Promoter-specific inhibition of transcription by daunorubicin in Saccharomyces cerevisiae

6 pages, 3 figures, 2 tables.-- PMID: 12164785 [PubMed].-- PMCID: PMC1222970.-- Available online Aug 7, 2002.Several anti-tumour drugs exert some of their cytotoxic effects by direct binding to DNA, thus inhibiting the transcription of certain genes. We analysed the influence of the anti-tumour antibiotic daunorubicin on the transcription of different genes in vivo using the budding yeast Saccharomyces cerevisiae. Daunorubicin only affected wild-type yeast strains at very high concentrations; however, erg6 mutant strains (but not pdr1, pdr3 or pdr5 strains) were sensitive to daunorubicin at low micromolar concentrations. In Delta erg6 strains, daunorubicin inhibited the galactose-induced transcription by Gal4p in a specific manner, since the transcription of identical reporters driven by other activators (either constitutive or inducible) was not inhibited. The drug concentrations at which Gal4p function was inhibited did not affect cell growth or viability. Furthermore, daunorubicin inhibited the growth in galactose and the transcriptional induction of resident Gal4p-driven genes upon galactose addition, two processes absolutely dependent on Gal4p function. We propose that daunorubicin and some transcription factors compete for DNA sequences encompassing CpG steps, and that this is the main determinant of the effects of the drug on transcription in vivo. Our approach may foster the development of anti-tumour drugs with more specific mechanisms of action.This study has been supported by grants PB98-0469, BMC2000-0898, BMC2001-0246 and GEN2001-4707-C8-08 from the Ministerio de Educación y Cultura (Spain) and Ministerio de Ciencia y Tecnología (Spain). S.M. is the recipient of a fellowship from the Generalitat de Catalunya. This study was carried out within the framework of the Centre de Referència en Biotecnologia of the Generalitat de Catalunya.Peer reviewe

Therapeutic potential of N-acetylcysteine in acrylamide acute neurotoxicity in adult zebrafish

Two essential key events in acrylamide (ACR) acute neurotoxicity are the formation of adducts with nucleophilic sulfhydryl groups on cysteine residues of selected proteins in the synaptic terminals and the depletion of the glutathione (GSx) stores in neural tissue. The use of N-acetylcysteine (NAC) has been recently proposed as a potential antidote against ACR neurotoxicity, as this chemical is not only a well-known precursor of the reduced form of glutathione (GSH), but also is an scavenger of soft electrophiles such as ACR. In this study, the suitability of 0.3 and 0.75 mM NAC to protect against the neurotoxic effect of 0.75 mM ACR has been tested in vivo in adult zebrafish. NAC provided only a mild to negligible protection against the changes induced by ACR in the motor function, behavior, transcriptome and proteome. The permeability of NAC to cross blood-brain barrier (BBB) was assessed, as well as the ACR-scavenging activity and the gamma-glutamyl-cysteine ligase (γ-GCL) and acylase I activities. The results show that ACR not only depletes GSx levels but also inhibits it synthesis from NAC/cysteine, having a dramatic effect over the glutathione system. Moreover, results indicate a very low NAC uptake to the brain, probably by a combination of low BBB permeability and high deacylation of NAC during the intestinal absorption. These results strongly suggest that the use of NAC is not indicated in ACR acute neurotoxicity treatment.This work was supported by the NATO SfP project MD.SFPP 984777 (D.R.) and the Spanish Government (CTM2017-83242-R; D.R.). M.F acknowledges financial support from the Beatriu de Pinós programme (Grant No. 2016 BP 00233) provided by the Secretariat of Universities and Research department of the Ministry for Business and Knowledge, Catalonia Government. Mention of specific products or trade names does not indicate endorsement by the US federal government.Peer reviewe

Alternative mechanisms of transcriptional activation by Rap1p

9 pages, 6 figures, 3 tables.-- PMID: 11358963 [PubMed].-- Printed version published Jul 13, 2001.Single Rap1p DNA-binding sites are poor activators of transcription of yeast minimal promoters, even when fully occupied in vivo. This low efficiency is due to two independent repression mechanisms as follows: one that requires the presence of histones, and one that requires Hrs1p, a component of the RNA polymerase II mediator complex. Both repression mechanisms were greatly reduced for constructs with tandemly arranged sites. In these constructs, UASrpg sequences (ACACCCATACATTT) activated better than telomere-like sequences (ACACCCACACACCC) in an orientation-dependent manner. Both mutations in the SWI/SNF complex and a deletion of amino acids 597--629 of Rap1p (Tox domain) decreased synergistic effects of contiguous telomeric sites. Conversely, deletion of amino acids 700--798 of Rap1p (Sil domain) made UASrpg and telomeric sites functionally indistinguishable. We propose that the Sil domain masks the main transactivation domain of Rap1p in Rap1p-telomere complexes, where the Tox domain behaves as a secondary activation domain, probably by interacting with chromatin-remodeling complexes. Rap1p DNA-binding sites in ribosomal protein gene promoters are mainly UASrpg-like; their replacement by telomeric sequences in one of these promoters (RPS17B) decreased transcription by two-thirds. The functional differences between UASrpgs and telomeric sequences may thus contribute to the differential expression of Rap1p-regulated promoters in vivo.This work was supported in part by Grants PB95-0433 and PB98-0469 from the Ministerio de Educación y Cultura (Spain).Peer reviewe

Estrogenic activity associated with organochlorine compounds in fish extracts from European mountain lakes

8 pages, 3 figures, 5 tables.-- PMID: 16997436 [PubMed].-- Online version available Sep 25, 2006.Issue title: Ozone at the Intensive Monitoring Plots in SW Europe.Fish muscle extracts from ten European mountain lakes were analyzed for organochlorine compounds (OCs) and estrogenic activity, the latter by a recombinant yeast assay based on the human estrogen receptor. Seventy percent of the samples showed estrogenic activity above detection limits and a subset of five samples showed estrogenic activities, equivalent to more than 10,000 pg/g of estradiol. These highly estrogenic samples occurred in two lakes, Velké Hinçovo in the Tatra Mountains and Redon in the Pyrenees. Principal component analysis correlated estrogenic activity of muscle extracts to fish age and concentrations of the more chlorinated polychlorobiphenyls (PCBs). This is consistent with previously observed correlations of these PCBs with fish age. In addition, most fish with high estrogenic activity were found in lakes containing high OC levels in the sediments, which gives further ground to atmospheric deposition of anthropogenic pollutants as main process leading to the observed endocrine disruption effects.Estrogen receptor agonists in lake fish are correlated with organochlorine compound content.I.V. is a recipient of a fellowship from Generalitat de Catalunya. This work has been supported by Ministerio de Ciencia y Tecnología, Spain (BMC2001-0246 and SAF2002-00371) and the EU programs (EMERGE, EVK1-CT-1999-00032 and EUROLIMPACS, GOCE-CT-2003-505540).Peer reviewe

Targeted Gene Expression in Zebrafish Exposed to Chlorpyrifos-Oxon Confirms Phenotype-Specific Mechanisms Leading to Adverse Outcomes

Zebrafish models for mild, moderate, and severe acute organophosphorus poisoning were previously developed by exposing zebrafish larvae to chlopyrifos-oxon. The phenotype of these models was characterized at several levels of biological organization. Oxidative stress and mitochondrial dysfunction were found to be involved in the development of the more severe phenotype. Here we used targeted gene expression to understand the dose-responsiveness of those two pathways and their involvement on generating the different zebrafish models. As the severe phenotype is irreversible after only 3 h of exposure, we also analyzed the response of the oxidative stress pathway at 3 and 24 h. Some of the genes related to oxidative stress were already differentially expressed at 3 h. There was an increase in differentially expressed genes related to both oxidative stress and mitochondrial function from the more mild to the more severe phenotype, suggesting the involvement of these mechanisms in increasing phenotype severity. Temporal data suggest that peroxynitrite leading to lipid peroxidation might be involved in phenotype transition and irreversibility. © 2016, The Author(s).This work was supported in part by the US Army ERDC-IRO (W912HZ-13-BAA-01; D.R., N.G.R.) and Environmental Quality Research Program (N.G.R.), the NATO SfP project MD.SFPP 984777 (D.R., N.G.R.), and the National Science Foundation EPSCoR Grant EPS-0903787 (N.G.R.).Peer reviewe

Multi-omic Analysis of Zebrafish Models of Acute Organophosphorus Poisoning With Different Severity

Organophosphorus compounds are acetylcholinesterase inhibitors used as pesticides and chemical warfare nerve agents. Acute organophosphorus poisoning (acute OPP) affects 3 million people, with 300 000 deaths annually worldwide. Severe acute OPP effects include overstimulation of cholinergic neurons, seizures, status epilepticus, and finally, brain damage. In a previous study, we developed 3 different chemical models of acute OPP in zebrafish larvae. To elucidate the complex pathophysiological pathways related to acute OPP, we used integrative omics (proteomic, transcriptomics, and metabolomics) on these 3 animal models. Our results show that these stochastic, apparently disparate morphological phenotypes can result from almost linear concentration-response variations in molecular levels. Results from the multiomics analysis strongly suggest that endoplasmic reticulum stress might play a central role in the pathophysiology of severe acute OPP, emphasizing the urgent need of further research on this molecular pathway. Endoplasmic reticulum stress could be an important therapeutic target to be included in the treatment of patients with severe acute OPP.NATO SfP project MD.SFPP 984777 (D.R.); the European Research Council under European Union’s Seven Framework Programme (FP/2007–2013)/ERC Grant Agreement No. 320737; the Spanish Government (CTM2017-83242-R and CTM2015-65691-R). M.F. acknowledges the financial support from the Government of Catalonia through a Beatriu de Pinos fellowship (2016 BP-B 00233)Peer reviewe

Use of vitellogenin mRNA as a biomarker for endocrine disruption in feral and cultured fish

6 pages, 3 figures, 2 tables.-- PMID: 14618290 [PubMed].-- Available online Nov 14, 2003.The presence of the female-specific yolk protein precursor vitellogenin in blood and liver from male fish is widely used as an indicator of endocrine disruption. We studied the induction of vitellogenin mRNA in liver from several species of fish, both maintained in fish tanks or captured in the wild. Our procedure requires minute amounts of liver samples (down to 50 mg), and can be applied to field samples if the appropriate RNA-stabilisation agent is used. We used reverse-transcriptase PCR and quantitative real-time PCR for detection and precise quantitation of vitellogenin mRNA levels. Male mummichog (Fundulus heteroclitus) exposed to 17beta-estradiol contained levels of vitellogenin mRNA up to 30 times higher than in untreated females and treatment with nonylphenol resulted in a weak but consistent induction of this transcript. We also studied levels of vitellogenin mRNA in a population of common carp (Cyprinus carpio) from the Anoia river, a river known for its high levels of estrogenic alkylphenols. The results were consistent with recorded data for fish from this sampling site. Finally, we also detected vitellogenin mRNA in barbs (Barbus graellsi), a species for which no vitellogenin sequence was available. The use of mRNA quantitation techniques for analysis of feral and cultured fish of different species opens the possibility of more precise detection and further control of the noxious effects of contaminants on the local fauna exposed to them.This work has been supported by Ministerio de Ciencia y Tecnología (Spain) (PPQ2001–1805-CO3–01, GEN2001–4707-C08–08, and BMC2001–0246) and by the EU Environment and Climate program through the Waste Water Cluster project EMERGE (EVK1-CT-1999–00032), and P-THREE (EVK1-CT-2002–00116).Peer reviewe

Estrogenic potential of halogenated derivatives of nonylphenol ethoxylates and carboxylates

7 páginas, 6 figuras, 3 tablas.Halogenated derivatives of nonylphenol and of its alkylates are generated during drinking water disinfection and treatment procedures. In this paper we analyze the potential of these compounds to interact with the estrogen receptor and to activate hormone-regulated gene promoters. We used the recombinant yeast assay (RYA) and the human breast cancer cell MCF7 proliferation assay for both estrogenic and antiestrogenic activities and the enzyme-linked receptor assay to examine in vitro binding to the receptor. Many nonylphenol derivatives were very weak estrogens in our functional tests when compared to nonylphenol while retaining a substantial affinity for the estrogen receptor in vitro. Antiestrogenicity tests demonstrated that brominated nonylphenol and most of the carboxylated compounds studied here behaved as estrogenic antagonists in the RYA. We also detected an increased cytotoxicity for the carboxylated derivatives in both yeast and mammalian cells. We conclude that derivatization may mask the apparent estrogenicity of nonylphenol, but the resulting compounds still represent a potential hazard since they are still able to bind the estrogen receptor and to influence the physiological response to estrogens. Our results also illustrate the advantage of combining different methods to assay estrogenicity of unknown substances.This work has been supported by the Ministerio de Ciencia y Tecnología (Spain) (PPQ2001-1805-CO3-01, PPQ2002-10945-E, and BMC2001-0246) and by the European Union Environment and Climate Program through the Waste Water Cluster Project SANDRINE (ENV4-CT98-0801), EMERGE (EVK1-CT-1999-00032), and P-THREE (EVK1-CT-2002-00116).Peer reviewe