Arno G. Siraki

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.

Anti-inflammatory and antioxidant properties of a novel resveratrol–salicylate hybrid analog

Resveratrol is a natural compound with a plethora of activities as well as limitations. We recently reported a series of resveratrol-salicylate analogs with potential chemopreventive activity. Herein, we report the anti-inflammatory and antioxidant properties of these resveratrol derivatives. Using an in vitro COX inhibition assay, and two in vivo protocols (carrageenan-induced peritonitis and paw edema), we identified a novel compound (C10) as a potent anti-inflammatory agent. The enhanced potency of C10 was associated with the ability of C10 to decrease the activity of myeloperoxidase (MPO) enzyme at 10mg/kg, whereas resveratrol and its natural analog (TMS) did not exert the same effect. Additionally, C10 significantly reduced the concentration of intracellular reactive oxygen species. Because of the proven association between cancer, inflammation, and oxidative stress, we believe that C10 is a promising chemopreventive molecule.

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

ETHNOPHARMACOLOGICAL RELEVANCEnRhazya 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.nnnAIM OF THE STUDYnThe 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.nnnMATERIALS AND METHODSnRhazya 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.nnnRESULTSnAF 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.nnnCONCLUSIONSnThe 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.

4-Aminobenzoic acid hydrazide inhibition of microperoxidase-11: Catalytic inhibition by reactive metabolites

Inhibition of human peroxidase enzymes such as myeloperoxidase or eosinophil peroxidase represents a novel therapeutic area, for which there are no current clinical therapeutics. We utilized 4-aminobenzoic acid hydrazide which was reported to be a potent irreversible inhibitor of myeloperoxidase to gain insight into the role of reactive metabolites in catalytic inhibition. In order to carry out detailed studies, we used a model peroxidase, microperoxidase-11 (MP-11). We investigated the heme spectrum of MP-11 in the presence of 4-ABAH and found that heme bleaching occurred that was irreversible. This coincided with an absence of catalytic activity. The spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was able to significantly prevent inactivation of peroxidase activity, therefore, we performed ESR spin trapping studies and detected a carbonyl carbon-centered radical of 4-ABAH. In order to determine if the free radical metabolites became bound to MP-11, we performed high-resolution MALDI with elemental analysis to determine the change in elemental composition that occurred in these reactions. These masses were assigned to free radical metabolites of 4-ABAH and were not observed in reactions containing DMPO. We conclude that the 4-ABAH free radical metabolites which were bound to MP-11 were involved in the catalytic inhibition and were scavenged by DMPO.

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.

Free Radical Metabolites in Arylamine Toxicity

Abstract Aromatic amines (also known as arylamines) form a very important class of xenobiotics. The arylamine substructure is found in pesticides, carcinogens, and drugs. It is therefore unsurprising that arylamine drugs possess varying degrees of toxicity which ultimately depend on dose, exposure, and the particular genetic makeup of the individual. Arylamines have been shown to undergo oxidation reactions to produce reactive metabolites. This chapter will focus on a subset of reactive metabolites which are the arylamine-free radical metabolites. A detailed discussion on how these free radical metabolites form is presented, and association between the latter and toxicity reactions are discussed. Particular emphasis is devoted to the subject of arylamine-induced blood dyscrasias and recent advances as well as future prospects in this area.

The role of cytochrome P450 1B1 and its associated mid-chain hydroxyeicosatetraenoic acid metabolites in the development of cardiac hypertrophy induced by isoproterenol

Numerous experimental studies have demonstrated the role of cytochrome P450 1B1 (CYP1B1) and its associated mid-chain hydroxyeicosatetraenoic acids (mid-chain HETEs) metabolite in the pathogenesis of cardiac hypertrophy. However, the ability of isoproterenol (ISO) to induce cardiac hypertrophy through mid-chain HETEs has not been investigated yet. Therefore, we hypothesized that ISO induces cardiac hypertrophy through the induction of CYP1B1 and its associated mid-chain HETE metabolites. To test our hypothesis, Sprague–Dawley rats were treated with ISO (5xa0mg/kg i.p.) for 12 and 72xa0h whereas, human ventricular cardiomyocytes RL-14 cells were exposed to 100xa0μM ISO in the presence and absence of 0.5xa0μM tetramethoxystilbene (TMS) a selective CYP1B1 inhibitor, or 25 nM CYP1B1-siRNA. Moreover, RL-14 cells were transiently transfected with the CRISPR-CYP1B1 plasmid. Thereafter, real-time PCR, western blot analysis, and liquid chromatography–electrospray ionization mass spectroscopy were used to determine the level of gene expression, protein expression, and mid-chain HETEs, respectively. Our results showed that ISO induced CYP1B1 protein expression and the level of cardiac mid-chain HETEs in vivo at pre-hypertrophic and hypertrophic stage. In vitro, inhibition of CYP1B1 using TMS or CYP1B1-siRNA significantly attenuates ISO-induced hypertrophy. Furthermore, overexpression of CYP1B1 significantly induced cellular hypertrophy and mid-chain HETEs metabolite. Mechanistically, the protective effect of TMS against cardiac hypertrophy was mediated through the modulation of superoxide anion, mitogen-activated protein kinases (MAPKs), and nuclear factor-κB (NF-κB). In conclusion, our study provides the first evidence that CYP1B1 and its associated mid-chain HETE metabolites are directly involved in the ISO-induced cardiac hypertrophy.

Screening of genotoxicity and mutagenicity in extractable organics from oil sands process–affected water

Large volumes of oil sands process-affected water (OSPW) are produced by the oil sands surface mining industry during alkaline hot-water extraction of bitumen. It is well documented that the acid extractable organics (AEOs) in OSPW, a highly complex mixture of acidic and polar neutral substances, are acutely toxic; but few studies have examined the genotoxicity or mutagenicity of this mixture. In the present study, the in vitro SOS Chromotest and the Ames test (TA98 and TA100 strains) were used to evaluate genotoxicity and mutagenicity for whole OSPW AEOs in the presence and absence of biotransformation by rat S9 liver enzymes. Two subfractions were also examined in the same assays: neutral extractable fraction (F1-NE), and the subsequent acid extractable fraction (F2-AE). In the SOS assay, whole AEO was cytotoxic when concentrated 2× (i.e., twice as concentrated as the environmental sample) and showed increasing genotoxic response above 6×. Co-exposure with S9 had a protective effect on the cell SOS-inducing factor and survival but did not eliminate genotoxicity above 6× concentrations. Most of the cytotoxicity was attributable to F2-AE, but both F1-NE and F2-AE had similar genotoxic dose-responses above 6×. In the Ames test without S9, whole AEO was mutagenic in both strains above 10× concentrations. Co-incubation with S9 had little effect on the TA100 strain but with TA98 resulted in bioactivation at midlevel doses (1.5-6.3×) and protection at higher doses (10-25×). The 2 subfractions were mutagenic in both strains but with different dose-responses. Further research in vivo or in more relevant cells is warranted to investigate the carcinogenic risks of OSPW. Environ Toxicol Chem 2017;36:1397-1404.

Differential eNOS-signalling by platelet subpopulations regulates adhesion and aggregation

AimsnIn addition to maintaining haemostasis, circulating blood platelets are the cellular culprits that form occlusive thrombi in arteries and veins. Compared to blood leucocytes, which exist as functionally distinct subtypes, platelets are considered to be relatively simple cell fragments that form vascular system plugs without a differentially regulated cellular response. Hence, investigation into platelet subpopulations with distinct functional roles in haemostasis/thrombosis has been limited. In our present study, we investigated whether functionally distinct platelet subpopulations exist based on their ability to generate and respond to nitric oxide (NO), an endogenous platelet inhibitor.nnnMethods and resultsnUtilizing highly sensitive and selective flow cytometry protocols, we demonstrate that human platelet subpopulations exist based on the presence and absence of endothelial nitric oxide synthase (eNOS). Platelets lacking eNOS (approximately 20% of total platelets) fail to produce NO and have a down-regulated soluble guanylate cyclase-protein kinase G (sGC-PKG)-signalling pathway. In flow chamber and aggregation experiments eNOS-negative platelets primarily initiate adhesion to collagen, more readily activate integrin αIIbβ3 and secrete matrix metalloproteinase-2, and form larger aggregates than their eNOS-positive counterparts. Conversely, platelets having an intact eNOS-sGC-PKG-signalling pathway (approximately 80% of total platelets) form the bulk of an aggregate via increased thromboxane synthesis and ultimately limit its size via NO generation.nnnConclusionnThese findings reveal previously unrecognized characteristics and complexity of platelets and their regulation of adhesion/aggregation. The identification of platelet subpopulations also has potentially important consequences to human health and disease as impaired platelet NO-signalling has been identified in patients with coronary artery disease.