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Biomedical subjects

E J Rauckman

Publications and source records attributed to E J Rauckman.

At least 19 recordsLinked to original sources
PubMed1991

Toxicity studies of acetone administered in the drinking water of rodents.

Two- and thirteen-week toxicity studies were conducted using male and female F344/N rats and B6C3F1 mice. Animals were exposed to the following concentrations of acetone in their drinking water: two-week studies 0; 5000; 10,000; 20,000; 50,000; or 100,000 ppm acetone. Thirteen-week rat and female mouse studies 0; 2500; 5000; 10,000; 20,000; or 50,000 ppm acetone. Thirteen week male mice were exposed to 0; 1250; 2500; 5000; 10,000; or 20,000 ppm acetone. Depressed body weight gain was restricted to the 50,000 and 100,000 ppm exposure groups. Male and female mice exposed respectively to 20,000 or 50,000 ppm acetone for 2 weeks developed hepatocellular hypertrophy. This change was not apparent after 13 weeks of exposure although relative and absolute liver weight was increased in high dose female mice. Bone marrow hypoplasia was observed in 5/5 high dose (100,000 ppm) male rats during the 2-week studies. Treatment of male rats for 13 weeks resulted in a variety of mild and subtle hematological changes that often occurred at relatively low levels of exposure (5000 ppm) and resembled those seen during the clinical condition of megaloblastic anemia. Changes characteristic of hypogonadism (depressed sperm motility and cauda epididymal and epididymal weight and elevated incidence of abnormal sperm) were observed in male rats receiving 50,000 ppm acetone for 13 weeks. The incidence and severity of a kidney lesion that is morphologically similar to the spontaneously occurring nephropathy among aging F-344 rats were increased at 20,000 and 50,000 ppm acetone, respectively, in 13-week male rats. In summary, the effects of acetone were either subtle in nature or occurred during very high levels of exposure confirming acetone's low level of toxicity. The daily levels of acetone exposure were often several-fold greater than possibly encountered by humans during the accidental consumption of contaminated groundwater (250 ppm; 5 mg/day) and frequently exceeded maximum levels reported following acute toxic exposures (2,500 mg/kg).

Acetone↗
PubMed1990

Stress initiated during isolation of rat renal proximal tubules limits in vitro survival.

The effects of oxidative damage were assessed in rat proximal tubule fragments (isolated by collagenase perfusion) by monitoring lactate dehydrogenase release (LDH-R) to measure cell viability and thiobarbituric acid (TBA) reactive material to follow oxidative damage. Increasing the oxygen content in the incubation atmosphere from 10 to 95% significantly increased LDH-R and TBA reactants. Addition of butylated hydroxytoluene or deferoxamine (DF) to the medium prevented these changes, but ascorbic acid or mannitol had no positive effect. Lima bean trypsin inhibitor also reduced LDH leakage significantly when added to the medium, but not when added to the perfusion buffers. In contrast, adding DF to the perfusate during tubule isolation produced the most pronounced benefit; net LDH-R after 4 hr was about 10% in tubules prepared this way compared to 20% when DF was omitted. Basal oxygen consumption declined to approximately the same extent as LDH-R increased. Maintenance of nystatin-stimulated respiration, ATP/ADP, GSH content and total adenine nucleotides indicated good cell function. These results suggest that oxidative damage initiated during the tubule isolation procedure limits cell survival but this effect can be counteracted substantially by the addition of DF to the perfusion buffer.

Animals↗
PubMed1987

Toxicological studies of chemical mixtures of environmental concern at the National Toxicology Program: health effects of groundwater contaminants.

In cooperation with the Agency for Toxic Substances and Disease Registry, the National Toxicology Program is participating in a Public Health Service activity related to the Comprehensive Environmental Response, Compensation and Liability Act (Superfund Act) by conducting toxicology studies on chemicals found in high-priority hazardous waste sites and for which adequate toxicological data are not available. As part of this effort, a project on the toxicology of chemical mixtures of groundwater contaminants was initiated. The first study, centered on the health effects of groundwater contaminants, is at the contractual stage. Nineteen organic and six inorganic chemicals, selected from more than 1000 known groundwater contaminants, will be given in drinking water to Fischer 344 rats and B6C3F1 mice for 3 or 6 months. Controls and five dose levels, based on average concentrations (i.e., baseline level) of individual component chemicals, or 0.1-, 10-, or 1000-fold of the baseline level, will be used. Toxicological end points include mortality, clinical signs, water and food consumption, body and organ weights, clinical pathology analytes (e.g., hematology, clinical chemistry, and urinalysis), gross and histopathology, neurobehavioral tests, sperm morphology and vaginal cytology evaluations (SMVCE), and cytogenetics. This paper summarizes the rationale behind our experimental design and the factors one must consider when designing studies of complex chemical mixtures.

Animals↗
PubMed1985

In vitro effects of acetaminophen and its analogues on human platelet aggregation and thromboxane B2 synthesis.

We examined the effect of acetaminophen and the structural analogues 2,6-dimethylacetaminophen, 3,5-dimethylacetaminophen, and N-acetyl-p-benzoquinone imine on human platelet aggregation, 14C-serotonin secretion, and thromboxane B2 synthesis. Preincubation with 1 mM acetaminophen for 2 min completely inhibited arachidonic acid- and collagen-stimulated platelet aggregation. Thromboxane B2 production and 14C-serotonin secretion by arachidonic acid-stimulated platelets also were completely inhibited. Preincubation of platelets with 1 mM 3,5-dimethylacetaminophen inhibited collagen and arachidonic acid-induced aggregation and arachidonic acid-stimulated thromboxane B2 synthesis, while treatment with 2,6-dimethylacetaminophen did not inhibit aggregation and blocked thromboxane B2 formation to a much lesser degree. Preincubation with 1 mM N-acetyl-p-benzoquinone imine inhibited arachidonic acid-induced aggregation and 14C-serotonin secretion but had no effect on arachidonic acid-induced thromboxane B2 formation and collagen-induced platelet aggregation.

Acetaminophen↗
PubMed1985

Effect of model trauma on the turnover of protein and hemoprotein components of hepatic microsomal membrane in immature rats.

Administration of 14C-leucine and delta-[3,5-3H]-aminolevulinic acid to immature male rats leads to the incorporation of radioactivity into microsomal protein, including the hemoprotein cytochrome P-450. Non-hepatic regional ischemic trauma results in an increase in the half-life of total microsomal protein, but does not exert the same effect on microsomal heme-associated protein. Loss of radioactivity from microsomal hemoprotein, primarily cytochrome P-450, from traumatized animals exhibits a biphasic pattern similar to that in control animals. The half-life of both the fast-phase component and the slow-phase component is unchanged by trauma. Trauma does, however, increase the ratio of the fast- to slow-phase components of microsomal heme. A significant increase in heme oxygenase activity after trauma suggests that the fast-phase component of hepatic microsomal cytochrome P-450 is more extensively degraded.

Animals↗
PubMed1984

Electron paramagnetic resonance studies of membrane proteins in hepatic microsomes.

Hepatic microsomal membranes, prepared under various conditions that yield either 'intact' or 'disrupted' microsomal vesicles, have been labeled via the sulfhydryl groups of intrinsic membrane proteins using nitroxide analogs of N-ethylmaleimide. Electron paramagnetic resonance spectra revealed the presence of two dominant classes of bound label corresponding to differing degrees of immobilization, the ratio of which were quantitated using a parameter designated the 'W/S' ratio. For latent microsomes, the value of this parameter was determined to be 0.65 +/- 0.02 and was influenced by factors such as label/protein ratio, incubation period, nitroxide structure, temperature and pH. The W/S ratio was also sensitive to the degree of membrane integrity as revealed by the latency of mannose 6-phosphate activity of glucose-6-phosphohydrolase. In addition, membrane disruption resulted in a corresponding decrease in the order parameter for nitroxide-labeled fatty acids intercalated within the lipid bilayer. The W/S ratio was observed to be dependent upon the method of microsome preparation yielding values of 1.02 +/- 0.02 for 'hypertonically disrupted' vesicles and 1.28 +/- 0.02 for 'mechanically disrupted' vesicles. Microsomal marker enzymes such as cytochrome P-450 and FAD-containing monooxygenase retained significant levels of functionality following nitroxide incorporation.

Affinity Labels↗
PubMed1984

Biotransformation of norcocaine to norcocaine nitroxide by rat brain microsomes.

In the mid 1970's, norcocaine was identified as a metabolite of cocaine in rat brain tissue. We extend these studies by demonstrating that rat brain FAD-containing monooxygenase metabolizes norcocaine to N-hydroxynorcocaine. This hydroxylamine is then further oxidized to the nitroxyl free radical norcocaine nitroxide by rat brain cytochrome P-450. Brain microsomal reduction of norcocaine nitroxide leads to the generation of superoxide. Finally, incubation of rat brain microsomes with either N-hydroxynorcocaine or norcocaine nitroxide leads to significant lipid peroxidation as monitored by spin-trapping techniques.

Animals↗
PubMed1984

Not all aromatic nitro compounds form free radicals.

One-electron reduction of the aromatic nitro-containing drug, clonazepam, by rat hepatic microsomes was found to produce a nitro anion radical which was observable by electron paramagnetic resonance (EPR) spectrometry under anaerobic conditions. It was determined that NADPH-cytochrome P-450 reductase may be the enzyme responsible for this reduction and that this free radical reacts rapidly with oxygen to produce superoxide. The vasodilator nifedipine, another aromatic nitro-containing drug, was found not to be reduced by rat hepatic microsomes to a free radical nor to stimulate superoxide production. Based on a series of experiments, we propose that the inability of nifedipine to be bioreduced to its nitro anion free radical is the result of geometric restrictions which prevent the transfer of an electron from cytochrome P-450 reductase to nifedipine.

Animals↗
PubMed1984

Proliferative response and renewal of hepatic function following cocaine administration in mice.

Experiments were undertaken to investigate the hepatic, temporal and spatial sequence of events following a single injection of cocaine, a known hepatotoxin. Centrilobular necrosis was induced in male mice (DBA/2Ha) 24 hr post-injection (PI). The time course of hepatic damage was monitored by assaying microsomal cytochrome P450 content, the activity of microsomal FAD-containing monooxygenase (FAD-M) and by determining the levels of serum glutamic pyruvic transaminase (SGPT). Kinetics of the onset of DNA synthesis were determined by autoradiography of thin liver sections and the incorporation of 3H-methyl thymidine into perchloric-acid-precipitable material. There was no increase in the labelling index (LI) and thymidine (TdR) incorporation in the first 24 hr PI. The LI rose to 14.6% and TdR incorporation showed a 5-fold increase over control values 48 hr PI. Both indices declined slightly at 72 hr PI and returned to control values by 96 hr PI. In contrast, the cytochrome P450 content declined by 69%, the FAD-M activity dropped by 40% and the SGPT levels showed an 18-fold increase at 24 hr PI, coincident with cytological signs of necrosis. Although the patterns of recovery differed between these selected enzymes, normal values were attained by 96 hr PI. These results demonstrate that cell damage and hepatic dysfunction precede the onset of DNA synthesis and subsequent proliferation.

Alanine Transaminase↗
PubMed1984

Production of superoxide during the metabolism of nitrazepam.

Nitrazepam is metabolized in both humans and rats to 7-amino-nitrazepam OFFicating that this drug is reduced to a number of metabolic intermediates including several free radical species. When rat-hepatic microsomes are incubated with NADPH in the presence of nitrazepam, its nitro anion free radical was observed under anaerobic conditions. In the presence of oxygen, this free radical reduced oxygen giving nitrazepam and superoxide. 7-Nitroxyl-nitrazepam was produced by the chemical oxidation of 7-amino-nitrazepam using m-chloroperbenzoic acid. Reaction of this reactive free radical with hepatic microsomes led to the covalent spin labelling of microsomal protein. This phenomenon was also observed by the enzymic oxidation of 7-amino-nitrazepam with hepatic microsomes, obtained from a phenobarbital-induced rat, in the presence of a NADPH-generating system. With the generation of superoxide and hydrogen peroxide (arising from the dismutation of superoxide), it is not surprising that nitrazepam-enhanced lipid peroxidation was demonstrated by monitoring the production of lipid peroxyl radicals using spin-trapping techniques.

Animals↗
PubMed1984

Reduction and glutathione conjugation reactions of N-acetyl-p-benzoquinone imine and two dimethylated analogues.

N-Acetyl-3,5-dimethyl-p-benzoquinone imine, N-acetyl-2,6-dimethyl-p-benzoquinone imine, and N-acetyl-p-benzoquinone imine were synthesized via the oxidation of 3,5-dimethylacetaminophen, 2,6-dimethylacetaminophen, and acetaminophen, respectively. All three quinone imines were rapidly reduced to their corresponding semiquinone imines by NADPH-cytochrome P-450 reductase. All three benzoquinone imines underwent comproportionation with their respective phenols to yield the corresponding semiquinone imines, which in the presence of oxygen gave superoxide. Identification of this latter free radical was based on spin-trapping techniques. Reduced GSH was found to be an excellent nucleophile toward N-acetyl-2,6-dimethyl-p-benzoquinone imine, whereas this thiol behaved as a one-electron reductant toward N-acetyl-3,5-dimethyl-p-benzoquinone imine. Finally, GSH was determined to act as both a nucleophile and a reductant toward N-acetyl-p-benzoquinone imine.

Animals↗
PubMed1983

Studies of the mobility of maleimide spin labels within the erythrocyte membrane.

We have confirmed a method yielding reproducible and reliable spectrometric parameters derived from spin-labeled erythrocyte ghosts using nitroxide derivatives of maleimide compounds. The disorder parameter, W/S, was shown to vary with changes in the structure of the label, the conditions utilized for labeling such as ionic strength and erythrocyte age and the presence of drugs such as alcohol and acetaminophen. The nitroxide spectrum was also found to change with increasing and decreasing temperature in an irreversible manner. These findings should permit increased reliance to be placed on the spin-labeling technique when used to monitor changes in membrane lipid or protein assembly.

Cyclic N-Oxides↗
PubMed1983

Acetaminophen hepatotoxicity. An alternative mechanism.

Alcohol-fed hamsters were used to study the mechanism by which acetaminophen initiates hepatotoxicity. Animals maintained on an ethanol-containing diet (Group B) exhibited an increased mortality rate after administration of acetaminophen (400 mg/kg) as compared to control hamsters (Group A). However, in those animals in which the ethanol-containing diet had been replaced by the control diet 24 hr before receiving acetaminophen (Group C), significant protection against acetaminophen toxicity was observed as compared to control animals (Group A). This observation correlates well with the finding that Group C hamsters had higher levels of glutathione and catalase than was found in either Group A or Group B animals. It was also demonstrated that acetaminophen was oxidized by cytochrome P-450, producing acetaminophen free radical and hydrogen peroxide. The free radical in the presence of oxygen was found to generate superoxide and presumably N-acetyl-p-benzoquinone imine. Microsomal lipid peroxidation was found to be stimulated markedly in the presence of acetaminophen. The role of glutathione in protecting hamsters from acetaminophen-mediated hepatotoxicity is discussed.

Acetaminophen↗
PubMed1983

Disruption of erythrocyte membranal organization by superoxide.

Human erythrocyte ghosts were covalently labeled with 4-maleimide-2,2,6,6-tetramethylpiperidinooxyl. Electron paramagnetic resonance (EPR) spectrometry revealed two major binding environments representing strongly (S) and weakly (W) immobilized species. The disorder parameter, W/S, determined from the respective peak amplitudes, was shown to be irreversibly elevated following treatment of the labeled ghosts with superoxide, indicating an increase in membrane fluidity. Labeled ghosts reduced with ascorbate showed no nitroxide EPR signals. However, following exposure of these membranes to superoxide, the nitroxide spectrum returned with a W/S ratio of 25. In contrast, the disorder parameter for spin-labeled ghosts decreased following exposure to hydroxyl radicals suggesting decreased fluidity, as a result of lipid peroxidation. This effect could be prevented by the inclusion of mannitol. These changes in membrane fluidity and/or protein mobility observed by EPR are compared with previous results obtained by other methods and provide additional evidence for physiologic alterations initiated by superoxide.

Cyclic N-Oxides↗
PubMed1983

Evidence of enhanced in vivo lipid peroxidation after acute cocaine administration.

An acute intraperitoneal dose (60 mg/kg) of cocaine to DBA/2Ha male mice results in enhanced lipid peroxidation in vivo, as measured by an increase in conjugated diene absorption in hepatic microsomal lipids. The initiation of this lipid peroxidation is an early consequence of cocaine administration; as early as 1 h after cocaine, peroxidized lipids are significantly greater in treated animals than in controls. This cocaine-induced lipid peroxidation remains at a maximal level from 2 to 4 h and returns approximately to control levels by 8 h. The metabolites of cocaine also produce lipid peroxidation in vitro. Liver microsomes from phenobarbital-treated DBA/2Ha male mice, incubated aerobically in the presence of NADPH, cocaine or the cocaine oxidative metabolites, norcocaine and norcocaine nitroxide, induced lipid peroxidation as measured by an increase in the production of thiobarbituric acid (TBA)-reactive products. The extent of lipid peroxidation is greater for the oxidative metabolites of cocaine than for cocaine itself.

Animals↗