Lung injury and repair: DNA synthesis following 1,1-dichloroethylene

Injury and cellular proliferation in the lung were examined following administration of 1,1-dichloroethylene (1,1-DCE) or vinylidene chloride. C57BL/6 male mice were treated orally with 200 mg/kg of 1,1-DCE prior to a single pulse of tritiated thymidine [( 3H]TdR). Necrosis and exfoliation of Clara cells of bronchiolar epithelium were evident by 1 day after chemical administration, and increased in severity by 2 days. A regenerative response was observed at 3 days after 1,1-DCE administration, and by 7 days the epithelium was substantially restored. At 30 days after 1,1-DCE, re-epithelization was achieved and areas devoid of epithelium were not observed. Changes in cellular proliferation were calculated from measurements of [3H]TdR incorporation into total pulmonary DNA. Activity of [3H]TdR was significantly inhibited at 1 day after chemical administration and thereafter increased: a peak of synthesis occurred between 3 and 5 days. At 7 days after 1,1-DCE administration, incorporation of [3H]TdR decreased to levels that were not significantly different from those of control animals. Autoradiographic examination of 0.5 micron thick plastic-embedded lung sections showed that [3H]TdR was incorporated into the DNA of bronchiolar epithelial cells, macrophages, interstitial, endothelial and Type II alveolar cells. However, the majority of the label was taken up by the nonciliated bronchiolar epithelial cells. The increased [3H]TdR incorporation into whole lung correlated with repopulation of bronchioles which was observed following injury. The results demonstrated that 1,1-DCE-induced damage to Clara cells of the bronchiolar epithelium was severe and rapid; re-epithelization was achieved in a relatively short time whereas differentiation was a prolonged process.     

Bioactivation of 1,1-dichloroethylene to its epoxide by CYP2E1 and CYP2F enzymes.

1,1-Dichloroethylene (DCE) exposure to mice elicits lung toxicity that selectively targets bronchiolar Clara cells. The toxicity is mediated by DCE metabolites formed via cytochrome P450 metabolism. The primary metabolites formed are DCE epoxide, 2,2-dichloroacetaldehyde, and 2-chloroacetyl chloride. The major metabolite detected is 2-S-glutathionyl acetate [C], a putative conjugate of DCE epoxide with glutathione. In this investigation, studies were undertaken to test the hypothesis that CYP2E1 and CYP2F2 are involved in bioactivation of DCE to the epoxide in murine lung. We have developed a method using liquid chromatography/mass spectrometry (LC/MS) to evaluate the kinetics of the rates of production of conjugate [C] by recombinant CYP2E1 and CYP2F enzymes and lung microsomes. Concentration-dependent formation of conjugate [C] was found in incubations of DCE with recombinant CYP2E1 and CYP2F enzymes and lung microsomes from CD-1, wild-type (mixed 129/Sv and C57BL), and CYP2E1-null mice. Recombinant rat CYP2E1 exhibited greater affinity and catalytic efficiency for DCE metabolism than did recombinant human CYP2E1, mouse CYP2F2, goat CYP2F3 or rat CYP2F4. In the lung microsomal incubations, the rates of conjugate [C] production were higher in CD-1 mice than in either wild-type or CYP2E1-null mice; the level of [C] in CYP2E1-null mice was about 66% of that in wild-type mice. These results demonstrated that LC/MS analysis is a suitable method for detection and quantitation of conjugate [C], and that CYP2E1 and CYP2F2 catalyze the bioactivation of DCE to the epoxide in murine lung. The results also demonstrated that CYP2E1 is the high-affinity enzyme involved in DCE bioactivation.     

Bioactivation of 1,1-dichloroethylene by CYP2E1 and CYP2F2 in murine lung

1,1-Dichloroethylene (DCE) exposure evokes lung toxicity with selective damage to bronchiolar Clara cells. Recent in vitro studies have implicated CYP2E1 and CYP2F2 in the bioactivation of DCE to 2-S-glutathionyl acetate [C], a putative conjugate of DCE epoxide with glutathione. An objective of this study was to test the hypothesis that bioactivation of DCE is catalyzed by both CYP2E1 and CYP2F2 in murine lung. Western blot analysis of lung microsomal proteins from DCE-treated CD-1 mice showed time-dependent loss of immunodetectable CYP2F2 and CYP2E1 protein. Dose-dependent formation of conjugate [C] was observed in the lungs of CD-1 mice treated with DCE (75-225 mg/kg), but it was not detected after pretreatment with 5-phenyl-1-pentyne (5-PIP). Treatment of mice with 5-PIP and also with diallyl sulfone (DASO2) significantly inhibited hydroxylation of p-nitrophenol (PNP) and chlorzoxazone (CHZX). Incubation of recombinant CYP2F3 (a surrogate for CYP2F2) and recombinant CYP2E1 with PNP and CHZX confirmed that they are substrates for both of the recombinant enzymes. Incubation of the recombinant enzymes with DASO2 or 5-PIP significantly inhibited hydroxylation of both PNP and CHZX. Bronchiolar injury was elicited in CD-1 mice treated with DCE (75 mg/kg), but it was abrogated with 5-PIP pretreatment. Bronchiolar toxicity also was manifested in the lungs of CYP2E1-null and wild-type mice treated with DCE (75 mg/kg), but protection ensued after pretreatment with 5-PIP or DASO2. These results showed that bioactivation of DCE in murine lung occurred via the catalytic activities of both CYP2E1 and CYP2F2 and that bioactivation by these enzymes mediated the lung toxicity.     

A new method to perform quantitative measurement of bronchoscopic images.

Bronchoscopy is a commonly used clinical tool that provides a direct image of the bronchial lumen. However, bronchoscopy has seen little use as a quantitative measurement tool, mainly because of the wide-angle lens which distorts the image. The present authors have tested the ability of numerical algorithms and commercial software to correct for this distortion. Test objects of known size were imaged with four different bronchoscopes. Commercial image analysis software was used to measure the size of features in the images before and after applying distortion correction algorithms. The technique was then applied by measuring airway narrowing in anaesthetized pigs during vagal stimulation. Without correction, object size near the edge of the field of view is underestimated by approximately 40%. The error in measured diameter of concentric circles was dependent on the radius of the circle, increasing to 25% for circles occupying 90% of the field. Third order polynomial functions were required to correct these errors. After correction, errors were independent of object size or location in the image. Correction for lens distortion was independent of the distance between bronchoscope and object. The authors conclude that modern image processing software can correct for the distortion produced by wide-angle bronchoscope lenses.     

1,1-Dichloroethylene-induced mitochondrial damage precedes apoptotic cell death of bronchiolar epithelial cells in murine lung

1,1-Dichloroethylene (DCE) causes pulmonary injury that is characterized by necrosis of bronchiolar Clara cells. Mitochondria have been identified as an early target in the toxic response. Because mitochondria have been implicated in both necrotic and apoptotic cell death, we have undertaken studies to test the hypothesis that DCE induces apoptosis, in addition to necrosis, in murine lung. A primary objective is to identify the biochemical events associated with pulmonary apoptosis. Groups of female CD-1 mice were treated with DCE (75 mg/kg i.p.) or corn oil. Using an antibody directed against DCE-cysteine conjugates, adducts were detected primarily in association with mitochondria in the apices of bronchiolar Clara cells. Furthermore, morphological studies demonstrated early mitochondrial alterations in Clara cells that included severe swelling and disruption of cristae. Western blotting of lung cytosolic proteins showed greater immunoreactivity for cytochrome c in fractions from mice treated with DCE for 4 h than in controls. Immunohistochemical studies with an antibody to activated caspase-3 and terminal deoxynucleotidyl transferase dUTP nick-end labeling were used to detect apoptotic cells. In both experiments, positive reactivities were observed in the bronchiolar epithelium at 12 and 24 h after DCE treatment, whereas reactivities were absent in tissues from control animals. Finally, bronchiolar epithelial cells showing morphological criteria of apoptosis (chromatin condensation and margination) were observed at 24 h after 75 and 125 mg/kg DCE. Apoptotic-like cells were more abundant in larger bronchioles. These data suggested that DCE produces pulmonary bronchiolar apoptosis by inducing mitochondrial perturbations, causing release of cytochrome c into the cytosol and caspase activation.     

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.     

1,1-Dichloroethylene elicits dose-dependent alterations in covalent binding and glutathione in murine liver.

Dose-response studies have been performed using the hepatotoxin 1,1-dichloroethylene (1,1-DCE) to investigate effects of its administration on covalent binding, content of reduced glutathione (GSH), and structural alterations in murine liver. Additionally, these studies have determined if the sites of cellular damage coincided preferentially with the sites of GSH depletion. Treatment with 1,1-DCE evoked dose-dependent alterations in both covalent binding and tissue GSH content. Progressive increases in covalent binding (2.8 +/- 0.8 to 12.5 +/- 1.9 nmol/mg protein) were found within the range of doses tested (75-225 mg/kg). Tissue GSH content (86.4 +/- 6.1 to 35.9 +/- 2.4 nmol/mg protein) declined significantly after 1,1-DCE treatment when compared to that in control mice (128.0 +/- 3.6 nmol/mg protein). Alterations in GSH content due to 1,1-DCE treatment included those associated with significant increases (34-81% of control) in blood volume. Concomitant increases in vascular GSH were observed, and at 225 mg/kg, GSH content comprised 190% of that found in controls. Histochemical staining for GSH exhibited dose-related decreases in staining intensities that were not concentrated in any particular region, but, rather, were uniformly distributed throughout the liver lobule. Liver injury involving centrilobular and midzonal hepatocytes was absent at 75 mg/kg, mild at 125 and 175 mg/kg, and was most severe at 225 mg/kg. The results from these experiments demonstrate dose-dependent relationships between the magnitude of covalent binding of 1,1-DCE metabolite(s), diminished levels of tissue GSH, and hepatocellular necrosis.     

Metabolism and toxicity of trichloroethylene in epididymis and testis

The widespread occupational exposure to trichloroethylene (TCE) led us to test the hypothesis that TCE causes toxicity in the male reproductive system. We also investigated mechanisms mediating the potential cytotoxic response. Mice were exposed to TCE (1000 ppm) by inhalation for 6 h/day for 5 days/week for a total of 19 days. Exposure after the first week was interspersed by a "weekend." To estimate internal exposure, we measured the TCE metabolites, trichloroacetic acid (TCA) and trichloroethanol (TCOH), in urine at Days 4, 9, 14, and 19. Urinary excretion of TCOH was significantly higher than TCA; levels of TCOH and TCA significantly increased by the second and third week, respectively. Cytochrome P450 2E1 (CYP2E1), an enzyme involved in TCE metabolism, was localized in the epididymal epithelium and testicular Leydig cells, and was found at higher levels in the former than the latter. Immunoblotting confirmed that CYP2E1 protein was present in greater amounts in epididymis than in testis. p-Nitrophenol hydroxylation, a CYP2E1 catalytic activity, was also higher in the epididymis than in the testis. Chloral, a major TCE metabolite, was generated in microsomal incubations at significantly higher levels in epididymis than in testis. Antibody inhibition of CYP2E1 reduced chloral formation, which was more pronounced in epididymis than in testis. After 4 weeks of TCE exposure, damage to the epididymis was manifested as sloughing of epithelial cells. These results indicated that TCE is metabolized in the male reproductive tract, leading to adverse effects that are more severe in the epididymis than in the testis.