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.

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.

Pharmacological activity of nitroxide analogues of dichloroisoproterenol and propranolol.

Spin-labeled analogues of dichloroisoproterenol and propranolol were synthesized. It was found that the KD's of both probes for the beta-adrenergic receptors of frog erythrocytes were about 30-fold higher than the KD's previously reported for the parent antagonists. Thus the introduction of a bulky nitroxide moiety in place of the isopropyl group on the amino nitrogen is associated with a decrease in affinity for the beta-adrenergic receptors. Nonetheless, the affinity of the spin-labeled propranolol would appear to be within a range compatible with EPR measurements.

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.

Pharmacokinetics of nitroxide NMR contrast agents.

Pharmacokinetics of the nitroxide stable free radical functionality of compounds containing this moiety were evaluated in the rat. The agents were injected i.v. at either high (1.75 mmoles/kg) or low (10 mumoles/kg) dose, and timed blood samples were drawn and assayed for nitroxide concentration by EPR spectrometry. Similarly, various organs and tissues were removed at specified times after injection and homogenized for determination of nitroxide concentration. Urine was collected by catheter for estimation of urinary excretion of the intact nitroxide free radical. At high doses, the various nitroxides exhibited an initial rapid disposition phase, followed by a terminal disposition phase with disappearance from the blood showing apparent log-linear half-lives of about 5 to 30 minutes. Generally, 20 to 60% of the dose was recovered in the urine. At low doses, dissimilar results were obtained. Blood levels again showed biphasic decay; however, blood concentrations at all times were much lower than those predicted by the high dose kinetics, indicating probably nonlinear pharmacokinetic behavior. Tissue homogenate studies showed low or nondetectable levels of nitroxide signal, demonstrating that the low blood concentrations could not be accounted for by a rapid uptake into specific tissues. Moreover, only 2 to 6% of the nitroxide could be recovered in the urine. Additional studies demonstrated that at the low dose a rapid in vivo bioreduction occurred which appeared to be saturable at the higher dose.

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.

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.

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.

Androgenic suppression of mouse hepatic FAD-containing monooxygenase activity.

Sex-related differences in the activity of hepatic FAD-containing monooxygenase (FAD-M) were found in C3H/St mice. Adult female mice had enzyme activities nearly two-fold greater than male mice and these differences, which were absent in sexually immature mice, became apparent at the onset of puberty. The sex differences in hepatic FAD-M appeared to be mediated through the suppressive effect of testosterone; castration of male mice enhanced enzyme activity, while androgenic replacement returned activities to control levels. Testosterone's suppressive effect was found to be relatively specific for hepatic FAD-M. Treatment of castrated male mice with both the anti-androgen flutamide and testosterone returned enzyme activity to control levels, suggesting that testosterone's regulation of hepatic microsomal FAD-M is receptor-mediated. Female gonadectomy had no effect on this enzyme's activity.

Carbon tetrachloride-induced lipid peroxidation: a spin trapping study.

It has been suggested that the active species responsible for carbon tetrachloride-induced lipid peroxidation is trichloromethyl radical ( . CCl3). Direct evidence for the existence of this reactive species can be obtained by spin trapping techniques, however, there are conflicting reports as to the identity of this free radical trapped. We have found that upon addition of carbon tetrachloride to a mixture of rat hepatic microsomes, NADPH and the spin trap, alpha (4-pyridinyl-1-oxide)-N-butyl nitrone (4-POBN) an electron paramagnetic resonance (epr) spectrum appeared. This spectrum was identical to that observed in the absence of carbon tetrachloride, except for enhanced rate of formation. We were able to identify this free radical, using model systems as a lipid peroxyl radical (LOO . ).

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.

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.

Effect of model vascular trauma on the hepatic drug-metabolizing enzymes of the neonatal rat.

Abdominal aorta ligation in neonatal rats was used as a model for pediatric traumatic injury. Short-term ischemic trauma (24 h) was found to have significant effect on plasma corticosterone levels, and on the specific activity of the drug-metabolizing enzymes FAD-containing monooxygenase and glucuronyl transferase in hepatic microsomes. Ischemic injury was determined to cause a decrease in FAD-containing monooxygenase activity in pre-weaning animals. Glucuronyl transferase activity was increased by this surgical procedure in animals less than 12 days age, and after weaning; however, glucuronyl transferase activity was decreased by this model in rats between 14 days of age and weaning.

A new class of irreversible muscarinic antagonists: beta-haloethylamine furoates.

The synthesis of a class of ultra-long acting muscarinic antagonists is described. Furthermore, it is noted that the stability of the, in situ, aziridinium ion is sufficiently great so that these agents can be used to study the effect of temperature upon the conformation of the muscarinic cholinoceptor. The inactivation kinetics of these receptors as well as the dissociation constants, Kd, for all probes are presented.