingly contradictory results of the two long-term carcinogenicity bioassays, in which DCE was found to be carcinogenic to Osborne-Mendel rats and B6C3F1, mice when administered by gavage but nontumorigenic to Sprague-Dawley rats and Swiss mice after chronic inhalation exposure. Therefore, our results provide additional evidence for the importance of a route of administration effect in the in vivo genotoxicity and carcinogenicity of DCE.

Report on carcinogenesis bioassay of 1,2-dichloroethane (EDC).

In a carcinogenesis bioassay of the halogenated solvent 1,2-dichloroethane (ethylene dichloride, or EDC), oral administration of the compound produced cancers in rats and mice. In male rats, dosage with EDC caused forestomach cancers, hemangiosarcomas (vascularized cancers) of multiple organs, and subcutaneous fibromas (cancers beneath the skin). Female rats exposed to EDC developed mammary (breast) cancers--in some high-dose animals as early as the 20th week of the study. The chemical also caused breast cancers as well as uterine cancers in female mice, and respiratory tract cancers in both male and female mice.