Influence of exposure route and oral dosage regimen on 1, 1-dichloroethylene toxicokinetics and target organ toxicity.

The objective of this investigation was to elucidate the effects of route of exposure and oral dosage regimen on the toxicokinetics (TK) of 1,1-dichloroethylene (DCE). Fasted male Sprague-Dawley rats that inhaled 100 or 300 ppm for 2 h absorbed total systemic doses of (10 or 30 mg/kg DCE, respectively. Other groups of rats received 10 or 30 mg/kg DCE by intravenous injection, bolus gavage (by mouth), or gastric infusion (g.i.) over a 2-h period. Serial microblood samples were taken from the cannulated, unanesthetized animals and analyzed for DCE content by gas chromatography to obtain concentration versus time profiles. Inhalation resulted in substantially higher peak blood concentrations and area under blood-concentration time curves (AUC(0)(2)) than did gastric infusion of the same dose over the same time frame at each dosage level, although inhalation (AUC(0)(infinity)) values were only modestly higher. Urinary N-acetyl-beta-D-glucosaminidase (NAG) and gamma-glutamyltranspeptidase (GGT) activities were monitored as indices of kidney injury in the high-dose groups. NAG and GGT excretion were much more pronounced after inhalation than gastric infusion. Administration of DCE by gavage also produced much higher Cmax and AUC(0)(2) values than did 2-h g.i., although AUC(0)(infinity) values were not very different. The 30 mg/kg bolus dose produced marked elevation in serum sorbitol dehydrogenase, an index of hepatocellular injury. Administration of this dose by inhalation and gastric infusion was only marginally hepatotoxic. These findings demonstrate the TK and target organ toxicity of DCE vary substantially between different exposure routes, as well as dosage regimens, making direct extrapolations untenable in health risk assessments.

Biotic and abiotic anaerobic transformations of trichloroethene and cis-1,2-dichloroethene in fractured sandstone.

A fractured sandstone aquifer at an industrial site in southern California is contaminated with trichloroethene (TCE) and cis-1,2-dichloroethene (cis-DCE) to depths in excess of 244 m. Field monitoring data suggest that TCE is undergoing reduction to cis-DCE and that additional attenuation is occurring. However, vinyl chloride (VC) and ethene have not been detected in significant amounts, so that if transformation is occurring, a process other than reductive dechlorination must be responsible. The objective of this study was to evaluate the occurrence of biotic and abiotic transformation processes at this site for TCE, cis-DCE, and VC. Anaerobic microcosms were constructed with site groundwater and sandstone core samples. 14C-labeled compounds were used to detect transformation products (e.g., CO2 and soluble products) that are not readily identifiable by headspace analysis. The microcosms confirmed the occurrence of biotic reduction of TCE to cis-DCE, driven by electron donor in the groundwater and/or sandstone. VC and ethene were not detected. Following incubation periods up to 22 months, the distribution of 14C indicated statistically significant transformation of [14C]TCE and [14C]cis-DCE in live microcosms, to as high as 10% 14CO2 from TCE and 20% 14CO2 from cis-DCE. In autoclaved microcosms, significant transformation of [14C]TCE and [14C]cis-DCE also occurred; although some 14CO2 accumulated, the predominant 14C product was soluble and could not be stripped by N2 from an acidic solution (referred to as nonstrippable residue, or NSR). Characterization of the NSR by high-performance liquid and ion chromatography identified glycolate, acetate, and formate as significant components. These results suggest that a combination of abiotic and biotic transformation processes is responsible for attenuation of TCE and cis-DCE in the fractured sandstone aquifer. Tracking the distribution of 14C during the microcosm study was essential for observing these phenomena.

Dose-dependent transitions in mechanisms of toxicity: case studies.

Experience with dose response and mechanisms of toxicity has shown that multiple mechanisms may exist for a single agent along the continuum of the full dose-response curve. It is highly likely that critical, limiting steps in any given mechanistic pathway may become overwhelmed with increasing exposures, signaling the emergence of new modalities of toxic tissue injury at these higher doses. Therefore, dose-dependent transitions in principal mechanisms of toxicity may occur, and could have significant impact on the interpretation of reference data sets for risk assessment. To illustrate the existence of dose-dependent transitions in mechanisms of toxicity, a group of academic, government, and industry scientists, formed under the leadership of the ILSI Health and Environmental Sciences Institute (HESI), developed a series of case studies. These case studies included acetaminophen, butadiene, ethylene glycol, formaldehyde, manganese, methylene chloride, peroxisome proliferator-activated receptor (PPAR), progesterone/hydroxyflutamide, propylene oxide, vinyl acetate, vinyl chloride, vinylidene chloride, and zinc. The case studies formed the basis for technical discourse at two scientific workshops in 2003.

Biologic markers of exposure to chlorinated solvents.

This article presents the current knowledge and clinical applications of the use of biomarkers of exposure to the halogenated solvents 1,1,1 trichloroethane (methylchloroform), trichloroethylene, tetrachloroethylene (perchloroethylene), and 1,1 dichloroethylene (vinylidene chloride). Although some studies have shown that protein and DNA adducts may form with chlorinated hydrocarbons, their application has not been validated sufficiently to justify their use as biologic markers of exposure.

Variability in carbon isotopic fractionation during biodegradation of chlorinated ethenes: implications for field applications.

Stable carbon isotopic analysis has the potential to assess biodegradation of chlorinated ethenes. Significant isotopic shifts, which can be described by Rayleigh enrichment factors, have been observed for the biodegradation of trichloroethlyene (TCE), cis-dichloroethylene (cDCE), and vinyl chloride (VC). However, until this time, no systematic investigation of isotopic fractionation during perchloroethylene (PCE) degradation has been undertaken. In addition, there has been no comparison of isotopic fractionation by different microbial consortia, nor has there been a comparison of isotopic fractionation by consortia generated from the same source, but growing under different conditions. This study characterized carbon isotopic fractionation during reductive dechlorination of the chlorinated ethenes, PCE in particular, for microbial consortia from two different sources growing under different environmental conditions in order to assess the extent to which different microbial consortia result in different fractionation factors. Rayleigh enrichment factors of -13.8@1000, -20.4@1000, and -22.4@1000 were observed for TCE, cDCE, and VC, respectively, for dechlorination by the KB-1 consortium. In contrast, isotopic fractionation during reductive dechlorination of perchloroethylene (PCE) could not always be approximated by a Rayleigh model. Dechlorination by one consortium followed Rayleigh behavior (epsilon = -5.2), while a systematic change in the enrichment factor was observed over the course of PCE degradation by two other consortia. Comparison of all reported enrichment factors for reductive dechlorination of the chlorinated ethenes shows significant variation between experiments. Despite this variability, these results demonstrate that carbon isotopic analysis can provide qualitative evidence of the occurrence and relative extent of microbial reductive dechlorination of the chlorinated ethenes.

Mechanisms of 1,1-dichloroethylene-induced cytotoxicity in lung and liver.

Exposure to 1,1-dichloroethylene (DCE) causes lung and liver toxicities in mice. The lesions are characterized by damage preferentially to bronchiolar Clara cells in the lung and necrosis of centrilobular hepatocytes in the liver. The primary metabolites formed from DCE in lung and liver microsomal incubations are the epoxide, 2,2-dichloroacetaldehyde and 2-chloroacetyl chloride, which undergo hydrolysis and/or conjugation with glutathione (GSH). The major products formed are the epoxide-derived GSH conjugates 2-(S-glutathionyl) acetyl glutathione [B] and 2-S-glutathionyl acetate [C]. The hydrate of 2,2-dichloroacetaldehyde (acetal) is also detected. These metabolites are detected in vivo in murine lung and liver cytosol and in bile, and importantly, also in human lung and liver microsomal incubations. Formation of the epoxide is mediated mainly by CYP2E1. Immunohistochemical studies localized the epoxide-derived GSH conjugate [C] and cysteine-containing proteins in Clara cells and centrilobular hepatocytes. These findings are consistent with the premise that the lung and liver cytotoxicities induced by DCE are associated with in situ formation of the epoxide within the target cells.

Conjugation of glutathione with the reactive metabolites of 1, 1-dichloroethylene in murine lung and liver.

Exposure to 1,1-dichloroethylene (DCE) elicits lung and liver cytotoxicities that are manifested in bronchiolar Clara cell injury and centrilobular necrosis, respectively. The tissue damage is associated with cytochrome P450-dependent bioactivation of DCE to reactive intermediates, and is consistent with the finding that the target cells coincided with the sites of high concentrations of cytochrome P450 enzymes. The metabolites formed from DCE bind covalently to cellular macromolecules, and the extent of binding and cell damage are inversely related to the content of intracellular glutathione (GSH). Histochemical studies showed that staining for GSH in the lung is localized in the bronchiolar epithelial and alveolar septal cells, with relatively strong staining in the Clara cells. In the liver, staining is observed rather uniformly in hepatocytes distributed across the hepatic lobule. Depletion of GSH correlates with the Clara cell damage and centrilobular necrosis observed in the lung and liver, respectively. The primary metabolites of DCE formed in lung and liver microsomal incubations have been identified as DCE-epoxide, 2,2-dichloroacetaldehyde and 2-chloroacetyl chloride. All are electrophilic metabolites that form secondary reactions including conjugation with GSH. Results of our studies indicated that the DCE-epoxide is the major metabolite forming conjugates with GSH, and this reaction is likely responsible for the depletion of GSH observed in vivo. Our findings support the premise that, following depletion of intracellular GSH, metabolites of DCE including the DCE-epoxide bind to cellular proteins, a process which leads to cell damage and suggests that conjugation with the thiol nucleophile represents a-detoxification mechanism.

Changes in cytochrome P450 enzymes by 1,1-dichloroethylene in rat liver and kidney.

We examined the effect of 1,1-dichloroethylene (1,1-DCE) on microsomal cytochrome P450 (P450) enzymes in rat liver and kidney. Rats were treated intraperitoneally with 1,1-DCE daily for 4 days, at doses of 200, 400, and 800 mg/kg. Among the P450-dependent monooxygenase activities in liver microsomes, testosterone 2alpha-hydroxylase (T2AH), which is associated with CYP2C11 activity, was remarkably decreased by 800 mg/kg 1,1-DCE. The level relative to control activity was < 10%. Furthermore, immunoblotting showed that 1,1-DCE (> or = 400 mg/kg) significantly decreased CYP2C11/6 protein levels in liver microsomes. In addition, 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), benzphetamine N-demethylase (BZND), chlorzoxazone 6-hydroxylase (CZ6H), and testosterone 6beta-hydroxylase (T6BH) activities were significantly decreased by the highest dose of 1,1-DCE (by 40-70%). However, the activities of other P450-dependent monooxygenases, namely 7-ethoxyresorufin O-deethylase (EROD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), erythromycin N-demethylase (EMND), lauric acid omega-hydroxylase (LAOH), and testosterone 7alpha-hydroxylase (T7AH) were not affected by 1,1-DCE at any dose. Immunoblotting showed CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A2/1 protein levels were significantly decreased by 60-66% by 1,1-DCE (800 mg/kg), whereas that of CYP4A1/2 was not affected by any dose of 1,1-DCE. By contrast, among the P450-dependent monooxygenase activities in kidney microsomes, only CZ6H activity was increased by 1,1-DCE (1.6-fold at 800 mg/kg). Also, it was observed that 1,1-DCE (800 mg/kg) significantly increased CYP2E1 protein levels by immunoblotting (approximately 1.5-fold). These results suggest that 1,1-DCE changes the constitutive P450 isoforms in the rat liver and kidney, and that these changes closely relate to the toxicity of 1,1-DCE.

Exploration of an interaction threshold for the joint toxicity of trichloroethylene and 1,1-dichloroethylene: utilization of a PBPK model.

Physiologically based pharmacokinetic (PBPK) modeling and gas uptake experiments were utilized to verify the competitive inhibition mechanism of interaction between trichloroethylene (TCE) and 1,1-dichloroethylene (DCE) and to investigate the presence of an interaction threshold between the two chemicals. Initially, gas uptake experiments were conducted on Fischer 344 rats where the initial concentrations of both DCE and TCE were 2000:0, 0:2000, 2000:2000, 1000:0, 1000:1000, and 500:500 ppm, respectively. When the different modes of inhibition interactions (competitive, uncompetitive and noncompetitive) were employed in the PBPK model, the model description of the competitive inhibition provided the best description of the declining concentrations in the gas uptake chamber. Furthermore, to predict the range at which the interaction threshold would be found, the PBPK model included a mathematical description of the percentage of enzyme sites occupied by either chemical in the presence or the absence of the other. By comparing the percentage of occupied sites by one chemical, in the presence of the other, to those sites occupied in the absence of the latter, the PBPK model predicted a range of concentrations (100 ppm or less) of either chemical where the competitive inhibition interaction would not be observed. Consequently, gas uptake experiments were designed where the initial concentration was selected at 2000 ppm for one chemical while the other chemical was set at 100 in one experiment and 50 ppm in another. Under these conditions, the best stimulation to the concentration depletion curves in the gas uptake system of the chemical in the higher concentration was obtained when the PBPK model was run under the assumption of no-interaction. This substantiated the model predictions of the presence of observable interaction only at concentrations higher than 100 ppm.

Pulmonary CYP2E1 bioactivates 1,1-dichloroethylene in male and female mice.

Pulmonary cytotoxicity induced by 1,1-dichloroethylene (DCE) has been linked to the generation of reactive intermediates through a cytochrome P450-dependent pathway. In the present studies, our objectives were to investigate and compare cytochrome P450 isozyme-selective bioactivation of DCE in vitro in the lungs of male and female mice. Our results showed that CYP2E1-dependent p-nitrophenol hydroxylation was significantly higher in microsomes from female (0.45 +/- 0.01 nmol/mg protein/min) than from male (0.38 +/- 0.02 nmol/mg protein/min) mice. Lung microsomes from male mice incubated in the presence of an NADPH-generating system and increasing amounts of DCE (5-20 mM) exhibited corresponding decreases in p-nitrophenol hydroxylase activity (19%-50%); however, greater decreases (26%-70%) were observed in lung microsomes from female mice incubated under the same conditions. In contrast, alterations in CYP2B1-dependent 7-pentoxyresorufin O-dealkylation and CYP1A1-dependent 7-ethoxyresorufin O-dealkylation were not detected in any microsomal preparation incubated with DCE. Reaction with an anti-CYP2E1 antibody abolished the inhibition of p-nitrophenol hydroxylation by DCE. Protein immunoblotting revealed significant decreases in the intensity of the bands of microsomal samples incubated previously with DCE; in contrast, alterations in heme content were not evoked by reaction with DCE. Our results have demonstrated that CYP2E1, and not CYP2B1 or CYP1A1, mediated the bioactivation of DCE. Furthermore, this bioactivation occurred to a greater extent in lung microsomes from female than from male mice, which suggests that females may be at slightly greater risk for DCE-induced pneumotoxicity.

Chloroethylene mixtures: pharmacokinetic modeling and in vitro metabolism of vinyl chloride, trichloroethylene, and trans-1,2-dichloroethylene in rat.

Environmental and occupational exposures are typically to mixtures of chemicals, although most toxicity information is for individual compounds. Interactions between chemicals may involve pharmacokinetic and/or pharmacodynamic effects resulting in modulation of toxicity. Therefore, physiologically based pharmacokinetic modeling has been used to analyze data describing the metabolism of vinyl chloride (VC) and trichloroethylene (TCE) mixtures in rats. A single saturable pathway was modeled, representing cytochrome P450 2E1. This was partially validated using preexposure to trans-1,2-dichloroethylene (tDCE) which virtually eliminated in vivo metabolism of both VC and TCE at low concentrations. Microsomes from tDCE-exposed animals showed inhibition of metabolism of P450 2E1 substrates (chlorzoxazone, p-nitrophenol, and TCE) and no effect on 7-ethoxycoumarin deethylation. Studies with liver microsomes from VC-exposed animals found that neither suicide inhibition nor induction occurred during 6-hr exposures to high concentrations. Therefore, these effects were not modeled. Modeling of mixtures of VC and TCE was successful only using competitive inhibition, as might be predicted for cytochrome P450 2E1 substrates, and not uncompetitive or noncompetitive inhibition. These results were further confirmed by determining the depletion of glutathione due to VC metabolism. The validation of a detailed model for the inhibition kinetics of metabolism of these two compounds permits better understanding of the implications of coexposures for toxicity. It is notable that competitive inhibition only becomes significant at relatively high concentrations (tens of ppm), while at typical low environmental concentrations (ppb), absorption is perfusion limited and enzyme is in excess so that the chemicals will be metabolized independently.

In vitro biotransformation of 1,1-dichloroethylene by hepatic cytochrome P-450 2E1 in mice.

1,1-Dichloroethylene (DCE) is hepatotoxic in mice and its cytotoxic effects are associated with cytochrome P-450 (P450)-dependent formation of metabolite(s) that bind covalently to tissue macromolecules. Our goal was to investigate effects of DCE on P450 in liver microsomes. Specific objectives were to examine 1) inactivation of P450 by DCE and to determine if during this inactivation the heme and/or apoprotein moieties are destroyed and 2) isozyme-selective biotransformation of DCE by P450. Our results showed significant reduction of P450 content in reactions containing DCE and microsomes from untreated (30%) or phenobarbital-treated (20%) mice. Maximal reduction (50%) of P450 was evoked by DCE in reactions catalyzed by microsomes from acetone-treated mice. Alterations in heme levels were not detected in any microsomal preparation incubated in the presence of DCE. Significant inhibition of p-nitro-phenol hydroxylation was found in microsomes incubated previously with DCE and was most pronounced in acetone-treated mice, as compared to control and phenobarbital-treated mice. DCE did not cause inhibition of 7-pentoxyresorufin-O-dealkylation in any microsomal preparation. Immunoinhibition with an anti-2E1 antibody abolished the observed inhibition of p-nitrophenol hydroxylation. Densitometric scanning of protein immunoblots using an anti-2E1 antibody revealed a 40% decrease in microsomes reacted with DCE, whereas no change was observed in immunoblots prepared with an anti-2B antibody. These results showed that 1) biotransformation of DCE is catalyzed by the 2E1 and not by the 2B enzyme and 2) DCE inactivates P450 by destruction of the apoprotein rather than the heme moieties.

Hyperthyroidism increases covalent binding and biliary excretion of 1,1-dichloroethylene in rats.

Distribution, covalent binding, and biliary excretion of 1,1-dichloroethylene (DCE) were examined in euthyroid (EuT) and hyperthyroid (HyperT) rats, which are more vulnerable to DCE hepatotoxicity. Male Sprague-Dawley rats were made hyperthyroid by 3 sc injections of thyroxine at 48-h intervals prior to experiments; euthyroid controls received vehicle injections. A time course study monitored the circulation and excretion of 14C-DCE label for 24 h after administration of 14C-labeled DCE (50 mg/kg in mineral oil) in serial blood and urine samples. At 24 h, total and covalently bound 14C-label were measured in liver, kidney, and lung. Hepatotoxicity of DCE was enhanced in the HyperT rats, as evidenced by elevated serum activities of aminotransferase and histopathology, and was associated with increases in circulating metabolite, and in metabolite bound to red blood cells and liver but not to kidney or lung. Hyperthyroidism had little effect on in vitro capacity of hepatic microsomes to convert DCE to reactive intermediates as reflected by covalent binding. A biliary excretion study in pentobarbital-anesthetized rats showed a striking, but transient, increase in toxicant metabolite excretion in bile of HyperT rats during the first 2 h after toxicant administration (14C-DCE, 100 mg/kg). During the next 2 h, biliary metabolite excretion by HyperT rats decreased while there was a rise in circulating amounts of total and bound 14C-label. Thus, although hyperthyroidism had little effect on the total extent of DCE metabolized, this hormonal disturbance may have transiently enhanced metabolite formation and definitely was associated with a lesser ability to detoxify reactive DCE metabolites capable of injuring hepatic cell constituents by covalent binding reactions.

Nephrotoxicity and covalent binding of 1,1-dichloroethylene in buthionine sulphoximine-treated mice.

Autoradiography of mice injected i.p. with 14C-labelled 1,1-dichloroethylene (vinylidene chloride, VDC) in C57B1/6 mice revealed a selective covalent binding of radioactivity in the proximal tubules, in the midzonal parts of the liver lobules and in the mucosa of the upper and lower respiratory tract. Since VDC is a renal carcinogen in male mice the effects of compounds modulating biotransformation and glutathione (GSH) levels on the renal covalent binding were examined following a single i.p. dose of 14C-VDC. Most pretreatments did not influence the level of binding but treatment with buthionine sulphoximine (BSO), an irreversible inhibitor of gamma-glutamylcysteine synthetase and glutathione (GSH)-depleting agent, increased the renal covalent binding of VDC three-fold. Histopathological examination of kidneys in BSO-pretreated male mice given single i.p. injections of subtoxic doses of VDC (25 and 50 mg/kg) showed necrosis in the proximal tubules (S1 and S2 segments) 24 h following administration. In mice given VDC only, no significant lesions in the kidneys were observed. The severe renal toxicity of VDC in BSO-pretreated mice is suggested to be related to metabolic activation of VDC in the proximal tubules, resulting in further GSH depletion and covalent binding.

1,1-Dichloroethylene hepatotoxicity: hypothyroidism decreases metabolism and covalent binding but not injury in the rat.

Our objective was to determine if the previously reported protective effect of hypothyroidism against 1,1-dichloroethylene hepatotoxicity was associated with a change in distribution and covalent binding. Sprague-Dawley male rats were made hypothyroid (HypoT) by surgical thyroidectomy 2 weeks prior to studies and compared to euthyroid (EuT) rats. Hypothyroidism decreased body weights and liver to body weight ratios while mitochondrial non-protein sulfhydryl groups and cytosolic alcohol dehydrogenase activities were increased by 50%. Rats received a single oral dose of 100 mg [14C]1,1-dichloroethylene (DCE)/kg in mineral oil and were killed at 2, 4, 12 or 24 h; controls received mineral oil only. More rapid liver injury, as measured by serum alanine aminotransferase activity and histology, was present at 2 and 4 h after DCE in HypoT than EuT rats, but a similar magnitude of injury was evident at 12 and 24 h. DCE decreased liver non-protein sulfhydryl groups to a comparable extent in HypoT and EuT rats. Cytosolic glutathione S-transferase and alcohol dehydrogenase activities were decreased only in HypoT rats after DCE. HypoT rats excreted approximately 30% less total [14C]DCE-derived label in urine and their livers, kidneys and lungs consistently contained slightly less covalently bound [14C]DCE-derived label. In contrast, between 1 and 4 h after DCE, greater amounts of acid-soluble and acid-precipitable [14C]DCE-derived label were recovered in red blood cells of HypoT rats. Our results indicate that hypothyroidism did not protect against oral DCE hepatotoxicity but was associated with a more rapid injury at early times. Concurrently, hypothyroidism was found to change the fate of [14C]DCE with higher amounts of 14C-label recovered at early times in red blood cells while less 14C-label was excreted in urine and bound to liver.

Physiologically based pharmacokinetic model for vinylidene chloride.

Vinylidene chloride (VDC), a potent hepatotoxin and suspected carcinogen, is metabolized by mixed-function oxidases into a reactive metabolite(s) which is responsible for its toxicity. The metabolite is detoxified by glutathione (GSH), and liver GSH status is an important factor in the expression of VDC toxicity. A physiologically based pharmacokinetic (PB-PK) model has been developed for VDC in the rat based on oxidative metabolism of VDC and subsequent GSH detoxification of metabolite. The model offers insight into the complex interrelationship between the processes of absorption, metabolism, and GSH conjugation, and simulates the manner in which these factors operate in regulating VDC toxicity. The PB-PK model successfully predicts blood, tissue, and exhaled air concentrations of VDC, and liver GSH levels as a function of dose and route of administration. The model also explains the complex dose-response mortality curves seen with VDC. Because of the low blood:air partition coefficient of VDC and its saturable metabolism, the amount of VDC dose that is metabolized is sensitive to the rate of absorption. After an intravenous bolus dose, most of the administered VDC is exhaled unchanged within a few minutes. Blood VDC half-life is not representative of metabolism rates but to reequilibration of VDC from fat. Rats with greater fat content, therefore, display longer VDC blood half-lives. Simulations are shown to demonstrate the strength of PB-PK modeling techniques in understanding the kinetic behavior of VDC in the rat under a variety of experimental conditions.