NADPH oxidases (NOX), catalyzing the reduction of molecular oxygen to form the superoxide radical anion (•O₂⁻) and hydrogen peroxide (H₂O₂), are involved in several pathological conditions, such as ...stroke, diabetes, atherosclerosis, but also in chronic neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, or multiple sclerosis. GKT136901 is a novel NOX-1/4 inhibitor with potential application in the areas of diabetic nephropathy, stroke, or neurodegeneration. In the present study, we investigated additional pharmacological activities of the compound with respect to direct free radical scavenging. GKT136901 did not interact with nitric oxide (•NO), •O₂⁻, or hydroxyl radicals (•OH), but it acted as selective scavenger of peroxynitrite (PON) already in the submicromolar concentration range. Alpha synuclein (ASYN) is a protein involved in the pathogenesis of Parkinson's disease and a known target for PON-dependent tyrosine nitration. Submicromolar concentrations of GKT136901 prevented tyrosine nitration and di-tyrosine-dependent dimer formation of ASYN by PON as indicated by Western blot and mass spectrometric analysis. GKT136901 itself was degraded when exposed to PON. In a human neuronal cell model, GKT136901 prevented both the depletion of reduced intracellular glutathione, and the degeneration of neurites when present during PON treatment of the cells. When GKT136901 was applied after PON treatment, no protective effect was observed, thus excluding an impact of GKT136901 on cellular death/survival pathways. In summary, selective scavenging of PON is an additional pharmacological property of the NOX-1/4 inhibitor GKT136901, and this may add to the efficiency of the drug in several disease models.
Assessment of the network of toxicity pathways by Omics technologies and bioinformatic data processing paves the road toward a new toxicology for the twenty-first century. Especially, the upstream ...network of responses, taking place in toxicant-treated cells before a point of no return is reached, is still little explored. We studied the effects of the model neurotoxicant 1-methyl-4-phenylpyridinium (MPP(+)) by a combined metabolomics (mass spectrometry) and transcriptomics (microarrays and deep sequencing) approach to provide unbiased data on earliest cellular adaptations to stress. Neural precursor cells (LUHMES) were differentiated to homogeneous cultures of fully postmitotic human dopaminergic neurons, and then exposed to the mitochondrial respiratory chain inhibitor MPP(+) (5 μM). At 18-24 h after treatment, intracellular ATP and mitochondrial integrity were still close to control levels, but pronounced transcriptome and metabolome changes were seen. Data on altered glucose flux, depletion of phosphocreatine and oxidative stress (e.g., methionine sulfoxide formation) confirmed the validity of the approach. New findings were related to nuclear paraspeckle depletion, as well as an early activation of branches of the transsulfuration pathway to increase glutathione. Bioinformatic analysis of our data identified the transcription factor ATF-4 as an upstream regulator of early responses. Findings on this signaling pathway and on adaptive increases of glutathione production were confirmed biochemically. Metabolic and transcriptional profiling contributed complementary information on multiple primary and secondary changes that contribute to the cellular response to MPP(+). Thus, combined 'Omics' analysis is a new unbiased approach to unravel earliest metabolic changes, whose balance decides on the final cell fate.
The hemodynamic and anti-ischemic effects of nitroglycerin (GTN) are rapidly blunted as a result of the development of nitrate tolerance. Hydralazine has been shown to prevent tolerance in ...experimental and clinical studies, all of which may be at least in part secondary to antioxidant properties of this compound. The antioxidant effects of hydralazine were tested in cell free systems, cultured smooth muscle cells, isolated mitochondria, and isolated vessels. Inhibitory effects on the formation of superoxide and/or peroxynitrite formation were tested using lucigenin and L-012 enhanced chemiluminescence as well as DHE-fluorescence. The peroxynitrite scavenging properties were also assessed by inhibition of nitration of phenol. Prevention of impairment of NO downstream signaling and GTN bioactivation was determined by measurement of P-VASP (surrogate parameter for the activity of the cGMP-dependent kinase-I, cGK-I) and mitochondrial aldehyde dehydrogenase (ALDH-2) activity. Hydralazine dose-dependently decreased the chemiluminescence signal induced by peroxynitrite from SIN-1 and by superoxide from HX/XO in a cell free system, by superoxide in smooth muscle cells and mitochondria acutely challenged with GTN. Moreover, hydralazine inhibited the peroxynitrite-mediated nitration of phenols as well as proteins in smooth muscle cells in a dose-dependent fashion. Finally, hydralazine normalized impaired cGK-I activity as well as impaired vascular ALDH-2 activity. Our results indicate that hydralazine is a highly potent radical scavenger. Thus, the combination with isosorbide dinitrate (ISDN) will favorably influence the nitroso-redox balance in the cardiovascular system in patients with congestive heart failure and may explain at least in part the improvement of prognosis in patients with chronic congestive heart failure.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We recently reported the preferential selection of the K65R resistance mutation in subtype C HIV-1 compared with subtype B and showed the underlying mechanism to be dependent on subtype C-specific ...silent nucleotide polymorphisms, i.e. genomic mutations that change the genotype but not the phenotype. The number of clinical reports demonstrating elevated numbers of K65R nevertheless suggests the existence of factors limiting the increased incidence of K65R mutations. Thus, we investigated the contributions of subtype C-specific silent nucleotide polymorphisms at thymidine analogue mutation (TAM) sites 70, 210 and/or 219 that might reduce the previously described preferential selection of K65R in subtype C HIV-1 associated with subtype C-specific nucleotide polymorphisms at sites 64/65.
Cell culture drug selections were performed with various drugs in MT2 cells.
The use of nucleoside/nucleotide reverse transcriptase inhibitors N(t)RTIs as single drugs or in combination confirmed the more frequent selection of K65R by multiple N(t)RTIs in a subtype B virus that contained the 64/65 nucleotide polymorphisms of subtype C than in a wild-type subtype B virus. This effect was attenuated in the presence of several silent TAM nucleotide polymorphisms, except when stavudine was employed in the selection protocol.
These results further demonstrate that stavudine can preferentially select for K65R in subtype C virus and also provide a basis for understanding the importance of silent nucleotide polymorphisms in regard to altered HIV drug resistance profiles.
Test systems to identify developmental toxicants are urgently needed. A combination of human stem cell technology and transcriptome analysis was to provide a proof of concept that toxicants with a ...related mode of action can be identified and grouped for read-across. We chose a test system of developmental toxicity, related to the generation of neuroectoderm from pluripotent stem cells (UKN1), and exposed cells for 6 days to the histone deacetylase inhibitors (HDACi) valproic acid, trichostatin A, vorinostat, belinostat, panobinostat and entinostat. To provide insight into their toxic action, we identified HDACi consensus genes, assigned them to superordinate biological processes and mapped them to a human transcription factor network constructed from hundreds of transcriptome data sets. We also tested a heterogeneous group of ‘mercurials’ (methylmercury, thimerosal, mercury(II)chloride, mercury(II)bromide, 4-chloromercuribenzoic acid, phenylmercuric acid). Microarray data were compared at the highest non-cytotoxic concentration for all 12 toxicants. A support vector machine (SVM)-based classifier predicted all HDACi correctly. For validation, the classifier was applied to legacy data sets of HDACi, and for each exposure situation, the SVM predictions correlated with the developmental toxicity. Finally, optimization of the classifier based on 100 probe sets showed that eight genes (F2RL2, TFAP2B, EDNRA, FOXD3, SIX3, MT1E, ETS1 and LHX2) are sufficient to separate HDACi from mercurials. Our data demonstrate how human stem cells and transcriptome analysis can be combined for mechanistic grouping and prediction of toxicants. Extension of this concept to mechanisms beyond HDACi would allow prediction of human developmental toxicity hazard of unknown compounds with the UKN1 test system.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Oxygen is involved in cell signaling through oxygenases and oxidases and this applies especially for the vascular system. Nitric oxide (
NO) and epoxyarachidonic acids are P450-dependent ...monooxygenase products and prostacyclin is formed via cyclooxygenase and a heme-thiolate isomerase. The corresponding vasorelaxant mechanisms are counteracted by superoxide which not only traps
NO but through the resulting peroxynitrite blocks prostacyclin synthase by nitration of an active site tyrosine residue. In a model of septic shock, this leads to vessel constriction by activation of the thromboxane A
2–prostaglandin endoperoxide H
2 receptor. This sequence of events is part of endothelial dysfunction in which the activated vascular smooth muscle counteracts and regenerates vessel tone by cyclooxygenase-2-dependent prostacyclin synthesis. Peroxynitrite was found to activate cyclooxygenases by providing the peroxide tone at nanomolar concentrations. Such new insights into the control of vascular function have allowed us to postulate a concept of redox regulation in which a progressive increase of superoxide production by NADPH-oxidase, mitochondria, xanthine oxidase, and even uncoupled NO-synthase triggers a network of signals originating from an interaction of
NO with superoxide.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
LUHMES cells are conditionally-immortalized non-transformed human fetal cells that can be differentiated to acquire a dopaminergic neuron-like phenotype under appropriate growth conditions. After ...differentiation by GDNF and cyclic adenosine monophosphate, LUHMES were sensitive to 1-methyl-4-phenylpyridinium (MPP
+) toxicity at ≤
5 μM, but resistant to the parental compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The high homogeneity and purity of the cultures allowed the detection of metabolic changes during the degeneration. Cellular ATP dropped in two phases after 24 and 48 h; cellular glutathione (GSH) decreased continuously, paralleled by an increase in lipid peroxidation. These events were accompanied by a time-dependent degeneration of neurites. Block of the dopamine transporter by GBR 12909 or mazindol completely abrogated MPP
+ toxicity. Inhibition of
de novo dopamine synthesis by α-methyl-
l-tyrosine or 3-iodo-
l-tyrosine attenuated toxicity, but did not reduce the initial drop in ATP. Inhibition of mixed lineage kinases by CEP1347 completely prevented the MPP
+-induced loss of viability and intracellular GSH, but failed to attenuate the initial drop of ATP. For the quantitative assessment of neurite degeneration, an automated imaging-based high content screening approach was applied and confirmed the findings made by pharmacological interventions in this study. Our data indicate that inhibition of mitochondrial ATP synthesis is not sufficient to trigger cell death in MPP
+-treated LUHMES.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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