An evaluation of myeloperoxidase-mediated bio-activation of NSAIDs in promyelocytic leukemia (HL-60) cells for potential cytotoxic selectivity.

Several lines of evidence have pointed towards the potential therapeutic benefit of NSAIDs in cancer therapy. In this study, we have investigated the acute bio-activation of NSAIDs and their metabolites via myeloperoxidase (MPO), a highly-expressed peroxidase enzyme in acute myeloid leukemia. As bio-activation involves the formation of reactive metabolites, we hypothesized that NSAIDs which produced reactive metabolites would be correlated with leukemia cell toxicity. We tested the enzymatic peroxidation of three NSAIDs, namely diclofenac, indomethacin, and naproxen in comparison with their hepatic metabolites, 4'- hydroxydiclofenac (4'-OHD), 5-hydroxydiclofenac (5-OHD), O-desmethyl-N-deschlorobenzoylindomethacin (DMBI), O-desmethylindomethacin (DMI) and O-desmethylnaproxen (ODN). Firstly, we used purified peroxidases in kinetic UV-vis kinetic spectrophotometry, and electron paramagnetic resonance (EPR) experiments to determine oxidation of ascorbic acid and glutathione (GSH), respectively. We then used HL-60 cells, as a model of acute myelogenous leukemia to carry out trypan blue exclusion, cellular ATP analysis, mitochondrial membrane potential (MMP) and cytofluorometric GSH assays. Our results present evidence that diclofenac, 4'-OHD, 5-OHD, DMBI and DMI demonstrated significant cytotoxic effect in the leukemic cells through oxidation by intracellular MPO. In the same vein, only diclofenac and its two metabolites caused a significant drop in the MMP and cellular ATP level; however, the cell death induced by indomethacin metabolites reflected a subtle effect on MMP or GSH content. Interestingly, only diclofenac and 4'-OHD (and not 5- OHD) caused a significant drop in HL-60 cells' GSH content. Among diclofenac compounds, only 4'-OHD also generated GS radical and caused a significant increase in ascorbate co-oxidation rate. Lastly, even though ODN also generated GS radical and potently cooxidized ascorbate, it showed no significant cytotoxicity. These results provide evidence of a correlation between acute cytotoxicity and MPO-bioactivated NSAIDs, though this was not correlated for all compounds (e.g., ODN). Further studies are required to determine both the MPO-dependent and MPO-independent mechanisms of cytotoxicity.

Metabolism of isoniazid by neutrophil myeloperoxidase leads to isoniazid-NAD(+) adduct formation: A comparison of the reactivity of isoniazid with its known human metabolites.

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD(+)) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD(+) adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages' cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV-Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD(+). Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV-Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product (λmax=326nm) in the presence of NAD(+). This adduct was confirmed to be isonicotinyl-NAD(+) using LC-MS analysis where the intact adduct was detected (m/z=769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity.

Proteomic profile of aminoglutethimide-induced apoptosis in HL-60 cells: Role of myeloperoxidase and arylamine free radicals.

In this study, the cellular effects resulting from the metabolism of aminoglutethimide by myeloperoxidase were investigated. Human promyelocytic leukemia (HL-60) cells were treated with aminoglutethimide (AG), an arylamine drug that has a risk of adverse drug reactions, including drug-induced agranulocytosis. HL-60 cells contain abundant amounts of myeloperoxidase (MPO), a hemoprotein, which catalyzes one-electron oxidation of arylamines using H2O2 as a cofactor. Previous studies have shown that arylamine metabolism by MPO results in protein radical formation. The purpose of this study was to determine if pathways associated with a toxic response could be determined from conditions that produced protein radicals. Conditions for AG-induced protein radical formation (with minimal cytotoxicity) were optimized, and these conditions were used to carry out proteomic studies. We identified 43 proteins that were changed significantly upon AG treatment among which 18 were up-regulated and 25 were down-regulated. The quantitative proteomic data showed that AG peroxidative metabolism led to the down-regulation of critical anti-apoptotic proteins responsible for inhibiting the release of pro-apoptotic factors from the mitochondria as well as cytoskeletal proteins such as nuclear lamina. This overall pro-apoptotic response was confirmed with flow cytometry which demonstrated apoptosis to be the main mode of cell death, and this was attenuated by MPO inhibition. This response correlated with the intensity of AG-induced protein radical formation in HL-60 cells, which may play a role in cell death signaling mechanisms.

Current status and future prospects of toxicogenomics in drug discovery.

In drug discovery and development (DDD), the efficacy, safety and cost of new chemical entities are the main concerns of the pharmaceutical industry. Continuously updated and stricter recommendations imposed by regulatory authorities result in greater challenges being faced by the industry. Reliable high-throughput techniques integrated with well-designed analytical tools at all stages of DDD (termed 'next-generation DDD') could be a possible approach to obtaining new drug approval by cutting costs as well as ensuring the highest level of patient safety. In this review, we describe the various components of holistic toxicogenomics with examples of applications, and discuss the various analytical tools and platforms to illustrate the current status and prospects of next-generation DDD.

Scavenging of free-radical metabolites of aniline xenobiotics and drugs by amino acid derivatives: toxicological implications of radical-transfer reactions.

We investigated a novel scavenging mechanism of arylamine free radicals by poly- and monoaminocarboxylates. Free radicals of arylamine xenobiotics and drugs did not react with oxygen in peroxidase-catalyzed reactions; however, they showed marked oxygen uptake in the presence of an aminocarboxylate. These free-radical intermediates were identified using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and electron paramagnetic resonance (EPR) spectrometry. Diethylenetriaminepentaacetic acid (DTPA), a polyaminocarboxylate, caused a concentration-dependent attenuation of N-centered radicals produced by the peroxidative metabolism of arylamines with the subsequent formation of secondary aliphatic carbon-centered radicals stemming from the cosubstrate molecule. Analogously, N,N-dimethylglycine (DMG) and N-methyliminodiacetate (MIDA), but not iminodiacetic acid (IDA), demonstrated a similar scavenging effect of arylamine-derived free radicals in a horseradish peroxidase/H2O2 system. Using human promyelocytic leukemia (HL-60) cell lysate as a model of human neutrophils, DTPA, MIDA, and DMG readily reduced anilinium cation radicals derived from the arylamines and gave rise to the corresponding carbon radicals. The rate of peroxidase-triggered polymerization of aniline was studied as a measure of nitrogen-radical scavenging. Although, IDA had no effect on the rate of aniline polymerization, this was almost nullified in the presence of DTPA and MIDA at half of the molar concentration of the aniline substrate, whereas a 20 molar excess of DMPO caused only a partial inhibition. Furthermore, the yield of formaldehyde, a specific reaction endproduct of the oxidation of aminocarboxylates by aniline free-radical metabolites, was quantitatively determined. Azobenzene, a specific reaction product of peroxidase-catalyzed free-radical dimerization of aniline, was fully abrogated in the presence of DTPA, as confirmed by GC/MS. Under aerobic conditions, a radical-transfer reaction is proposed between aminocarboxylates and arylamine free radicals via the carboxylic group-linked tertiary nitrogen of the deprotonated amino acid derivatives. These findings may have significant implications for the biological fate of arylamine xenobiotic and drug free-radical metabolites.

The effect of bicarbonate on menadione-induced redox cycling and cytotoxicity: potential involvement of the carbonate radical.

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.

The interaction of diamines and polyamines with the peroxidase-catalyzed metabolism of aromatic amines: a potential mechanism for the modulation of aniline toxicity.

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.

High-performance liquid chromatographic determination of memantine in human urine following solid-phase extraction and precolumn derivatization.

A validated HPLC-UV method is presented for the quantification of urinary memantine hydrochloride, a novel medication approved to treat moderate and advanced cases of Alzheimer's disease. The drug and amantadine hydrochloride, the internal standard, were extracted from human urine using SPE. The extract was then buffered and derivatized at room temperature using o-phthalaldehyde in the presence of N-acetyl-L-cyteine. Chromatographic separation of the formed derivatives was achieved on a C18 column using methanol-water mobile phase adjusted to pH 7 and pumped isocratically at 1 mL/min. The UV detector was set at 340 nm. The chromatographic run time did not exceed 10 min. The LOD and LOQ were 8 and 20 ng/mL, respectively. The RSDs for intraday and interday precisions did not exceed 5.5%. The method was used to monitor memantine hydrochloride in human urine in order to determine an appropriate sampling interval for future noninvasive therapeutic drug monitoring. The assay could also be applied to the determination of amantadine. The described assay showed that a postdosing time interval of 25-75 h seems adequate for sampling and monitoring memantine in urine.

Induction of quinone oxidoreductase 1 enzyme by Rhazya stricta through Nrf2-dependent mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhazya stricta Decne. (Apocynaceae) is a common medicinal plant in the Arabian Peninsula, Pakistan and India. Rhazya stricta has been used traditionally to treat several diseases including tumors; however, the underlying mechanism is still not fully elucidated. AIM OF THE STUDY: The aim of this study is to examine the ability of Rhazya stricta to induce a key enzyme involved in cancer chemoprevention, NAD(P)H:quinone oxidoreductase 1 (Nqo1) in murine and human hepatoma cells. Nqo1 is regulated by the nuclear factor erythroid 2-related factor 2 (Nrf2) and the aryl hydrocarbon receptor (AhR) transcription factors. MATERIALS AND METHODS: Rhazya stricta leaves were extracted using ethanol, the strong basic alkaloid fraction (AF) was isolated according to a bioassay-guided fractionation and its mass spectrum was used as a fingerprint for its identity. The effect of increasing concentrations of AF on Nqo1 was tested in murine hepatoma Hepa 1c1c7 and human HepG2 cells. The role of Nrf2-dependent mechanism was tested by using Nrf2-dependent luciferase assay and by determining the Nrf2 nuclear accumulation in Hepa 1c1c7 cells. The role of AhR-dependent mechanism was assessed by using an AhR-deficient version of murine hepatoma c12 cells. RESULTS: AF significantly induced the Nqo1 at mRNA, protein and catalytic activity levels in murine hepatoma Hepa 1c1c7 cells. Moreover, the induction of Nqo1 by AF was completely abolished by using the transcriptional inhibitor, actinomycin D, implying a role of transcriptional regulation. In addition, the role of Nrf2 signaling pathway was confirmed by the induction of Nrf2-dependent luciferase activity and the induced Nrf2 nuclear accumulation in Hepa 1c1c7 cells. Interestingly, AF induced Nqo1 at mRNA and catalytic activity in c12 and HepG2 cells. Finally, the AF induced the Nrf2-dependent luciferase activity in HepG2 cells, confirming the role of Nrf2 in its regulation. CONCLUSIONS: The present study presents the first evidence that Rhazya stricta and its active strongly basic alkaloid fraction induce the chemopreventative enzyme, Nqo1 through Nrf2-dependent mechanism.

Post-trapping derivatization of radical-derived EPR-silent adducts: application to free radical detection by HPLC/UV in chemical, biochemical, and biological systems and comparison with EPR spectroscopy.

Free radicals are conventionally detected by electron paramagnetic resonance (EPR) spectroscopy after being trapped as spin adducts. Albeit this technique has demonstrated utmost efficacy in studying free radicals, its application to biological settings is intrinsically hampered by the inevitable bioreduction of radical-derived paramagnetic adducts. Herein, we describe a reliable technique to detect and quantify free radical metabolites, wherein reduced alkyl- and phenyl-5,5-dimethyl-1-pyrroline N-oxide (DMPO) adducts are converted into ultrastable N-naphthoate esters. To mimic the ubiquitous in vivo microenvironment, bioreductants, exogenous thiols, and sodium borohydride were studied. Nitroxyl reduction was confirmed using EPR and triphenyltetrazolium chloride. The formation of the N-naphthoyloxy derivatives was established by liquid chromatography/mass spectrometry (LC/MS). The derivatives were chromatographed using a binary eluent. HPLC and internal standards were synthesized using Grignard addition. The labeled DMPO adduct is (1) fluorescent, (2) stable as opposed to nitroxyl radical adducts, (3) biologically relevant, and (4) excellently chromatographed. Applications encompassed chemical, biochemical, and biological model systems generating C-centered radicals. Different levels of phenyl radicals produced in situ from whole blood were successfully determined. The method is readily applicable to the detection of hydroxyl radical. Analogously, DMPO, the spin trap, could be detected with extreme sensitivity suitable for in vivo applications. The developed method proved to be a viable alternative to EPR, where for the first time the reductive loss of paramagnetic signals of DMPO-trapped free radicals is transformed into fluorescence emission. We believe the proposed methodology could represent a valuable tool to probe free radical metabolites in vivo using DMPO, the least toxic spin trap.

Evaluating the reliability of analytical results using a probability criterion: a Bayesian perspective.

Methods validation is mandatory in order to assess the fitness of purpose of the developed analytical method. Of core importance at the end of the validation is the evaluation of the reliability of the individual results that will be generated during the routine application of the method. Regulatory guidelines provide a general framework to assess the validity of a method, but none address the issue of results reliability. In this study, a Bayesian approach is proposed to address this concern. Results reliability is defined here as "the probability (π) of an analytical method to provide analytical results (X) within predefined acceptance limits (±λ) around their reference or conventional true concentration values (µ(T)) over a defined concentration range and under given environmental and operating conditions." By providing the minimum reliability probability (π(min)) needed for the subsequent routine application of the method, as well as specifications or acceptance limits (±λ), the proposed Bayesian approach provides the effective probability of obtaining reliable future analytical results over the whole concentration range investigated. This is summarised in a single graph: the reliability profile. This Bayesian reliability profile is also compared to two frequentist approaches, the first one derived from the work of Dewé et al. [W. Dewé, B. Govaerts, B. Boulanger, E. Rozet, P. Chiap, Ph. Hubert, Chemometr. Intell. Lab. Syst. 85 (2007) 262-268] and the second proposed by Govaerts et al. [B. Govaerts, W. Dewé, M. Maumy, B. Boulanger, Qual. Reliab. Eng. Int. 24 (2008) 667-680]. Furthermore, to illustrate the applicability of the Bayesian reliability profile, this approach is also applied here to a bioanalytical method dedicated to the determination of ketoglutaric acid (KG) and hydroxymethylfurfural (HMF) in human plasma by SPE-HPLC-UV.

Drug-induced protein free radical formation is attenuated by unsaturated fatty acids by scavenging drug-derived phenyl radical metabolites.

Aromatic amine drugs like aminoglutethimide (AG) and related congeners have been shown to produce phenyl radicals through metabolism by myeloperoxidase (MPO)/H(2)O(2), which has been proposed to play a role in drug-induced agranulocytosis. AG has also been shown to induce MPO protein radical formation, but the ultimate fate of these metabolically generated phenyl radicals is still unknown. We tested the reactivity of linoleic acid (LA) and GSH with aniline-based compounds in the presence of horseradish peroxidase (HRP)/H(2)O(2) by measuring oxygen consumption. We found a qualitative correlation between drugs or xenobiotics that formed phenyl radical metabolites with the cooxidation of LA. Most compounds that reacted with LA did not react with GSH. Furthermore, an AG-derived phenyl radical was detected by EPR spin-trapping with MNP (2-methyl-2-nitrosopropane), in a reaction containing AG and HRP/H(2)O(2); these spectra were attenuated in the presence of LA and docosahexaenoic acid (DHA) indicating that phenyl radical scavenging occurred. Since it has been proposed that the phenyl radical metabolite leads to protein radical formation on MPO, we investigated the effect of LA and DHA in immuno-spin trapping experiments with MPO-containing HL-60 cell lysate. Using anti-DMPO, a protein radical was detected on a putative MPO fragment from the reaction of DMPO, AG, and glucose/glucose oxidase. When LA or DHA was included in this reaction, protein radical formation was significantly inhibited. Our results show that certain polyunsaturated fatty acids (PUFAs) act as scavengers of aromatic amine drug-derived phenyl radicals which in turn prevent protein radical formation. However, the interaction of phenyl radical drug metabolites with PUFAs will be dictated by their relative concentrations compared to those of other targets. Most importantly, it is possible to differentiate peroxidase substrates that generate phenyl radical metabolites from N-centered radicals on the basis of their reactivity toward GSH vs PUFAs, and PUFAs are targets for metabolically generated phenyl radicals.

Determination of methotrexate and indomethacin in urine using SPE-LC-DAD after derivatization.

A validated HPLC-DAD assay is presented for the simultaneous quantification of methotrexate and indomethacin in a drug combination which is used synergistically to intervene with tumoral or inflammatory tissue microenvironments. The analytes were isolated from urine via solid phase extraction. The method is based on derivatizing both analytes with a soluble carbodiimide coupler and 2-nitrophenylhydrazine directed to their commonly occurring carboxylate functions. The chromatographic separation was accomplished on an octylsilica column in less than 15 min using acetate buffer (pH 4; 10 mM)-methanol (60:40, v/v) as eluent at 1.5 ml/min and monitored at 400 nm. The selectivity of the method was demonstrated in a pre-dosed rheumatoid arthritis patient. Quality control samples were prepared and analyzed to reveal the validity of the method. Life samples collected from a healthy volunteer were monitored for both drugs and their urinary levels were determined.

Using tolerance intervals in pre-study validation of analytical methods to predict in-study results. The fit-for-future-purpose concept.

It is recognized that the purpose of validation of analytical methods is to demonstrate that the method is suited for its intended purpose. Validation is not only required by regulatory authorities, but is also a decisive phase before the routine use of the method. For a quantitative analytical method the objective is to quantify the target analytes with a known and suitable accuracy. For that purpose, first, a decision about the validity of the method based on prediction is proposed: a method is declared proper for routine application if it is considered that most of the future results generated will be accurate enough. This can be achieved by using the "beta-expectation tolerance interval" (accuracy profile) as the decision tool to assess the validity of the analytical method. Moreover, the concept of "fit-for-purpose" is also proposed here to select the most relevant response function as calibration curve, i.e. choosing a response function based solely on the predicted results this model will allow to obtain. This paper reports four case studies where the results obtained with quality control samples in routine were compared to predictions made in the validation phase. Predictions made using the "beta-expectation tolerance interval" are shown to be accurate and trustful for decision making. It is therefore suggested that an adequate way to conciliate both the objectives of the analytical method in routine analysis and those of the validation step consists in taking the decision about the validity of the analytical method based on prediction of the future results using the most appropriate response function curve, i.e. the fit-for-future-purpose concept.