Systems analysis of the effect of hydrogen sulfide on the growth of Methylococcus capsulatus Bath.

Methanotrophs are bacteria capable on growing on methane as their sole carbon source. They may provide a promising route for upgrading natural gas into more valuable fuels and chemicals. However, natural gas may contain significant quantities of hydrogen sulfide. Little is known about how hydrogen sulfide affects the growth and physiology of methanotrophs aside from a few studies showing that it is inhibitory. This study investigated how hydrogen sulfide affects the growth and physiology of the model methanotroph, Methylococcus capsulatus Bath. Growth studies demonstrated that hydrogen sulfide inhibits the growth of M. capsulatus Bath when the concentration exceeds 0.5% (v/v). To better understand how hydrogen sulfide is inhibiting the growth of M. capsulatus Bath, transcription and metabolite concentrations were profiled using RNA sequencing and gas chromatography-mass spectrometry, respectively. Our analysis of the differentially expressed genes and changes in metabolite concentrations suggests that hydrogen sulfide inhibits cellular respiration. The cells respond to sulfide stress in part by increasing the rate of sulfide oxidation and by increasing the expression of sulfide quinone reductase and a putative persulfide dioxygenase. In addition, they reduce the expression of the native calcium-dependent methanol dehydrogenase and increase the expression of XoxF, a lanthanide-dependent methanol dehydrogenase. While the reason of this switch in unknown, XoxF has previously been shown to be induced by lanthanides or nitric oxide in methanotrophs. Collectively, these results further our understanding of how methanotrophs respond to sulfide stress and may aid in the engineering of strains resistant to hydrogen sulfide. KEY POINTS: • Hydrogen sulfide inhibits growth of Methylococcus capsulatus Bath • Sulfide stress inhibits cellular respiration • Sulfide stress induces XoxF, a lanthanide-dependent methanol dehydrogenase.

Epoxinnamide: An Epoxy Cinnamoyl-Containing Nonribosomal Peptide from an Intertidal Mudflat-Derived Streptomyces sp.

Cinnamoyl-containing nonribosomal peptides (CCNPs) form a unique family of actinobacterial secondary metabolites and display various biological activities. A new CCNP named epoxinnamide (1) was discovered from intertidal mudflat-derived Streptomyces sp. OID44. The structure of 1 was determined by the analysis of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) data along with a mass spectrum. The absolute configuration of 1 was assigned by the combination of advanced Marfey's method, 3JHH and rotating-frame overhauser effect spectroscopy (ROESY) analysis, DP4 calculation, and genomic analysis. The putative biosynthetic pathway of epoxinnamide (1) was identified through the whole-genome sequencing of Streptomyces sp. OID44. In particular, the thioesterase domain in the nonribosomal peptide synthetase (NRPS) biosynthetic gene cluster was proposed as a bifunctional enzyme, which catalyzes both epimerization and macrocyclization. Epoxinnamide (1) induced quinone reductase (QR) activity in murine Hepa-1c1c7 cells by 1.6-fold at 5 µM. It also exhibited effective antiangiogenesis activity in human umbilical vein endothelial cells (IC50 = 13.4 µM).

Journal of pineal research guideline for authors: Defining and characterizing melatonin targets.

A multitude of effects has been attributed to melatonin at pmol/L to mmol/L concentrations. More than fifteen targets have been proposed for melatonin but only few of them are well characterized. The current guidelines intend to provide a framework to improve and rationalize the characterization of melatonin targets and effects. They should be considered as mandatory guidelines and minimum requirements for manuscripts submitted to the Journal of Pineal Research.

Svalbamides A and B, Pyrrolidinone-Bearing Lipodipeptides from Arctic Paenibacillus sp.

Two new secondary metabolites, svalbamides A (1) and B (2), were isolated from a culture extract of Paenibacillus sp. SVB7 that was isolated from surface sediment from a core (HH17-1085) taken in the Svalbard archipelago in the Arctic Ocean. The combinational analysis of HR-MS and NMR spectroscopic data revealed the structures of 1 and 2 as being lipopeptides bearing 3-amino-2-pyrrolidinone, d-valine, and 3-hydroxy-8-methyldecanoic acid. The absolute configurations of the amino acid residues in svalbamides A and B were determined using the advanced Marfey's method, in which the hydrolysates of 1 and 2 were derivatized with l- and d- forms of 1-fluoro-2,4-dinitrophenyl-5-alanine amide (FDAA). The absolute configurations of 1 and 2 were completely assigned by deducing the stereochemistry of 3-hydroxy-8-methyldecanoic acid based on DP4 calculations. Svalbamides A and B induced quinone reductase activity in Hepa1c1c7 murine hepatoma cells, indicating that they represent chemotypes with a potential for functioning as chemopreventive agents.

Physalin A regulates the Nrf2 pathway through ERK and p38 for induction of detoxifying enzymes.

BACKGROUND: Physalin A isolated from Physalis alkekengi var. franchetii has been known to have many pharmacological properties. However, its effect through the Nrf2 pathway has not yet been elucidated. In the present study, we determined the effects of physalin A on cancer chemoprevention via the Nrf2 pathway. METHODS: Experiments were performed in Hepa-1c1c7 and HepG2 cells. The quinone reductase (QR) activity assay was used to assess the activity of physalin A and other compounds isolated from P. alkekengi. The antioxidant response element (ARE) reporter assay was used to determine physalin A induced transcription of Nrf2 target genes, whereas the oligonucleotide pull-down assay was used to investigate Nrf2 binding to the AREs post physalin A treatment. Real-time PCR and western blotting were performed to determine the expression of Nrf2 target genes. Immunocytochemistry was used to determine Nrf2 localization after treatment with physalin A. Kinase inhibitors were used to test the involvement of Nrf2-targeting kinases and the role of ERK and p38 phosphorylation was confirmed using western blotting. RESULTS: Physalin A significantly induced QR activity. As an upstream effector of QR, Nrf2 induced genes containing the ARE, which encode various antioxidants and detoxification enzymes. We observed that physalin A increased the expression of Nrf2 and its target genes in HepG2 cells. Moreover, we observed that physalin A-induced Nrf2 activation was regulated by ERK and p38 kinase in HepG2 cells. CONCLUSIONS: Taken together, we showed that physalin A increased detoxifying enzyme expression via activation of Nrf2 and its target genes. These results imply that physalin A could be a potential chemopreventive agent for liver diseases, as well as cancer.

Tenebrionid secretions and a fungal benzoquinone oxidoreductase form competing components of an arms race between a host and pathogen.

Entomopathogenic fungi and their insect hosts represent a model system for examining invertebrate-pathogen coevolutionary selection processes. Here we report the characterization of competing components of an arms race consisting of insect protective antimicrobial compounds and evolving fungal mechanisms of detoxification. The insect pathogenic fungus Beauveria bassiana has a remarkably wide host range; however, some insects are resistant to fungal infection. Among resistant insects is the tenebrionid beetle Tribolium castaneum that produces benzoquinone-containing defensive secretions. Reduced fungal germination and growth was seen in media containing T. castaneum dichloromethane extracts or synthetic benzoquinone. In response to benzoquinone exposure, the fungus expresses a 1,4-benzoquinone oxidoreductase, BbbqrA, induced >40-fold. Gene knockout mutants (ΔBbbqrA) showed increased growth inhibition, whereas B. bassiana overexpressing BbbqrA (Bb::BbbqrA(O)) displayed increased resistance to benzoquinone compared with wild type. Increased benzoquinone reductase activity was detected in wild-type cells exposed to benzoquinone and in the overexpression strain. Heterologous expression and purification of BbBqrA in Escherichia coli confirmed NAD(P)H-dependent benzoquinone reductase activity. The ΔBbbqrA strain showed decreased virulence toward T. castaneum, whereas overexpression of BbbqrA increased mortality versus T. castaneum. No change in virulence was seen for the ΔBbbqrA or Bb::BbbqrA(O) strains when tested against the greater wax moth Galleria mellonella or the beetle Sitophilus oryzae, neither of which produce significant amounts of cuticular quinones. The observation that artificial overexpression of BbbqrA results in increased virulence only toward quinone-secreting insects implies the lack of strong selection or current failure of B. bassiana to counteradapt to this particular host defense throughout evolution.

Activity-guided isolation of phase II enzyme inducers from buckwheat flour methanolic extracts.

BACKGROUND: Buckwheat is an important alternative crop and a raw material for functional food formulation. Phase II detoxification proteins/enzymes provide cytoprotective roles against oxidative stress and inflammation originating from various stressors. We aimed to identify and characterize potential phase II enzyme inducers from methanolic extracts of buckwheat flour, using an activity-guided fractionation based on the induction of quinone reductase (QR) (EC 1.6.5.2) in Hepa 1c1c7 cells. RESULTS: We isolated the QR inducers N-trans-feruloyltyramine (I), syringic acid (II), quercetin (III) and myricetin (IV). The relative QR-inducing ability, as well as the concentration required to double QR specific activity (CD values, in parentheses), decreased in the order: quercetin (3.0 µmol L-1 ) > N-trans-feruloyltyramine (24 µmol L-1 ) > myricetin (58 µmol L-1 ) > syringic acid (5.4 mmol L-1 ). Quercetin and N-trans-feruloyltyramine exhibited the greatest extent of QR induction of an approximately four-fold maximum induction and these compounds also exhibited the greatest values for the ratio of IC50 (i.e. level to reduce viability by 50%): CD values of 11 and > 8.3, respectively, among the four QR inducers isolated. Isobologram analyses for binary combinations of compounds I-IV revealed primarily antagonistic interactions for QR induction. CONCLUSION: These findings add to our understanding of the nutraceutical potential of buckwheat as a chemoprophylactic dietary component. © 2018 Society of Chemical Industry.

Positive feedback during sulfide oxidation fine-tunes cellular affinity for oxygen.

UNLABELLED: Sulfide (H2S in the gas form) is the third gaseous transmitter found in mammals. However, in contrast to nitric oxide (NO) or carbon monoxide (CO), sulfide is oxidized by a sulfide quinone reductase and generates electrons that enter the mitochondrial respiratory chain arriving ultimately at cytochrome oxidase, where they combine with oxygen to generate water. In addition, sulfide is also a strong inhibitor of cytochrome oxidase, similar to NO, CO and cyanide. The balance between the electron donor and the inhibitory role of sulfide is likely controlled by sulfide and oxygen availability. The present study aimed to evaluate if and how sulfide release and oxidation impacts on the cellular affinity for oxygen. RESULTS: i) when sulfide delivery approaches the maximal sulfide oxidation rate cells become exquisitely dependent on oxygen; ii) a positive feedback makes the balance between sulfide-releasing and -oxidizing rates the relevant parameter rather than the absolute values of these rates, and; iii) this altered dependence on oxygen is detected with sulfide concentrations that remain in the low micromolar range. CONCLUSIONS: i) within the context of continuous release of sulfide stemming from cellular metabolism, alterations in the activity of the sulfide oxidation pathway fine-tunes the cell's affinity for oxygen, and; ii) a decrease in the expression of the sulfide oxidation pathway greatly enhances the cell's dependence on oxygen concentration.

Structure-activity relationships and docking studies of synthetic 2-arylindole derivatives determined with aromatase and quinone reductase 1.

In our ongoing effort of discovering anticancer and chemopreventive agents, a series of 2-arylindole derivatives were synthesized and evaluated toward aromatase and quinone reductase 1 (QR1). Biological evaluation revealed that several compounds (e.g., 2d, IC50 = 1.61 µM; 21, IC50 = 3.05 µM; and 27, IC50 = 3.34 µM) showed aromatase inhibitory activity with half maximal inhibitory concentration (IC50) values in the low micromolar concentrations. With regard to the QR1 induction activity, 11 exhibited the highest QR1 induction ratio (IR) with a low concentration to double activity (CD) value (IR = 8.34, CD = 2.75 µM), while 7 showed the most potent CD value of 1.12 µM. A dual acting compound 24 showed aromatase inhibition (IC50 = 9.00 µM) as well as QR1 induction (CD = 5.76 µM) activities. Computational docking studies using CDOCKER (Discovery Studio 3.5) provided insight in regard to the potential binding modes of 2-arylindoles within the aromatase active site. Predominantly, the 2-arylindoles preferred binding with the 2-aryl group toward a small hydrophobic pocket within the active site. The C-5 electron withdrawing group on indole was predicted to have an important role and formed a hydrogen bond with Ser478 (OH). Alternatively, meta-pyridyl analogs may orient with the pyridyl 3'-nitrogen coordinating with the heme group.

Role of Quinone Reductase 2 in the Antimalarial Properties of Indolone-Type Derivatives.

Indolone-N-oxides have antiplasmodial properties against Plasmodium falciparum at the erythrocytic stage, with IC50 values in the nanomolar range. The mechanism of action of indolone derivatives involves the production of free radicals, which follows their bioreduction by an unknown mechanism. In this study, we hypothesized that human quinone reductase 2 (hQR2), known to act as a flavin redox switch upon binding to the broadly used antimalarial chloroquine, could be involved in the activity of the redox-active indolone derivatives. Therefore, we investigated the role of hQR2 in the reduction of indolone derivatives. We analyzed the interaction between hQR2 and several indolone-type derivatives by examining enzymatic kinetics, the substrate/protein complex structure with X-ray diffraction analysis, and the production of free radicals with electron paramagnetic resonance. The reduction of each compound in cells overexpressing hQR2 was compared to its reduction in naïve cells. This process could be inhibited by the specific hQR2 inhibitor, S29434. These results confirmed that the anti-malarial activity of indolone-type derivatives was linked to their ability to serve as hQR2 substrates and not as hQR2 inhibitors as reported for chloroquine, leading to the possibility that substrate of hQR2 could be considered as a new avenue for the design of new antimalarial compounds.

Systemic structure-activity relationship study of phenyl polyyne diols as potential chemopreventive agents.

The present study reports the cancer chemopreventive activities of phenyl polyyne diols derived from polyacetylene triol. Thirty-seven analogues based on a 1-phenylhexa-2,4-diyne-1,6-diol scaffold were prepared and their effects on QR activity were elucidated, as well as their cytotoxicities. We found that most of the derivatives based on phenylhexa-2,4-diyne-1,6-diol exhibited good QR induction activity and relatively low cytotoxicity and systemic structure-activity relationship was revealed. In particular, 4-fluorophenyl, 3-chlorophenyl, and 3,4-dioxolophenyl derivatives showed the best profiles in terms of QR induction, cytotoxicity, and CI.

Naphtho[1',2':4,5]imidazo[1,2-a]pyridine-5,6-diones: Synthesis, enzymatic reduction and cytotoxic activity.

Naphtho[1',2':4,5]imidazo[1,2-a]pyridine-5,6-diones (NPDOs), a new type of N-heterocycle-fused o-quinones, have been synthesized. They have been found to be efficient electron-accepting substrates of NADPH-dependent single-electron-transferring P-450R and two-electron transferring NQO1, generating reactive oxygen species (ROS) with a concomitant decrease in NADPH, which is consistent with redox-cycling. The reactivity of NPDOs toward P-450R (in terms of kcat/Km) varied in the range of 10(6)-10(7)M(-1)s(-1), while their reduction by NQO1 proceeded much faster, approaching the diffusion control limit (kcat/Km∼10(8)-10(9)M(-1)s(-1)). NPDOs exhibited relatively high cytotoxic activity against human lung carcinoma (A-549) and breast tumor (MCF-7) cell lines (LC50=0.1-8.3µM), while promyelocytic leukemia cells (HL-60) were less sensitive to NPDOs (LC50⩾10µM). 3-Nitro-substituted NPDO (11) revealed the highest potency against both A-549 and MCF-7 cell lines, with LC50 of 0.12±0.03µM and 0.28±0.08µM, respectively. Dicoumarol partly suppressed the activity of the compounds against A-594 and MCF-7 cell lines, suggesting that their cytotoxic action might be partially influenced by NQO1-mediated bioreductive activation.

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol.

Microbes in contaminated environments often evolve new metabolic pathways for detoxification or degradation of pollutants. In some cases, intermediates in newly evolved pathways are more toxic than the initial compound. The initial step in the degradation of pentachlorophenol by Sphingobium chlorophenolicum generates a particularly reactive intermediate; tetrachlorobenzoquinone (TCBQ) is a potent alkylating agent that reacts with cellular thiols at a diffusion-controlled rate. TCBQ reductase (PcpD), an FMN- and NADH-dependent reductase, catalyzes the reduction of TCBQ to tetrachlorohydroquinone. In the presence of PcpD, TCBQ formed by pentachlorophenol hydroxylase (PcpB) is sequestered until it is reduced to the less toxic tetrachlorohydroquinone, protecting the bacterium from the toxic effects of TCBQ and maintaining flux through the pathway. The toxicity of TCBQ may have exerted selective pressure to maintain slow turnover of PcpB (0.02 s(-1)) so that a transient interaction between PcpB and PcpD can occur before TCBQ is released from the active site of PcpB.

Statistical Correlations between HPLC Activity-Based Profiling Results and NMR/MS Microfraction Data to Deconvolute Bioactive Compounds in Mixtures.

Recent approaches in natural product (NP) research are leading toward the discovery of bioactive chemical entities at the microgram level. In comparison to classical large scale bioassay-guided fractionation, the use of LC-MS metabolite profiling in combination with microfractionation for both bioactivity profiling and NMR analysis, allows the identification of bioactive compounds at a very early stage. In that context, this study aims to assess the potential of statistic correlation analysis to enable unambiguous identification of features related to bioactive compounds in mixtures, without the need for complete isolation. For that purpose, a mixture of NPs was microfractionated by rapid small-scale semi-preparative HPLC for proof-of-concept. UHPLC-ESI-TOFMS profiles, micro-flow CapNMR spectra and a cancer chemopreventive assay carried out on every microfraction were analysed by statistical correlations.

Correlation Between Phytochemical and Mineral Contents and Antioxidant Activity of Black Glutinous Rice Bran, and Its Potential Chemopreventive Property.

In this work total anthocyanin content (TAC), total flavonoid content (TFC), total phenolic content (TPC) and minerals found in five black glutinous rice cultivars (MS, SK, PY, PC and KK) from Thailand were analyzed. The antioxidant activity of anthocyanin-rich black glutinous rice bran extracts against nitric oxide radical (NO˙), superoxide radical (O2˙Z) and lipid peroxyl radical (LOO˙) was also determined. Potential chemopreventive property of rice bran extract was screened based on cellular bioassays for phase II detoxification enzyme induction. Quinone reductase (QR) induction in murine hepatoma cells was used as a marker for this effect. Rice bran extract of cultivar KK had the highest TAC, of SK the highest TFC and of PC the highest TPC. The best antioxidants against NO˙, O2˙Z and LOO˙ were cultivars KK, MS, and SK, respectively. Overall, TAC, TFC and TPC had a combinatorial effect on the antioxidant activities of all extracts; none of them dominated. Minerals may not play a role in the antioxidant activity of the extracts because most correlations between them and the antioxidant activity were unpredictable. However, rice bran contained high mass fractions of some essential minerals on dry mass basis, including Zn (103-133 µg/g), Se (11-18 µg/g) and Cu (3.8-7.1 µg/g). Chemopreventive study indicated that PC cultivar was the most potent chemopreventor with the lowest concentration of an inducer needed to double the QR activity (CD value) of 0.7 µg/mL. These findings showed that black glutinous rice bran is rich in phytochemicals and some essential minerals, and has a potential chemopreventive property.

Endophytic fungi associated with Taxus fuana (West Himalayan Yew) of Pakistan: potential bio-resources for cancer chemopreventive agents.

CONTEXT: Endophytic fungi, being a prolific source of bioactive secondary metabolites, are of great interest for natural product discovery. OBJECTIVE: Isolation and partial characterization of endophytic fungi inhabiting the leaves and woody parts of Taxus fuana Nan Li & R.R. Mill. (Taxaceae) and evaluation of biological activity. MATERIALS AND METHODS: Endophytic fungal isolates were identified by molecular analysis of internal transcribed spacer (ITS) regions of 18S rDNA. Extracts of the endophytic fungi cultured on potato dextrose agar and modified medium were evaluated using cancer chemoprevention bioassays [inhibition of TNF-α-induced NFκB, aromatase and inducible nitric oxide synthase (iNOS); induction of quinone reductase 1 (QR1)] and growth inhibition with MCF-7 cells. RESULTS: Nine of 15 fungal isolates were identified as belonging to Epicoccum, Mucor, Penicillium, Chaetomium, Paraconiothriym, Plectania or Trichoderma. Five of the 15 extracts inhibited NFκB activity (IC50 values ranging between 0.18 and 17 µg/mL) and five inhibited iNOS (IC50 values ranging between 0.32 and 12.9 µg/mL). In the aromatase assay, only two isolates mediated inhibition (IC50 values 12.2 and 10.5 µg/mL). With QR1 induction, three extracts exhibited significant activity (concentrations to double activity values ranging between 0.20 and 5.5 µg/mL), and five extracts inhibited the growth of MCF-7 cells (IC50 values ranging from 0.56 to 17.5 µg/mL). Six active cultures were derived from woody parts of the plant material. CONCLUSION: The endophytic fungi studied are capable of producing pharmacologically active natural compounds. In particular, isolates derived from the wood of Taxus fuana should be prioritized for the isolation and characterization of bioactive constituents.

Identification of dialkyl diacetylene diols with potent cancer chemopreventive activity.

An increasing importance of chemoprevention for controlling cancer risks prompted the discovery of new active cancer chemopreventive agents. In this study, we designed and synthesized substituted hexa-2,4-diyne-1,6-diols, more structurally simplified, tunable, and easily preparable than natural gymnasterkoreaynes, and evaluated their cancer chemopreventive activities by measuring concentration of doubling quinone reductase activity (CD), cell viability, and chemopreventive index (CI). Most of the diols exhibited good CD activity and low cytotoxicity. In particular, tetradeca-5,7-diyne-4,9-diol and 2-methyltetradeca-5,7-diyne-4,9-diol showed the best cancer chemopreventive activity, approximately equipotent to that of sulforaphane. And, by synthesizing optically active stereoisomers of selected active compounds, the effect of stereochemistry was also studied. Eventually, we produced a chemopreventive compound for in vivo study.

Respiratory complex III dysfunction in humans and the use of yeast as a model organism to study mitochondrial myopathy and associated diseases.

The bc1 complex or complex III is a central component of the aerobic respiratory chain in prokaryotic and eukaryotic organisms. It catalyzes the oxidation of quinols and the reduction of cytochrome c, establishing a proton motive force used to synthesize adenosine triphosphate (ATP) by the F1Fo ATP synthase. In eukaryotes, the complex III is located in the inner mitochondrial membrane. The genes coding for the complex III have a dual origin. While cytochrome b is encoded by the mitochondrial genome, all the other subunits are encoded by the nuclear genome. In this review, we compile an exhaustive list of the known human mutations and associated pathologies found in the mitochondrially-encoded cytochrome b gene as well as the fewer mutations in the nuclear genes coding for the complex III structural subunits and accessory proteins such as BCS1L involved in the assembly of the complex III. Due to the inherent difficulties of studying human biopsy material associated with complex III dysfunction, we also review the work that has been conducted to study the pathologies with the easy to handle eukaryotic microorganism, the yeast Saccharomyces cerevisiae. Phenotypes, biochemical data and possible effects due to the mutations are also discussed in the context of the known three-dimensional structure of the eukaryotic complex III. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes.

Isorhamentin is a 3'-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes.

NQO2 is a reactive oxygen species generating off-target for acetaminophen.

The analgesic and antipyretic compound acetaminophen (paracetamol) is one of the most used drugs worldwide. Acetaminophen overdose is also the most common cause for acute liver toxicity. Here we show that acetaminophen and many structurally related compounds bind quinone reductase 2 (NQO2) in vitro and in live cells, establishing NQO2 as a novel off-target. NQO2 modulates the levels of acetaminophen derived reactive oxygen species, more specifically superoxide anions, in cultured cells. In humans, NQO2 is highly expressed in liver and kidney, the main sites of acetaminophen toxicity. We suggest that NQO2 mediated superoxide production may function as a novel mechanism augmenting acetaminophen toxicity.