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J L Poyer

Publications and source records attributed to J L Poyer.

At least 19 recordsLinked to original sources
PubMed1998

Clarification of the relationship between free radical spin trapping and carbon tetrachloride metabolism in microsomal systems.

It has been proposed that the C-phenyl-N-tert-butylnitrone/trichloromethyl radical adduct (PBN/.CCl3) is metabolized to either the C-phenyl-N-tert-butylnitrone/carbon dioxide anion radical adduct (PBN/.CO2-) or the glutathione (GSH) and CCl4-dependent PBN radical adduct (PBN/[GSH-.CCl3]). Inclusion of PBN/.CCl3 in microsomal incubations containing GSH, nicotinamide adenine dinucleotide phosphate (NADPH), or GSH plus NADPH produced no electron spin resonance (ESR) spectral data indicative of the formation of either the PBN/[GSH-.CCl3] or PBN/.CO2- radical adducts. Microsomes alone or with GSH had no effect on the PBN/.CCl3 radical adduct. Addition of NADPH to a microsomal system containing PBN/.CCl3 presumably reduced the radical adduct to its ESR-silent hydroxylamine because no ESR signal was observed. The Folch extract of this system produced an ESR spectrum that was a composite of two radicals, one of which had hyperfine coupling constants identical to those of PBN/.CCl3. We conclude that PBN/.CCl3 is not metabolized into either PBN/[GSH-.CCl3] or PBN/.CO2- in microsomal systems.

Animals↗
PubMed1997

The structure of free radical metabolites detected by EPR spin trapping and mass spectroscopy from halocarbons in rat liver microsomes.

Electron impact (EI) tandem mass spectrometry (MS/MS) combined with EPR spin trapping was used to detect and identify the free radical metabolites of various halocarbons in rat liver microsomal dispersions. EPR spectra of the spin adducts of radical metabolites derived from fluorine-containing halocarbons display fluorine hyperfine splitting, which can be used as proof for the identification of this kind of halocarbon-derived free radical spin adduct. For halocarbons without fluorine atoms, MS/MS was found to be a very useful and simple method for the detection and identification of the structures of halocarbon-derived spin adducts from radical metabolites. The molecular ions from spin adducts of these halocarbon-derived free radical intermediates were observed for the first time by scanning the precursor ion spectrum of m/z 57. These assignments were further confirmed by the use of perdeuterated tert-butyl PBN which provides the precursor ion spectrum of m/z 66.

Animals↗
PubMed1996

Blood chemistry changes in the rat induced by high doses of nitronyl free radical spin traps.

For greatest efficacy, it is desirable to use spin trapping agents in the highest concentrations possible. Fifty-four male Sprague-Dawley rats were used to explore the relative toxicity of four representative nitronyl spin traps at doses chosen on the basis of earlier lethality studies. Most studies were confined to the 3- to 6-h period following drug injection, because the behavioral signs of toxicity are most evident early after injection and because spin trapping studies would typically be performed within this time frame. Doses of spin trap were dissolved in a corn oil/buffer vehicle and injected intraperitoneally (i.p.). Toxic signs were recorded periodically, and at the time of euthanasia or spontaneous death a blood sample was collected by cardiac puncture for clinical chemistry analysis and a necropsy was performed. Both gross pathology and histopathological examination of the major organs were essentially negative in all cases, with no obvious evidence of cellular damage being observed. Neither DMPO (232 mg/100 g b.wt.) nor PBN (100 mg/100 g b.wt.) were lethal in the present study, while both M4PO (20 and 40 mg/ 100 g b.wt.) and PyOBN (100 and 200 mg/100 g b.wt.) were lethal. Abnormal clinical chemistry findings were generally confined to those animals that died spontaneously or were euthanized early for humane reasons. In most cases, death was associated with marked seizure activity and impaired respiration, and deaths occurred within a few min to a few hours. The mechanism of toxicity was unclear due to the lack of histopathological evidence and the wide range of abnormal serum analytes in those rats killed by either M4PO or PyOBN. In conclusion, during the first 6 h after IP administration there is little indication of tissue damage by the nitrone spin traps until the dose is increased to a lethal level, at which point an acute, rapidly occurring, wide-spread disruption of tissue integrity seems to occur.

Animals↗
PubMed1995

Biological spin trapping. II. Toxicity of nitrone spin traps: dose-ranging in the rat.

To obtain the strongest possible free radical spin adduct signal using the electron paramagnetic resonance spectroscopy-spin trapping technique, it is desirable to load an animal with the highest dose of spin trap possible. One hundred and twenty six male Sprague-Dawley rats were used to establish the toxic dose range for PBN (alpha-phenyl N-tert butyl nitrone) and 18 other similar spin traps. The lethal dose of PBN was found to be approximately 100 mg/100 g BW (0.564 mmol/100 g. The 18 other compounds were then tested, and their toxicities were gauged in terms of molar equivalents to PBN. Of these spin traps, DMPO (5,5-dimethyl-1-pyrroline-N-oxide) was found to be the least toxic (no toxic signs at twice the lethal dose for PBN) while 2,6-difluoro-PBN and M4PO (3,3,5,5-tetramethyl-1-pyrroline-N-oxide) were the most toxic, both causing death at one eighth the PBN-equivalent lethal dose. Nine of the 18 nitrones appeared non-toxic at the 0.25 PBN-equivalent lethal dose level.

Animals↗
PubMed1995

The role of free radicals in paraquat-induced corneal lesions.

Paraquat is a synthetic bipyridylium salt widely used as herbicide and defoliant. Enzyme-catalyzed redoxcycling of paraquat generates oxygen radicals. The toxic, even lethal, effects of paraquat are due to free radical-mediated tissue injury. Ocular lesions, sometimes quite severe, have been observed following accidental splashing of paraquat solutions onto the eyes. These studies were designed to document the generation of paraquat free radicals in corneal tissue, and to describe the histological nature of the corneal injuries in experimental animals (rabbits and monkeys). The EPR spectrum of rabbit corneas, 30 min. after intrastromal injection of paraquat, showed the signal of the free radical of paraquat. Ultrastructural studies of corneas 8 days after intrastromal injections (100 microliters) of paraquat solutions showed that the initial lesions occur at the epithelium/basement membrane interface. In rabbit cornea, dose dependent lesions were observed, i.e. whereas 50 mM paraquat caused only minimal damage to the epithelial basement membrane, 75 mM caused complete dissolution to the basement membrane with some damage to stromal collagen, and loss of epithelium with stromal ulceration and severe inflammatory response were observed with 150 mM paraquat. Monkey corneas were less susceptible than those of rabbits to the effects of paraquat. No lesions were observed following intrastromal injections of 50 mM or 75 mM paraquat. With higher concentrations of paraquat (100 mM and 150 mM) the primary injuries were to the proximal and lateral plasma membranes of basal epithelial cells; basement membrane alterations were detected only adjacent to areas of significant plasma membrane damage. The underlying Bowman's membrane and stroma were not affected. Anatomical differences between the corneas of rabbit and monkeys as well as possible biochemical differences may account for the species differences observed.

Animals↗
PubMed1994

Study of reproducibility of spin trapping results in the use of C-phenyl-N-tert-butyl nitrone (PBN) for trichloromethyl radical detection in CCl4 metabolism by rat liver microsomal dispersions. Biological spin trapping I.

The well-known metabolism of CCl4 to trichloromethyl radicals in rat liver microsomal dispersions has been reinvestigated with the goal to determine the repeatability and reproducibility of the EPR signal intensity of the EPR spectrum of the CCl3 adduct of PBN. It was found that at least eight repeat experiments were needed under identical conditions to obtain an average value with an error of +/- 10%. When the effect of changing the concentrations of CCl4, PBN or NADPH-generating system was investigated, the plots of EPR signal intensity vs. the variable selected showed initial smooth increases in signal strength with respect to an increase in concentrations of CCl4, PBN or NADPH-generating system. However, considerable scatter was found after the initial slope and only general trends could be recognized. It is concluded that with CCl4, no increase in EPR signal is found after 10 mM concentration. For PBN, the optimum concentration is about 30 mM. The signal strength seems to increase with increased amounts of NADPH generating system although with diminishing slope.

Animals↗
PubMed1992

Acetylene, a mammalian metabolite of 1,1,1-trichloroethane.

1,1,1-Trichloroethane (TCE) is a widely used industrial solvent of low acute toxicity. It is slowly oxidized to trichloroethanol and trichloroacetic acid by cytochrome P-450-dependent mono-oxygenases. Increased inhalative uptake by rats under hypoxia and spin-trapping experiments indicate that TCE is also reductively metabolized to a radical intermediate. Acetylene is formed as a metabolite, suggesting transfer of an additional electron to form the corresponding carbene. Hypoxia and induction of mixed-function mono-oxygenases accelerate the formation of acetylene. Experiments performed in vitro with rat liver microsomal fractions yield analogous results.

Acetylene↗
PubMed1990

HPLC procedure for the pharmacokinetic study of the spin-trapping agent, alpha-phenyl-N-tert-butyl nitrone (PBN).

Considerable progress has been made in the use of spin-trapping agents for the trapping of free radicals in biological systems. Radicals have been detected in both in vitro and in vivo systems using this methodology. Free radicals have not only been identified by this procedure, but also the intensity of radical generation and the duration of their production has been assessed as well. One of the most widely used spin-trapping agents in biological systems is PBN. This spin trap appears to be relatively nontoxic at the levels required for successful trapping experiments, but there is no information concerning the possible fate of PBN in such biological systems. Metabolism of PBN could alter the concentration of PBN at the site of trapping which may affect the efficiency of radical capture, especially in in vivo systems. In this study, PBN was administered intraperitoneally to rats and the concentration of the spin trap in various organs was determined by high pressure liquid chromatography as a function of time (15 min to 12 h). The concentration of PBN in plasma peaked at 15 min while the maximum in all organs tested occurred at 30 min. The time course of PBN concentrations in all tissues followed similar curves, and declined rather steeply after the 30-min maximum with a biological half-life of 134 min. However, the amount of PBN per gram of tissue was always higher in liver and kidney than in the brain, heart, and lung. PBN was detected in the urine for as long as 24 h after injection of the compound.

Animals↗
PubMed1990

Structure identification of free radicals by ESR and GC/MS of PBN spin adducts from the in vitro and in vivo rat liver metabolism of halothane.

Free radicals were detected from the in vitro metabolism of halothane (rat liver microsomes) by the PBN spin trapping method. The detected radical species include the 1-chloro-2,2,2-trifluoro-1-ethyl radical (I), as determined by mass spectral analysis, and lipid-type radicals assigned by high resolution ESR spectroscopy with the use of d14-deuterated PBN. The lipid-derived radicals are a carbon-centred radical with the partially assigned structure CH2R and an oxygen-centred radical of the OR' type. From the mass spectral analysis of the spin adduct mixture there is also evidence for a halocarbon double adduct of PBN of the type I-PBN-I.

Animals↗
PubMed1990

Mass spectroscopy and chromatography of the trichloromethyl radical adduct of phenyl tert-butyl nitrone.

Positive structural identification of the PBN-trichloromethyl spin adduct in vitro was accomplished with the use of high pressure liquid chromatography and/or gas chromatography coupled with mass spectrometry. Both thin layer and liquid chromatography were used to separate a complex mixture of compounds from rat liver extracts treated with CCl4 in vitro and in vivo. Deuterated PBN's (PBN-d9; tert-butyl deuteration, or PBN-d14; both phenyl and tert-butyl deuteration) were also used to aid in the mass spectral analysis of spin adducts from liver extracts of CCl4 exposed rat livers, since the tert-butyl group fragment ion. C4D9+ (m/z = 66) is always present for PBN and PBN spin adducts. In addition, the masses of the ion peaks increase by the amount of deuteration, i.e. an increase of 9 for PBN-d9 or PBN-d14 in comparison to normally synthesized PBN.

Animals↗
PubMed1988

Hydralazine-dependent carbon dioxide free radical formation by metabolizing mitochondria.

The addition of hydralazine (1-hydrazinophthalazine) to rat liver mitochondria metabolizing malate/glutamate causes formation of a carbon-centered free radical which was spin-trapped with phenyl-t-butylnitrone (PBN) or dimethylpyrrolidine-N-oxide (DMPO). The coupling constants of the spin-trapped free radical were AN = 16.1, AH beta = 4.6 G for PBN and AN = 15.9, AH beta = 18.9 G for DMPO-trapped radical in aqueous solution. The spin-trapped free radical was shown to be the carbon dioxide anion free radical by independent synthesis, high pressure liquid chromatography separation, and electron paramagnetic resonance characterization. The amount of carbon dioxide anion free radical produced was absolutely dependent upon the presence of hydralazine and varied depending on mitochondrial substrate, with by far the highest amount produced by pyruvate. Studies with 13C-labeled pyruvate demonstrated that the carbon dioxide free radical came from C-1 of this compound.

Animals↗
PubMed1985

Chemistry and biology of spin-trapping radicals associated with halocarbon metabolism in vitro and in vivo.

The spin-trapping method is introduced and discussed. Some chemistry of nitroxides and nitrones is reviewed. Pattern recognition of ESR spectra of nitroxides is outlined. Factors controlling the magnitude of hyperfine splitting constants are mentioned. Methods of assigning spin adducts are listed. Review articles in the literature are referenced. Results in the electrochemical reduction of halocarbons are presented and some parallels with superoxide chemistry shown. Various speculative reactions are given. The in vitro and in vivo experiments where halocarbon radicals have been detected by spin trapping are reviewed and some new results reported. A comparison for different animals is added.

Animals↗
PubMed1984

Oxygen- and carbon-centered free radical formation during carbon tetrachloride metabolism. Observation of lipid radicals in vivo and in vitro.

Free radical reactions involved in the metabolism of carbon tetrachloride by rat liver have been considered to be a cause of at least part of the injury resulting from exposure to this halocarbon. In an earlier study employing electron spin resonance and spin-trapping techniques, we demonstrated that trichloromethyl (13.CCl3) radicals are readily observed in rat liver microsomes metabolizing 13CCl4, and that the same radical could be shown to form in vivo in the liver of intact rats given a single dose of 13CCl4. This report describes the production of lipid dienyl (L.) and oxygen-centered lipid radicals (LO. or LOO., or both) in in vitro systems metabolizing 13CCl4, and also the formation of lipid dienyl radicals (L.) in liver of intact animals exposed to CCl4. The radicals appear to be produced in a sequence of reactions governed among other things by the oxygen tension in the system. The lipid radicals (L.) which form in intact liver of CCl4-treated rats are apparently the result of an attack on lipids of the endoplasmic reticulum by 13.CCl3 radicals formed by reductive cleavage to CCl4 and are the initial intermediates in the process of lipid peroxidation. These investigations demonstrate that while the events occurring in liver microsomes in vitro appear to parallel those which take place in intact liver in vivo, the conditions in vivo make the spin-trapping studies of radicals in intact animals much more selective than it is in vitro for a given spin trap, and requires the use of more than one type of spin-trapping agent to detect different radical species in vivo.

Animals↗
PubMed1982

Specificity of a phenobarbital-induced cytochrome P-450 for metabolism of carbon tetrachloride to the trichloromethyl radical.

Evidence is presented which demonstrates that the first polypeptide to disappear in liver microsomes of phenobarbital-induced rats treated with CC14 was the 52,000 dalton p-450 cytochrome. Data are also presented which show that this form of cytochrome P-450 was capable of generating the trichloromethyl radical from CCl4 in a reconstituted system containing the purified cytochrome, NADPH-cytochrome P-450 reductase, NADPH, CCl4, and the spin-trapping agent, phenyl-t-butyl nitrone. Other cytochrome P-450 fractions not containing the 52,000 dalton form did not produce this radical. The formation of this highly reactive radical may have resulted in localized damage to the cytochrome, causing the cytochrome either to be released from the microsomal membrane or to form large aggregates which did not migrate in the gel electrophoretic procedures employed.

Animals↗
PubMed1978

Spin-trapping of the trichloromethyl radical produced during enzymic NADPH oxidation in the presence of carbon tetrachloride or bromotrichloromethane.

Utilizing the spin-trapping agent phenyl-t-butyl nitrone, a free radical has been detected which is produced from carbon tetrachloride or bromotrichloromethane during the enzymic oxidation of NADPH by rat liver microsomes. The presence of NADPH is obligatory for generation of the radical. The formation of the trichloromethyl radical-phenyl-t-butyl nitrone adduct is an enzymic process, as evidenced by the inhibition of its formation in systems containing heated microsomes and in systems containing p-hydroxymercuribenzoate. A computer-simulated ESR spectrum for the trichloromethyl adduct of phenyl-t-butyl nitrone can reproduce the essential features of the spectrum of the spin-trapped radical produced enzymically from CCl4. A mechanism is proposed for the formation of the trichloromethyl radical from CCl4 or BrCCl3.

Animals↗