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We have investigated the effect of NaHC[O.sub.3] on menadione redox cycling and cytotoxicity. A cell-free system utilized menadione and ascorbic acid to catalyze a redox cycle, and we utilized murine hepatoma (Hepa 1c1c7) cells for in vitro experiments. Experiments were performed using low (2 mmol/L) and physiological (25 mmol/L) levels of NaHC[O.sub.3] in buffer equilibrated to physiological pH. Using oximetry, ascorbic acid oxidation, and ascorbyl radical detection, we found that menadione redox cycling was enhanced by NaHC[O.sub.3]. Furthermore, Hepa 1c1c7 cells treated with menadione demonstrated cytotoxicity that was significantly increased with physiological concentrations of NaHC[O.sub.3] in the media, compared with low levels of NaHC[O.sub.3]. Interestingly, the inhibition of superoxide dismutase (SOD) with 2 different metal chelators was associated with a protective effect against menadione cytotoxicity. Using isolated protein, we found a significant increase in protein carbonyls with menadione-ascorbate-SOD with physiological NaHC[O.sub.3] levels; low NaHC[O.sub.3] or SOD-free reactions produced lower levels of protein carbonyls. In conclusion, these findings suggest that the hydrogen peroxide generated by menadione redox cycling together with NaHC[O.sub.3]-CO2 are potential substrates for SOD peroxidase activity that can lead to carbonate-radical-enhanced cytotoxicity. These findings demonstrate the importance of NaHC[O.sub.3] in menadione redox cycling and cytotoxicity. Key words: quinones, menadione, redox cycling, ascorbic acid, cytotoxicity, carbonate radical, bicarbonate, superoxide dismutase, Hepa 1c1c7 cells. Nous avons examine l'effet du NaHC[O.sub.3] sur le cycle redox et la cytotoxicite de la menadione. La catalyse du cycle redox par la menadione et l'acide ascorbique s'est deroulee en systeme acellulaire alors que les experiences in vitro ont ete realisees avec les cellules d'hepatome de souris (Hepa 1c1c7). Les experiences ont ete realisees avec un niveau faible (2 mmol/L) ou physiologique (25 mmol/L) de NaHC[O.sub.3] dans un tampon equilibre a pH physiologique. Nous avons trouve au moyen de l'oxymetrie, de l'oxydation d'acide ascorbique, et de la detection des radicaux ascorbyles que le cycle redox de la menadione etait accru par le NaHC[O.sub.3].De plus, le traitement des cellules Hepa 1c1c7 a la menadione montrait que la cytotoxicite etait significativement accrue dans le milieu contenant un niveau physiologique de NaHC[O.sub.3] comparativement au milieu contenant un niveau faible de NaHC[O.sub.3]. Fait interessant, l'inhibition de la superoxyde dismutase (SOD) par deux chelateurs de metaux differents protegeait les cellules contre la cytotoxicite de la menadione. En utilisant la proteine isolee, nous avons trouve une augmentation significative de proteines carbonyles dans le melange menadione-ascorbate-SOD a niveau physiologique de NaHC[O.sub.3]; un niveau faible de NaHC[O.sub.3] ou l'absence de SOD produisaient moins de proteines carbonyles. En conclusion, ces donnees suggerent que le peroxyde d'hydrogene genere par le cycle redox de la menadione et le NaHC[O.sub.3]-CO2 sont des substrats potentiels de l'activite peroxydase de la SOD qui peut conduire a une augmentation de la cytotoxicite provoquee par le radical carbonate. Ces donnees demontrent l'importance du NaHC[O.sub.3] dans le cycle redox et la cytotoxicite de la menadione. [Traduit par la Redaction] Mots-cles: quinones, menadione, cycle redox, acide asorbique, cytotoxicite, radical carbonate, bicarbonate, superoxyde dismutase, cellules Hepa 1c1c7., Introduction Quinones are virtually ubiquitous compounds that are found both exogenously and endogenously. As such, they can be classified as xenobiotics and endobiotics, respectively. Menadione (2-methyl-1,4-naphtoquinone) is a well-studied quinone [...]

Synthetic and biological amines such as ethylenediamine (EDA), spermine, and spermidine have not been previously investigated in free-radical biochemical systems involving aniline-based drugs or xenobiotics. We aimed to study the influence of polyamines in the modulation of aromatic amine radical metabolites in peroxidase-mediated free radical reactions. The aniline compounds tested caused a relatively low oxidation rate of glutathione in the presence of horseradish peroxidase (HRP), and [H.sub.2][O.sub.2]; however, they demonstrated marked oxygen consumption when a polyamine molecule was present. Next, we characterized the free-radical products generated by these reactions using spin-trapping and electron paramagnetic resonance (EPR) spectrometry. Primary and secondary but not tertiary polyamines dose-dependently enhanced the N-centered radicals of different aniline compounds catalyzed by either HRP or myeloperoxidase, which we believe occurred via charge transfer intermediates and subsequent stabilization of aniline-derived radical species as suggested by isotopically labeled aniline. Aniline/peroxidase reaction product(s) were monitored at 435 nm by kinetic spectrophotometry in the presence and absence of a polyamine additive. Using gas chromatography--mass spectrometry, the dimerziation product of aniline, azobenzene, was significantly amplified when EDA was present. In conclusion, di- and poly-amines are capable of enhancing the formation of aromatic-amine-derived free radicals, a fact that is expected to have toxicological consequences. Key words: aniline drugs and xenobiotics, polyamines, free radicals, spin trapping, EPR, GC-MS. Les amines synthetiques et biologiques comme l'ethylene diamine (EDA), la spermine et la spermidine n'ont pas encore ete etudiees dans des systemes biochimiques de radicaux libres impliquant des medicaments ou des substances xenobiotiques a base d'aniline. Nous visions a etudier l'influence des polyamines comme modulateurs des metabolites de radicaux d'amines aromatiques, dans des reactions produisant des radicaux libres par l'action de peroxydases. Les composes a base d'aniline testes oxydaient le glutathion a un taux relativement faible en presence de peroxydase de raifort (HRP) et de [H.sub.2][O.sub.2]; toutefois, ils consommaient de l'oxygene de facon marquee en presence d'une polyamine. Nous avons ensuite caracterise les radicaux libres generes par ces reactions a l'aide de la spectrometrie par capture d'intermediaires de reaction et de la spectrometrie de resonance paramagnetique electronique (RPE). Les polyamines primaires et secondaires, contrairement aux polyamines tertiaires, accroissaient de maniere dependante de la concentration les radicaux libres d'azote sur les differents composes a base d'aniline sous la catalyse soit de la HRP ou de la myeloperoxydase, ce que nous croyons survenir via des intermediaires de transfert de charges et la stabilisation subsequente d'especes de radicaux derivees de l'aniline comme le suggere l'aniline marquee de maniere isotopique. Les produits de la reaction aniline/peroxydase ont ete suivis a 435 nm par spectrometrie cinetique en presence ou en absence d'une amine. Selon la GC-MS, le produit de dimerisation de l'aniline, l'azobenzene, etait significativement amplifie lorsque l'EDA etait presente. En conclusion, les diamines et les polyamines sont capables d'accroitre la formation de radicaux libres derives d'amines, un fait qui devrait avoir une importance toxicologique. [Traduit par la Redaction] Mots-cles: medicaments et xenobiotiques a base d'aniline, polyamines, radicaux libres, capture d'intermediaires de reaction, RPE, GC-MS., Introduction Endogenous free radical intermediates constantly occur in the living cell as waste or bioactive molecules that might behave as potent exterminators or regulators of cellular function (Droge 2002). Amid [...]

We investigated a novel scavengingmechanism of arylamine freeradicals by poly- and monoaminocarboxylates. Free radicals of arylaminexenobiotics and drugs did not react with oxygen in peroxidase-catalyzedreactions; however, they showed marked oxygen uptake in the presenceof an aminocarboxylate. These free-radical intermediates were identifiedusing the spin trap 5,5-dimethyl-1-pyrroline-N-oxide(DMPO) and electron paramagnetic resonance (EPR) spectrometry. Diethylenetriaminepentaaceticacid (DTPA), a polyaminocarboxylate, caused a concentration-dependentattenuation of N-centered radicals produced by the peroxidative metabolismof arylamines with the subsequent formation of secondary aliphaticcarbon-centered radicals stemming from the cosubstrate molecule. Analogously, N,N-dimethylglycine (DMG) and N-methyliminodiacetate (MIDA), but not iminodiacetic acid (IDA), demonstrateda similar scavenging effect of arylamine-derived free radicals ina horseradish peroxidase/H2O2system. Usinghuman promyelocytic leukemia (HL-60) cell lysate as a model of humanneutrophils, DTPA, MIDA, and DMG readily reduced anilinium cationradicals derived from the arylamines and gave rise to the correspondingcarbon radicals. The rate of peroxidase-triggered polymerization ofaniline was studied as a measure of nitrogen-radical scavenging. Although,IDA had no effect on the rate of aniline polymerization, this wasalmost nullified in the presence of DTPA and MIDA at half of the molarconcentration of the aniline substrate, whereas a 20 molar excessof DMPO caused only a partial inhibition. Furthermore, the yield offormaldehyde, a specific reaction endproduct of the oxidation of aminocarboxylatesby aniline free-radical metabolites, was quantitatively determined.Azobenzene, a specific reaction product of peroxidase-catalyzed free-radicaldimerization of aniline, was fully abrogated in the presence of DTPA,as confirmed by GC/MS. Under aerobic conditions, a radical-transferreaction is proposed between aminocarboxylates and arylamine freeradicals via the carboxylic group-linked tertiary nitrogen of thedeprotonated amino acid derivatives. These findings may have significantimplications for the biological fate of arylamine xenobiotic and drugfree-radical metabolites. [ABSTRACT FROM AUTHOR]

We have investigated the effect of NaHCO3 on menadione redox cycling and cytotoxicity. A cell-free system utilized menadione and ascorbic acid to catalyze a redox cycle, and we utilized murine hepatoma (Hepa 1c1c7) cells for in vitro experiments. Experiments were performed using low (2 mmol/L) and physiological (25 mmol/L) levels of NaHCO3 in buffer equilibrated to physiological pH. Using oximetry, ascorbic acid oxidation, and ascorbyl radical detection, we found that menadione redox cycling was enhanced by NaHCO3. Furthermore, Hepa 1c1c7 cells treated with menadione demonstrated cytotoxicity that was significantly increased with physiological concentrations of NaHCO3 in the media, compared with low levels of NaHCO3. Interestingly, the inhibition of superoxide dismutase (SOD) with 2 different metal chelators was associated with a protective effect against menadione cytotoxicity. Using isolated protein, we found a significant increase in protein carbonyls with menadione-ascorbate-SOD with physiological NaHCO3 levels; low NaHCO3 or SOD-free reactions produced lower levels of protein carbonyls. In conclusion, these findings suggest that the hydrogen peroxide generated by menadione redox cycling together with NaHCO3-CO2 are potential substrates for SOD peroxidase activity that can lead to carbonate-radical-enhanced cytotoxicity. These findings demonstrate the importance of NaHCO3 in menadione redox cycling and cytotoxicity. [ABSTRACT FROM AUTHOR]

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Synthetic and biological amines such as ethylenediamine (EDA), spermine, and spermidine have not been previously investigated in free-radical biochemical systems involving aniline-based drugs or xenobiotics. We aimed to study the influence of polyamines in the modulation of aromatic amine radical metabolites in peroxidase-mediated free radical reactions. The aniline compounds tested caused a relatively low oxidation rate of glutathione in the presence of horseradish peroxidase (HRP), and H2O2; however, they demonstrated marked oxygen consumption when a polyamine molecule was present. Next, we characterized the free-radical products generated by these reactions using spin-trapping and electron paramagnetic resonance (EPR) spectrometry. Primary and secondary but not tertiary polyamines dose-dependently enhanced the N-centered radicals of different aniline compounds catalyzed by either HRP or myeloperoxidase, which we believe occurred via charge transfer intermediates and subsequent stabilization of aniline-derived radical species as suggested by isotopically labeled aniline. Aniline/peroxidase reaction product(s) were monitored at 435 nm by kinetic spectrophotometry in the presence and absence of a polyamine additive. Using gas chromatography - mass spectrometry, the dimerziation product of aniline, azobenzene, was significantly amplified when EDA was present. In conclusion, di- and poly-amines are capable of enhancing the formation of aromatic-amine-derived free radicals, a fact that is expected to have toxicological consequences. [ABSTRACT FROM AUTHOR]

Copyright of Canadian Journal of Physiology & Pharmacology is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)