Edward S. Reynolds

1,1-Dichloroethylene-Induced Pulmonary Injury

Oral administration of 1,1-dichloroethylene (1,1-DCE) produces acute injury to the lungs of C57Bl/6 mice. The bronchiolar epithelium is most severely affected with damage selective for Clara cells. After a 100 mg kg/dose of 1.1-DCE. Clara cells show extensive dilatation of cisternae and degeneration of the endoplasmic reticulum. At 6 hr after administration of 200 mg 1,1-DCE/kg, both ciliated and Clara cells are necrotic, and bronchiolar epithelial lining exfoliates. By 24 hr. pulmonary edema, hemorrhage, and focal atelectasis are also present. Pulmonary injury, at 24 hr after the high dose, is associated with a significant hypoxia as demonstrated through a decrease in the partial pressure of oxygen in arterial blood. In spite of the severe injury, recovery occurs and airways display an intact epithelial lining with normal parenchymal elements by 7 days.

Allylamine cardiotoxicity: I. Sequence of pathologic events

Allylamine, given to adult male rats in drinking water at a concentration of 0.005, 0.05, or 0.1% for 21–104 days, caused dose-dependent myocardial and vascular lesions, although no consistent histopathologic changes were observed in other organs. Treated animals also demonstrated decreased weight gain and diminished fluid consumption which were dose dependent. An “H2O match” experiment in which control rats were given amounts of water equivalent to the decreased volume of fluid ingested by animals given 0.1% allylamine indicated that dehydration alone does not cause the degree of decreased weight gain or the cardiovascular lesions observed in allylamine-treated animals. When animals given 0.1% allylamine were sacrificed at appropriate intervals, acute myocardial lesions were observed as early as 4 days, and progressive fibrous lesions consistently developed by 21 days. The myocardial lesion began as small areas of multifocal subendocardial myocardial necrosis; extension of necrosis continued and was followed by fibrosis which, when extensive, resulted in an apical aneurysmal scar which frequently was grossly evident. Vascular arterial lesions consisting of medial thickening and hyalinization occurred predominantly within scarred areas but were not observed before 21 days of allylamine consumption. Coronary arteries were studied by morphometry in rats given 0.1% allylamine for 21 days; these studies indicated that a subtle medial hypertrophy occurs in medium and large sized vessels in hearts which do not display an evident morphologic vascular lesion. Serum creatine phosphokinase activity in rats given allylamine rose sporadically with occasional four-to fivefold increases in individual animals, most likely reflecting this focal and progressive necrotizing process of the myocardium. When a single dose of allylamine (50, 100, or 150 mg/kg) was given by gavage, acute morphologic myocardial lesions were observed 24 hr later at the two higher but not the lowest dose; serum creatine phosphokinase activity appeared elevated at the lower dose levels. These studies demonstrate that myocardial necrosis and fibrosis begin in the first few days of allylamine consumption and precede significant morphologic vascular lesions.

Effect of administrative vehicle on oral 1,1-dichloroethylene toxicity

Abstract The relationship between the acute toxicity and biologic fate of 1,1-dichloroethylene (1,1-DCE) was examined in fasted and fed male Sprague-Dawley rats given 200 mg 1,1-DCE/kg orally in a mineral oil, a corn oil, or an aqueous Tween-80 vehicle. Exhalation of unchanged 1,1-DCE by individual rats was monitored at selected 15-min intervals for 5 hr and all rats were sacrificed at 6 hr. The administrative vehicle affected the magnitude of liver injury in fasted rats; with mineral oil or corn oil, injury was massive [> 100-fold elevation of glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT)] whereas with the aqueous Tween-80 vehicle, injury was moderate t 15-fold elevation of GOT and GPT). In contrast, in all fed groups liver injury was slight (two- to threefold elevation of GOT and GPT). Rats with massive liver injury also had an ∼50-fold increase in plasma free hemoglobin and their kidneys exhibited numerous granular “heme” casts in Henles loop without apparent degenerative changes in either glomeruli or tubular epithelium. No pathologic changes were observed in heart, lungs, spleen, adrenals, or duodenum. The administrative vehicle did not affect the total amount of 1,1-DCE exhaled, which was approximately half the given dose, nor did the vehicle affect the initial rapid phase of 1,1-DCE exhalation, which lasted ∼1 hr and had t 1 2 values ranging from 15 to 21 min for the fasted groups and from 10 to 13 min for the fed groups. In contrast, the later slow phase of 1,1-DCE exhalation was predictably affected by the administrative vehicle; t 1 2 values for the fasted and fed groups, respectively, were most prolonged with the poorly absorbed mineral oil vehicle (257 and 280 min), intermediate with the more digestable corn oil vehicle (73 and 103 min), and briefest with the more absorbable aqueous Tween vehicle (22 and 42 min). Further studies are needed to determine if the relative resistance of the fed animals to hepatic injury is due to the capacity of these animals to detoxify 1,1-DCE for a longer duration than the fasted animals.

Halothane hepatotoxicity: enhancement by polychlorinated biphenyl pretreatment.

Pretreatment of rats with the potent mixed-function oxidase system inducer, Aroclor 1254 (150 μmol/kg for seven days), provides a model system for the study of halothane hepatotoxicity. Within two hours of the end of anesthesia (0.85 per cent for five hours), values of the serum transaminases (SGOT

Metabolism of [14C]carbon tetrachloride to exhaled, excreted and bound metabolites: Dose-response, time-course and pharmacokinetics

Fasted male rats were given six doses of 14CCl4 ranging from non-hepatotoxic (0.1 mmole/kg) to severely hepatotoxic (26 mmoles/kg). Time-course and pharmacokinetics of CCl4, 14CO2 and CHCl3 elimination by exhalation were monitored by measuring amounts recovered in breath during discrete 15-min intervals for 8-12 hr. Amounts of 14C-labeled metabolite recovered bound to liver macromolecules at 24 hr and excreted in urine or feces for 24 hr were also determined. Comparison pharmacokinetic studies were done with 14CHCl3 and Na(2)14CO3. After all doses of 14CCl4, the major metabolite was CO2, twenty to thirty times less metabolite was recovered bound to liver macromolecules, and intermediate amounts of metabolite were excreted in urine and feces. CHCl3 was the least abundant metabolite at low CCl4 doses, but the second most abundant at high doses. Stronger associations were found between the magnitude of liver injury at 24 hr (quantitated as serum glutamate-pyruvate transaminase activity) and the extent or rate of CCl4 metabolism by pathways leading to CO2 and CHCl3 than by pathways leading to 14C-metabolites bound in liver or excreted in urine. Time-course and pharmacokinetic data indicated that a major pathway of CCl4 metabolism leading to CO2 became impaired within 2 hr after administration of hepatotoxic doses of CCl4.

Adsorption and reversed-phase high-performance liquid chromatography of p-nitrobenzyl esters of monohydroxy fatty acids

To enhance the UV detectability of hydroxy fatty acids, p-nitrobenzyl (PNB) esters of twenty-two different monohydroxy fatty acids of various chain-lengths (C16-C22) and differing positional isomers were formed using O-(p-nitrobenzyl)-N,N-(diisopropyl)-isourea (PNBDI) as alkylating agent. Reversed-phase and adsorption high-performance liquid chromatography (HPLC) of the twenty-two monohydroxy fatty acid PNB esters were studied. The PNB group did not dominate the chromatographic properties of monohydroxy fatty acids and it did not interfere with the HPLC separation of positional isomers. PNBDI was, however, found to be less than ideal for formation of PNB derivatives of monohydroxy fatty acids because UV absorbing contaminants of PNBDI interfered with the HPLC analyses.

Early effects of 1,1-dichloroethylene on canalicular and plasma membranes: ultrastructure and stereology

The pathogenesis of 1,1-dichloroethylene (1,1-DCE)-induced hepatotoxicity was investigated in fasted male rats by identifying the earliest morphological alterations in organelles. In situ perfusion-fixed liver tissue was examined by light and electron microscopy at 1, 2, or 3 hr after oral administration of 25, 50, and 100 mg 1,1-DCE/kg in mineral oil. The earliest morphological alterations, which occurred within 1 to 2 hr after 1,1-DCE administration, were dilation of bile canaliculi with an increase in the number of microvilli or membrane fragments in canaliculi and the formation of canalicular diverticuli in centrolobular hepatocytes. Subsequently, microvilli on sinusoidal surfaces were disrupted or lost. Membrane whorls were frequently found in bile canaliculi, the space of Disse, and between the lateral membranes of hepatocytes at early times. As injury progressed, centrolobular hepatocytes retracted from endothelial cells and sinusoidal plasma membranes invaginated to form cytoplasmic vacuoles. Stereological analysis of centrolobular hepatocytes at the 25 mg/kg dose showed a significant increase in canalicular volume density by 3 hr and no detectable alteration in mitochondrial volume density. These results indicate that changes in canalicular shape and microvilli configuration are the earliest morphological alterations following 1-DCE ingestion.

Isopropanol enhancement of carbon tetrachloride metabolism in vivo

We examined the effects of isopropanol (ISOP) pretreatment on the metabolism of 14CCl4 to 14CO2 and CHCl3 exhaled in the breath, to 14C metabolite excreted in 24 hr urine and feces from 0 to 24 hr, and to 14C metabolite bound to liver at 24 hr. Fasted male rats were given 0.1 or 2.0 mmoles 14CCl4/kg. ISOP pretreatment, which markedly enhanced the hepatotoxicity of CCl4, selectively enhanced the rate and total extent of 14CO2 and CHCl3 metabolite exhalation. The pathways of CCl4 metabolism leading to CO2 and CHCl3 metabolite formation may be more relevant to the hepatotoxicity of CCl4 than the pathways leading to urinary, fecal or covalently bound metabolites.

Relationships between the pharmacokinetics of carbon tetrachloride conversion to carbon dioxide and chloroform and liver injury.

Rate and extent of CCl4 metabolism by pathways leading to CO2 and CHCl3 were evaluated by measuring the amounts of these metabolites exhaled during discrete intervals following six different doses of CCl4. Pulmonary pharmacokinetics of 14CO2 and CHCl3 exhalation after CCl4 administration were compared with those after Na214CO3 and 14CHCl3 administration. Exhalation of 14CO2 metabolite declined more rapidly than expected after hepatotoxic doses of CCl4. This decline could be due to injury associated changes in the metabolism of CCl4.

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.