Drug repurposing is a versatile strategy to improve current therapies. Disulfiram has long been used in the treatment of alcohol dependency and multiple clinical trials to evaluate its clinical value in oncology are ongoing. We have recently reported that the disulfiram metabolite diethyldithiocarbamate, when combined with copper (CuET), targets the NPL4 adapter of the p97VCP segregase to suppress the growth of a spectrum of cancer cell lines and xenograft models in vivo. CuET induces proteotoxic stress and genotoxic effects, however important issues concerning the full range of the CuET-evoked tumor cell phenotypes, their temporal order, and mechanistic basis have remained largely unexplored. Here, we have addressed these outstanding questions and show that in diverse human cancer cell models, CuET causes a very early translational arrest through the integrated stress response (ISR), later followed by features of nucleolar stress. Furthermore, we report that CuET entraps p53 in NPL4-rich aggregates leading to elevated p53 protein and its functional inhibition, consistent with the possibility of CuET-triggered cell death being p53-independent. Our transcriptomics profiling revealed activation of pro-survival adaptive pathways of ribosomal biogenesis (RiBi) and autophagy upon prolonged exposure to CuET, indicating potential feedback responses to CuET treatment. The latter concept was validated here by simultaneous pharmacological inhibition of RiBi and/or autophagy that further enhanced CuET's tumor cytotoxicity, using both cell culture and zebrafish in vivo preclinical models. Overall, these findings expand the mechanistic repertoire of CuET's anti-cancer activity, inform about the temporal order of responses and identify an unorthodox new mechanism of targeting p53. Our results are discussed in light of cancer-associated endogenous stresses as exploitable tumor vulnerabilities and may inspire future clinical applications of CuET in oncology, including combinatorial treatments and focus on potential advantages of using certain validated drug metabolites, rather than old, approved drugs with their, often complex, metabolic profiles.

• MeSH
• dánio pruhované metabolismus   MeSH
• disulfiram * metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• nádory * metabolismus   MeSH
• ribozomy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In this work, we carried out neurochemical and behavioral analysis of zebrafish (Danio rerio) treated with rotenone, an agent used to chemically induce a syndrome resembling Parkinson's disease (PD). Dopamine release, measured with fast-scan cyclic voltammetry (FSCV) at carbon-fiber electrodes in acutely harvested whole brains, was about 30% of that found in controls. Uptake, represented by the first order rate constant (k) and the half-life (t1/2) determined by nonlinear regression modeling of the stimulated release plots, was also diminished. Behavioral analysis revealed that rotenone treatment increased the time required for zebrafish to reach a reward within a maze by more than 50% and caused fish to select the wrong pathway, suggesting that latent learning was impaired. Additionally, zebrafish treated with rotenone suffered from diminished locomotor activity, swimming shorter distances with lower mean velocity and acceleration. Thus, the neurochemical and behavioral approaches, as applied, were able to resolve rotenone-induced differences in key parameters. This approach may be effective for screening therapies in this and other models of neurodegeneration.

• MeSH
• dánio pruhované metabolismus   MeSH
• dopamin metabolismus   MeSH
• kognice MeSH
• modely nemocí na zvířatech MeSH
• Parkinsonova nemoc * MeSH
• rotenon * farmakologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Adenylosuccinate lyase (ADSL) functions in de novo purine synthesis (DNPS) and the purine nucleotide cycle. ADSL deficiency (ADSLD) causes numerous neurodevelopmental pathologies, including microcephaly and autism spectrum disorder. ADSLD patients have normal serum purine nucleotide levels but exhibit accumulation of dephosphorylated ADSL substrates, S-Ado, and SAICAr, the latter being implicated in neurotoxic effects through unknown mechanisms. We examined the phenotypic effects of ADSL depletion in human cells and their relation to phenotypic outcomes. Using specific interventions to compensate for reduced purine levels or modulate SAICAr accumulation, we found that diminished AMP levels resulted in increased DNA damage signaling and cell cycle delays, while primary ciliogenesis was impaired specifically by loss of ADSL or administration of SAICAr. ADSL-deficient chicken and zebrafish embryos displayed impaired neurogenesis and microcephaly. Neuroprogenitor attrition in zebrafish embryos was rescued by pharmacological inhibition of DNPS, but not increased nucleotide concentration. Zebrafish also displayed phenotypes commonly linked to ciliopathies. Our results suggest that both reduced purine levels and impaired DNPS contribute to neurodevelopmental pathology in ADSLD and that defective ciliogenesis may influence the ADSLD phenotypic spectrum.

• MeSH
• adenylsukcinátlyasa nedostatek   metabolismus   MeSH
• aminoimidazolkarboxamid analogy a deriváty   metabolismus   MeSH
• autistická porucha metabolismus   MeSH
• buněčné linie MeSH
• buněčný cyklus MeSH
• ciliopatie metabolismus   MeSH
• dánio pruhované metabolismus   MeSH
• fenotyp MeSH
• fosfoproteiny metabolismus   MeSH
• kur domácí metabolismus   MeSH
• lidé MeSH
• mikrocefalie metabolismus   MeSH
• neurogeneze * MeSH
• poruchy autistického spektra metabolismus   MeSH
• poruchy metabolismu purinů a pyrimidinů metabolismus   MeSH
• poškození DNA MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• puriny metabolismus   MeSH
• ribonukleotidy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH

The aim of this study was to assess the impact of sulfamethoxazole (SMX) on oxidative stress indices in zebrafish (Danio rerio). The test was completed after 14 days. The tested concentrations were 50, 100 and 500 µg/L of SMX. Glutathione peroxidase, glutathione reductase, glutathione S-transferase and lipid peroxidation were investigated to determine the effects of SMX on oxidative stress in zebrafish. Lipid peroxidation gradually increased slightly (but non-significantly) at all tested concentrations during the test as compared to the control. The evaluation of oxidative stress biomarkers showed no significant changes in the activity of antioxidant enzymes in any experimental group exposed to SMX as compared to the control. The gradual increase in lipid peroxidation after 3 and 14 days in the SMX treated groups as compared to the control group indicates increasing cell membrane damage.

• MeSH
• antibakteriální látky toxicita   MeSH
• antioxidancia metabolismus   MeSH
• časové faktory MeSH
• dánio pruhované metabolismus   MeSH
• oxidační stres účinky léků   MeSH
• peroxidace lipidů účinky léků   MeSH
• sulfamethoxazol toxicita   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Cyanobacteria are known for their ability to produce and release mixtures of up to thousands of compounds into the environment. Recently, the production of novel metabolites, retinoids, was reported for some cyanobacterial species along with teratogenic effects of samples containing these compounds. Retinoids are natural endogenous substances derived from vitamin A that play a crucial role in early vertebrate development. Disruption of retinoid signalling- especially during the early development of the nervous system- might lead to major malfunctions and malformations. In this study, the toxicity of cyanobacterial biomass samples from the field containing retinoids was characterized by in vivo and in vitro bioassays with a focus on the potential hazards towards nervous system development and function. Additionally, in order to identify the compounds responsible for the observed in vitro and in vivo effects the complex cyanobacterial extracts were fractionated (C18 column, water-methanol gradient) and the twelve obtained fractions were tested in bioassays. In all bioassays, all-trans retinoic acid (ATRA) was tested along with the environmental samples as a positive control. Retinoid-like activity (mediated via the retinoic acid receptor, RAR) was measured in the transgenic cell line p19/A15. The in vitro assay showed retinoid-like activity by specific interaction with RAR for the biomass samples. Neurotoxic effects of selected samples were studied on zebrafish (Danio rerio) embryos using the light/dark transition test (Viewpoint, ZebraLab system) with 120 hpf larvae. In the behavioural assay, the cyanobacterial extracts caused significant hyperactivity in zebrafish at 120 hpf after acute exposure (3 h prior to the measurement) at concentrations below the teratogenicity LOEC (0.2 g dw L-1). Similar effect was observed after exposure to fractions of the extracts with detected retinoid-like activity and additive effect was observed after combining the fractions. However, the effect on behaviour was not observed after exposure to ATRA only. To provide additional insight into the behavioural effects and describe the underlying mechanism gene expression of selected biomarkers was measured. We evaluated an array of 28 genes related to general toxicity, neurodevelopment, retinoid and thyroid signalling. We detected several affected genes, most notably, the Cyp26 enzymes that control endogenous ATRA concentration, which documents an effect on retinoid signalling.

• MeSH
• biomasa MeSH
• biotest MeSH
• chemické látky znečišťující vodu metabolismus   toxicita   MeSH
• chování zvířat účinky léků   MeSH
• dánio pruhované růst a vývoj   metabolismus   MeSH
• embryo nesavčí účinky léků   metabolismus   MeSH
• exprese genu účinky léků   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• receptory kyseliny retinové genetika   metabolismus   MeSH
• sinice růst a vývoj   metabolismus   MeSH
• tretinoin metabolismus   toxicita   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Herbicides and their metabolites are often detected in water bodies where they may cause adverse effects to non-target organisms. Their effects at environmentally relevant concentrations are often unclear, especially concerning mixtures of pesticides. This study thus investigated the impacts of one of the most used herbicides: S-metolachlor and its two metabolites, metolachlor oxanilic acid (MOA) and metolachlor ethanesulfonic acid (MESA) on the development of zebrafish embryos (Danio rerio). Embryos were exposed to the individual substances and their environmentally relevant mixture until 120 hpf (hours post-fertilization). The focus was set on sublethal endpoints such as malformations, hatching success, length of fish larvae, spontaneous movements, heart rate and locomotion. Moreover, expression levels of eight genes linked to the thyroid system disruption, oxidative stress defense, mitochondrial metabolism, regulation of cell cycle and retinoic acid (RA) signaling pathway were analyzed. Exposure to S-metolachlor (1 μg/L) and the pesticide mixture (1 μg/L of each substance) significantly reduced spontaneous tail movements of 21 hpf embryos. Few rare developmental malformations were observed, but only in larvae exposed to more than 100 μg/L of individual substances (craniofacial deformation, non-inflated gas bladder, yolk sac malabsorption) and to 30 μg/L of each substance in the pesticide mixture (spine deformation). No effect on hatching success, length of larvae, heart rate or larvae locomotion were found. Strong responses were detected at the molecular level including induction of p53 gene regulating the cell cycle (the pesticide mixture - 1 μg/L of each substance; MESA 30 μg/L; and MOA 100 μg/L), as induction of cyp26a1 gene encoding cytochrome P450 (pesticide mixture - 1 μg/L of each substance). Genes implicated in the thyroid system regulation (dio2, thra, thrb) were all overexpressed by the environmentally relevant concentrations of the pesticide mixture (1 μg/L of each substance) and MESA metabolite (1 μg/L). Zebrafish thyroid system disruption was revealed by the overexpressed genes, as well as by some related developmental malformations (mainly gas bladder and yolk sac abnormalities), and reduced spontaneous tail movements. Thus, the thyroid system disruption represents a likely hypothesis behind the effects caused by the low environmental concentrations of S-metolachlor, its two metabolites and their mixture.

• MeSH
• acetamidy metabolismus   toxicita   MeSH
• chemické látky znečišťující vodu metabolismus   toxicita   MeSH
• dánio pruhované metabolismus   MeSH
• embryo nesavčí účinky léků   metabolismus   MeSH
• embryonální vývoj účinky léků   MeSH
• herbicidy metabolismus   toxicita   MeSH
• larva MeSH
• štítná žláza účinky léků   embryologie   MeSH
• synergismus léků MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Among the benzotriazole ultraviolet stabilizers (BUVSs), UV-234 and UV-320 are frequently detected in aquatic ecosystem. Despite the fact that these chemicals are present in low ng/L levels in surface water, they show high bio-accumulation potential and pose exposure risks to aquatic organisms. However, there are limited toxicological data available in fish. In this study, zebrafish embryos were exposed to 0.01, 0.1 and 1 μM UV-234 or UV-320 for up to 6 days. Developmental toxicity as well as effects on mitochondrial bioenergetics, immune system responses, and locomotor activity in zebrafish were measured. After UV-234 treatment (0.1-1 μM), hatching time of embryos was increased compared to controls. There was also a ∼20-40% reduction in non-mitochondrial respiration and oligomycin-dependent mitochondrial respiration in embryos treated with 1 μM UV-234 for 24 and 48 h respectively; conversely basal respiration and non-mitochondrial respiration were increased ∼20-30% in embryos treated with 1 μM UV-320 at 48 h. Transcript levels of sod1 were down-regulated with BUVSs while sod2 mRNA was highly up-regulated with both UV-234 and UV-320, suggesting an oxidative damage response. Considering that mitochondrial signaling regulates innate immune pathways, we measured the expression of immune related transcripts (tlr5a, tlr5b, mmp9, il8, tnfa, cxcl-C1c, nfkb1, and ifng). Of these, only il8 and cxcl-C1c mRNA were decreased in response to 0.1 μM UV-320. To associate early molecular events with behavior, locomotor activity was assessed. UV-234 reduced larval activity in a dark photokinesis assay by ∼15%, however behavioral responses at environmentally-relevant concentrations of BUVSs were not consistent across experiments nor BUVSs. These data suggest that BUVSs can perturb mitochondrial bioenergetics, embryonic development, and locomotor activity of zebrafish, but these responses appear to be dose-, time- and BUVSs dependent, suggesting these chemicals may have unique modes of action.

• MeSH
• dánio pruhované embryologie   metabolismus   MeSH
• embryonální vývoj účinky léků   MeSH
• energetický metabolismus fyziologie   MeSH
• larva účinky léků   MeSH
• lokomoce fyziologie   MeSH
• mitochondrie metabolismus   MeSH
• oxidační stres účinky léků   MeSH
• přirozená imunita účinky léků   MeSH
• triazoly farmakologie   MeSH
• ultrafialové záření MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Ziram is a broad spectrum pesticide that belongs to the class of dimethyl-dithiocarbamate (DTC) fungicides. The objectives of this study were to assess the effects of ziram in developing zebrafish. Ziram was highly toxic to zebrafish embryos, with a 96-h LC50 value of 1082.54 nM (∼0.33 mg/L). Zebrafish embryos at 6 h post-fertilization (hpf) were exposed to solvent control (0.1% DMSO), or one dose of 1, 10, 100, and 1000 nM ziram for 96 h. Ziram induced lethality in a dose-dependent manner, decreased hatching rate and heartbeat, and caused wavy deformities at 72 and 96 hpf at 100 and 1000 nM. Basal oxygen consumption rates of zebrafish at 24 hpf were decreased with 1000 nM, suggesting that ziram affects oxidative phosphorylation. We also measured the expression of transcripts associated with the oxidative stress response (sod1 and sod2) and dopamine receptor signaling at ∼96 h of exposure. There was no difference in the expression of genes related to oxidative stress, nor those related to the dopamine system. Locomotor activity was also assessed in larval zebrafish (7 dpf), and ziram increased total activity, the velocity in light zone, and total distance moved at 10 nM, while it decreased the mean time spent in the dark zone at 1 and 10 nM. Behavioral responses were dependent upon the time point and clutch examined. These data demonstrate that ziram negatively impacts embryonic development (i.e. mortality, hatching, heartbeat and notochord development) of zebrafish, decreases basal respiration of embryos, and alters behavioral responses in larvae.

• MeSH
• chování zvířat účinky léků   MeSH
• dánio pruhované růst a vývoj   metabolismus   MeSH
• dopamin genetika   MeSH
• embryo nesavčí účinky léků   MeSH
• embryonální vývoj účinky léků   MeSH
• fungicidy průmyslové metabolismus   toxicita   MeSH
• larva účinky léků   MeSH
• lokomoce účinky léků   MeSH
• oxidační stres genetika   MeSH
• spotřeba kyslíku účinky léků   MeSH
• ziram toxicita   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi. Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix.

• MeSH
• dánio pruhované genetika   růst a vývoj   metabolismus   MeSH
• dospělí MeSH
• exom MeSH
• fenotyp MeSH
• fibroblasty metabolismus   patologie   MeSH
• fyziologická kalcifikace * MeSH
• genomika * MeSH
• glykomika * MeSH
• glykopeptidasa nedostatek   MeSH
• glykosylace MeSH
• Golgiho aparát metabolismus   patologie   MeSH
• kohortové studie MeSH
• kojenec MeSH
• kultivované buňky MeSH
• lidé MeSH
• mladý dospělý MeSH
• mutace * MeSH
• přenašeče organických aniontů závislé na sodíku genetika   metabolismus   MeSH
• rodokmen MeSH
• symportéry genetika   metabolismus   MeSH
• transport proteinů MeSH
• vrozené poruchy glykosylace komplikace   MeSH
• vývojové onemocnění kostí etiologie   metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• kojenec MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Notch signalling is critical for the development of the nervous system. In the zebrafish mindbomb mutants, disruption of E3 ubiquitin ligase activity inhibits Notch signalling. In these mutant embryos, precocious development of primary neurons leading to depletion of neural progenitor cells results in a neurogenic phenotype characterized by defects in neural patterning and brain development. Cyclin-dependent kinase 5 (Cdk5), a predominant neuronal kinase, is involved in a variety of essential functions of the nervous system. Most recently, mammalian studies on Notch and Cdk5 regulating each other's function have been emerging. The status of Cdk5 in the mindbomb mutant embryos with excessive primary neurons is not known. In situ hybridization of the zebrafish mindbomb mutant embryos uncovered a robust upregulation in Cdk5 expression but with a reduced Cdk5 activity. The implications of these findings in both the mammalian system and zebrafish are discussed in this mini-review to provide a glimpse into the relationship between Notch and Cdk5 that may explain certain neurodevelopmental defects associated with either mutations in ubiquitin ligase or altered expression of Cdk5.

• MeSH
• biologické modely MeSH
• cyklin-dependentní kinasa 5 metabolismus   MeSH
• dánio pruhované metabolismus   MeSH
• mutace genetika   MeSH
• proteiny dánia pruhovaného genetika   MeSH
• receptory Notch metabolismus   MeSH
• upregulace genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH