Mary Treinen Moslen

Reactive Oxygen Species in Normal Physiology, Cell Injury and Phagocytosis

Formation of free radicals and other ROS is a continuous aspect of life. Examples include the free radical intermediates which are formed by the nonenzymatic peroxidation of polyunsaturated fatty acids of membrane lipids in a destructive process and which are also formed by the enzymatic peroxidation of arachidonic acid in the biosynthesis of potent chemoattractants. Organisms cope with these reactive species by a variety of strategies that limit formation of ROS or remove cytotoxic products. Oxidative burst reactions that yield ROS provide an effective, vital process for killing invading organisms. Research on why ROS formation is impaired in phagocytic cells of people with chronic granulomatous disease has provided new insights into the complexity of the factors that prevent inadvertent activation of this destructive force.

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.

Genotoxic effects of a sub-acute low-level inhalation exposure to a mixture of carcinogenic chemicals

A study was conducted using a combined testing protocol (CTP), to determine whether short-term biological end-points, singly or in combination, are sufficiently sensitive to identify damage induced by exposure to ambient levels of industrial chemicals. A small-scale inhalation set-up which is both economical and easy to assemble was designed. Mice were exposed to 4 concentrations of a custom-blend mixture of benzene, chloroprene, epichlorohydrin and xylene in a ratio of 2:2:1:2, respectively. The concentrations for benzene, chloroprene and xylene were 0, 0.1, 1.0 and 10 ppm each. Concentrations for epichlorohydrin were half those for the other components. Groups of 22 males and 22 female mice were exposed to each concentration of the mixture for 3 and 6 weeks. Selected biological end-points including urine mutagenesis, bone marrow cell aberrations and micronuclei, spleen lymphocyte aberrations and liver enzyme induction were monitored. The spleen lymphocyte aberrations and liver enzyme induction were the most sensitive end-points. The lymphocytes showed a significant induction of chromosome aberrations from exposure for 3 weeks to all 3 concentrations of the mixtures. After 6 weeks of exposure, significant induction of aberrations was observed after exposure to low and medium concentrations but not to the high concentration. This lack of response at the high concentration after 6 weeks exposure, appeared to correlate with a significant induction of glutathione S-transferase in the liver. Since this enzyme is known to detoxify 3 of the 4 chemicals in our mixture, it may indicate a detoxification mechanism after enzyme induction. The present study indicates that the CTP is sufficiently sensitive to identify toxicological effects after exposure to ambient levels of a gas mixture.

Species differences in testicular and hepatic biotransformation of 2-methoxyethanol

Biotransformation of 2-methoxyethanol (2-ME) by alcohol and aldehyde dehydrogenases is an established factor in the toxicity of this useful solvent. Little is known about potential capacity for 2-ME biotransformation by testis or other target tissues. We detected appreciable capacity for 2-ME biotransformation by alcohol dehydrogenase in testes from Sprague-Dawley rats. However, kinetic analysis showed a 6-fold lower affinity for 2-ME by alcohol dehydrogenase of testis compared to liver. 2-ME biotransformation was also detected in testes from Wistar rats and one strain of mice but not in testes from hamsters, guinea pigs, rabbits, dogs, cats or humans. Testes from all these species readily converted the aldehyde metabolite of 2-ME to 2-methoxyacetic acid. Hepatic capacities for 2-ME biotransformation by alcohol dehydrogenase varied from 22 to 2.5 mumol/mg prot/min with a species rank order of: hamsters >> rats = mice > guinea pigs = rabbits. There was no consistent concordance between activities for 2-ME versus ethanol, the prototype substrate for alcohol dehydrogenase, which could reflect substrate preferences of different isozymes. Species differences between rats and hamsters were also found for testicular and hepatic biotransformation of the glycol ethers, 2-ethoxyethanol and 2-butoxyethanol. Although species differences in capacity for 2-ME biotransformation were found, the observations do not provide an explanation for reported species and strain differences in susceptibility to 2-ME toxicity.

IDENTIFICATION OF AN ANION-TRANSPORT ATPASE THAT CATALYZES GLUTATHIONE CONJUGATE-DEPENDENT ATP HYDROLYSIS IN CANALICULAR PLASMA-MEMBRANES FROM NORMAL RATS AND RATS WITH CONJUGATED HYPERBILIRUBINEMIA (GY MUTANT)

Rat liver canalicular plasma membranes were found to contain a 37-kDa protein that is immunologically cross-reactive with the dinitrophenyl glutathione-stimulated ATPase previously identified in human tissues. The protein, which was partially purified by affinity chromatography, exhibited ATPase activity dependent on dinitrophenyl glutathione, bilirubin ditaurate, and other dianionic compounds. The localization of this protein in the canalicular membrane and its measured enzymatic activity indicate that it is involved in the transport of glutathione derivatives and other dianionic organic compounds. A rat mutant in which the above transport activities are impaired contained the protein in amounts similar to those in a normal control.

A stable colorimetric assay to measure toxin elevation of inorganic phosphate in bile

A modified assay for the measurement of nanomole amounts of Pi in 50 microliters of bile is described. The assay is based on the formation of a complex between malachite green dye and phosphomolybdate under acidic conditions. Only three simple steps are required to produce a colored complex which remains stable for at least 3 h; these steps are precipitation with acid, addition of color reagent containing dye and surfactant, and incubation at 37 degrees C. Analysis of bile samples collected from a rat given the toxin 1,1-dichloroethylene demonstrated that the assay is well suited to routine assays of biliary Pi as an endogenous indicator of aberrant hepatobiliary function.

Biliary function studies during multiple time periods in freely moving rats a useful system and set of marker solutes

Biliary output of endogenous and exogenous compounds is altered by anesthesia, depletion of bile salts, and hydrostatic pressure. The described system for bile function studies minimizes these confounding factors by substantially modifying existing methods. Experiments were conducted in freely moving rats which eliminates effects of anesthesia or restraint-induced stress. Depletion of bile salts was prevented by intraduodenal infusion of taurocholate which maintains bile volume. Bile was collected in containers taped to the rats back which minimizes hydrostatic forces induced by lengthy or elevated biliary cannulas. Animals were prepared for hepatobiliary function studies 1 week before experiments by placement and exteriorization of a jugular cannula and a bile duct to duodenal fistula. Experiments involved monitoring biliary outputs of marker solutes for various pathways of bile formation during three sequential time periods of 120 min, that is, a basal period in the morning and two experimental periods in the afternoon. We found similar patterns of biliary output in each time period for small i.v. doses of conventional exogenous markers [3H-taurocholate, phenolphthalein glucuronide, indocyanine green, and horseradish peroxidase] and for less commonly studied endogenous markers [glucose, inorganic phosphate (Pi), total protein, and leucine aminopeptidase]. This temporal stability indicates a lack of confounding circadian variability for these markers during the course of the biliary function study. Biliary excretion patterns of these marker solutes (e.g., rapid high recoveries of phenolphthalein glucuronide and low concentrations of Pi and glucose) demonstrated that our system for bile function studies is associated with intactness of the examined pathways of bile formation. These results validate our system and set of marker solutes for in vivo biliary function studies.(ABSTRACT TRUNCATED AT 250 WORDS)