The pulmonary disposition and metabolism of 5-hydroxytryptamine

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Hemin rescues adrenodoxin, heme a and cytochrome oxidase activity in frataxin-deficient oligodendroglioma cells

AbstractMutations in the frataxin gene cause neurodegeneration and demyelination in Friedreich's ataxia. We showed earlier that frataxin deficiency causes primary iron–sulfur cluster defects, and later causes defects in heme and cytochrome c hemoprotein levels. Iron–sulfur (Fe/S) clusters are required in two enzymes of heme biosynthesis in humans i.e. in ferrochelatase and adrenodoxin. However, decreases in ferrochelatase activity have not been observed in frataxin-deficient HeLa cells or patient lymphoblasts. We knocked down frataxin in oligodendroglioma cells using siRNA, which produced significant defects in the activity of the Fe/S cluster enzymes adrenodoxin and aconitase, the adrenodoxin product heme a, and cytochrome oxidase, for which heme a serves as a prosthetic group. Exogenous hemin produced a significant rescue of adrenodoxin, aconitase, heme a levels and cytochrome oxidase activity. Thus hemin rescues iron–sulfur cluster defects that are the result of frataxin-deficiency, perhaps as a consequence of increasing the pool of bioavailable iron, and thus should be more fully tested for beneficial effects in Friedreich's ataxia models

Simultaneous quantification of multiple urinary naphthalene metabolites by liquid chromatography tandem mass spectrometry.

Naphthalene is an environmental toxicant to which humans are exposed. Naphthalene causes dose-dependent cytotoxicity to murine airway epithelial cells but a link between exposure and human pulmonary disease has not been established. Naphthalene toxicity in rodents depends on P450 metabolism. Subsequent biotransformation results in urinary elimination of several conjugated metabolites. Glucuronide and sulfate conjugates of naphthols have been used as markers of naphthalene exposure but, as the current studies demonstrate, these assays provide a limited view of the range of metabolites generated from the parent hydrocarbon. Here, we present a liquid chromatography tandem mass spectrometry method for measurement of the glucuronide and sulfate conjugates of 1-naphthol as well as the mercapturic acids and N-acetyl glutathione conjugates from naphthalene epoxide. Standard curves were linear over 2 log orders. On column detection limits varied from 0.91 to 3.4 ng; limits of quantitation from 1.8 to 6.4 ng. The accuracy of measurement of spiked urine standards was -13.1 to + 5.2% of target and intra-day and inter-day variability averaged 7.2 (± 4.5) and 6.8 (± 5.0) %, respectively. Application of the method to urine collected from mice exposed to naphthalene at 15 ppm (4 hrs) showed that glutathione-derived metabolites accounted for 60-70% of the total measured metabolites and sulfate and glucuronide conjugates were eliminated in equal amounts. The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide. The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up

Simultaneous Quantification of Multiple Urinary Naphthalene Metabolites by Liquid Chromatography Tandem Mass Spectrometry

<div><p>Naphthalene is an environmental toxicant to which humans are exposed. Naphthalene causes dose-dependent cytotoxicity to murine airway epithelial cells but a link between exposure and human pulmonary disease has not been established. Naphthalene toxicity in rodents depends on P450 metabolism. Subsequent biotransformation results in urinary elimination of several conjugated metabolites. Glucuronide and sulfate conjugates of naphthols have been used as markers of naphthalene exposure but, as the current studies demonstrate, these assays provide a limited view of the range of metabolites generated from the parent hydrocarbon. Here, we present a liquid chromatography tandem mass spectrometry method for measurement of the glucuronide and sulfate conjugates of 1-naphthol as well as the mercapturic acids and N-acetyl glutathione conjugates from naphthalene epoxide. Standard curves were linear over 2 log orders. On column detection limits varied from 0.91 to 3.4 ng; limits of quantitation from 1.8 to 6.4 ng. The accuracy of measurement of spiked urine standards was -13.1 to + 5.2% of target and intra-day and inter-day variability averaged 7.2 (± 4.5) and 6.8 (± 5.0) %, respectively. Application of the method to urine collected from mice exposed to naphthalene at 15 ppm (4 hrs) showed that glutathione-derived metabolites accounted for 60-70% of the total measured metabolites and sulfate and glucuronide conjugates were eliminated in equal amounts. The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide. The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up.</p></div

Covalent interactions of reactive naphthalene metabolites with proteins

Naphthalene produces selective necrosis of Clara cells in the mouse but not in the rat. The pulmonary toxicity depends on cytochrome P450-mediated metabolism; however, the selective pulmonary toxicity of naphthalene in the mouse does not correspond to tissue-selective covalent binding of reactive naphthalene metabolites in vivo. These studies compare reactive metabolite binding in target and nontarget cells and in various subcompartments of mouse lung and characterize, by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the proteins to which arylating metabolites are bound. Reactive metabolite binding was substantially higher in incubations of [H-3]-naphthalene with distal bronchioles and isolated Clara cells than with explants of trachea or bronchus from the mouse. Likewise, binding was substantially higher in incubations of murine Clara cells than in identical incubations with mouse hepatocytes (nontarget cells) or rat trachea cells (nonsusceptible species). These data show a good correlation between cellular Susceptibility to toxicity and the amount of reactive metabolite bound in vitro. Concentrations of adduct were highest in the medium and the nuclear/cell debris fraction (1000 x g pellet) of isolated Clara cells incubated with naphthalene; very small amounts of adduct were noted in pellets isolated at 20,000 or at 100,000 x g (mitochondrial and microsomal fractions) or in cytosol. These observations were consistent with the finding that adduct concentrations in bronchoalveolar lavage were substantially higher than in the lung at low doses of naphthalene and suggest that monitoring adducts in lavage may serve as a useful biomarker of exposure and effect. [H-3]-Naphthalene metabolites were bound primarily to three proteins: relative molecular weight, 14 to 15 (major), 30 and 45 kD (minor), in mouse Clara cell incubations; binding in hepatocyte incubations was solely to a protein of 14 to 15 kD. The chromatographic characteristics of the 14-kD adduct differed substantially from those of rat surfactant protein B or a secretory protein elaborated by Clara cells with a M(r) of 10 to 12 kD. Reactive metabolite binding to proteins in both Clara cells and hepatocytes appears to be highly selective. These studies suggest that the amount of metabolite bound to the 14 to 15 kD protein may be an important determinant in cellular susceptibility to naphthalene and that the nature of the major protein targeted in murine Clara cells (susceptible) and hepatocytes (nonsusceptible) is similar

Overview of naphthalene metabolism and excretion.

<p>(A) Naphthalene metabolism showing the formation of both naphthol- and glutathione-derived urinary metabolites. (B) Chemical structures of targeted urinary naphthalene metabolites for LC/ESI-MS/MS analysis.</p