Toxicity of Dithiothreitol (DTT) to Drosophila melanogaster
ABSTRACT: Graphical abstract Highlights • Contact to Dithiothreitol (DTT), a widely used antioxidant, is lethal to the fruit fly Drosophila melanogaster.• At low concentrations, DTT contact induces the expression of some apoptosis genes.• In parallel, DTT triggers the expression of a cuticle barrier gene.• Expression of some genes involved in detoxification is intensified with increasing DTT concentrations.• Toxicity of DTT to the exnvironment should be tested. The thiol-containing compound Dithiothreitol (DTT) has been shown to be toxic to cultured cells by inducing the generation of reactive oxygen species that ultimately cause cell death. However, its effects on multicellular organisms and the environment have not been investigated yet in detail. In this work, we tested the toxicity of DTT to the model insect Drosophila melanogaster. We show that DTT is lethal to D. melanogaster by topical application but not through feeding. DTT treatment triggers the transcription of the canonical apoptosis regulators grim, hid and rpr at low amounts. The amplitude of this induction declines with elevating DTT amounts. By live microscopy, we observe apoptotic cells especially in the gut of DTT treated flies. In parallel, low DTT amounts also activate the expression of the cuticle barrier component gene snsl. This indicates that a physical defence response is launched upon DTT contact. This combined measure is seemingly successful in preventing fly death. The expression of a number of known detoxification genes including cyp6a2, cyp6a8, cyp12d1 and GstD2 is also enhanced through DTT contact. The degree of upregulation of these genes is proportional to the applied DTT amounts. Despite this effort, flies exposed to high amounts of DTT eventually die. Together, D. melanogaster is able to sense DTT toxicity and adjust the defence response successfully at least at low concentrations. This is the first time to analyse the molecular consequences of DTT exposure in a multicellular organism. Our work provides a new model to discuss the physiological response of animals against thiol toxins and to resurvey the effect of redox agents on the environment.
Project description:Cuticle barrier efficiency in insects depends largely on cuticular lipids. To learn about the evolution of cuticle barrier function, we compared the basic properties of the cuticle inward and outward barrier function in adults of the fruit flies Drosophila suzukii and Drosophila melanogaster that live on fruits sharing a similar habitat. At low air humidity, D. suzukii flies desiccate faster than D. melanogaster flies. We observed a general trend indicating that in this respect males are less robust than females in both species. Xenobiotics penetration occurs at lower temperatures in D. suzukii than in D. melanogaster. Likewise, D. suzukii flies are more susceptible to contact insecticides than D. melanogaster flies. Thus, both the inward and outward barriers of D. suzukii are less efficient. Consistently, D. suzukii flies have less cuticular hydrocarbons (CHC) that participate as key components of the cuticle barrier. Especially, the relative amounts of branched and desaturated CHCs, known to enhance desiccation resistance, show reduced levels in D. suzukii. Moreover, the expression of snustorr (snu) that encodes an ABC transporter involved in barrier construction and CHC externalization, is strongly suppressed in D. suzukii. Hence, species-specific genetic programs regulate the quality of the lipid-based cuticle barrier in these two Drosophilae. Together, we conclude that the weaker inward and outward barriers of D. suzukii may be partly explained by differences in CHC composition and by a reduced Snu-dependent transport rate of CHCs to the surface. In turn, this suggests that snu is an ecologically adjustable and therefore relevant gene in cuticle barrier efficiency.
Project description:The interactions between entomopathogenic fungi and insects serve a classic example of a co-evolutionary arms race between pathogens and their target host. The cuticle, site of the first contact between insects and entomopathogenic fungus, is an important defensive barrier against pathogens. It is covered by a layer of lipids that appears to play a key role in these processes and cuticular free fatty acid (FFA) profiles are consider as a determinant of susceptibility, or resistance, to fungal infections. These profiles are species-specific. The cockroaches Blattella germanica (Blattodea: Blattidae) and Blatta orientalis (Blattodea: Ectobiidae) are unsusceptible to the soil fungus Conidiobolus coronatus (Entomophthorales: Ancylistaceae) infection, therefore we studied the profiles of FFAs in order to understand the defensive capabilities of the cockroaches. The fungus was cultivated for three weeks in minimal medium. Cell-free filtrate was obtained, assayed for elastase, N-acetylglucosaminidase, chitobiosidase and lipase activity, and then used for in vitro hydrolysis of the cuticle from wings and thoraces of adults and oothecae. The amounts of amino acids, N-glucosamine and FFAs released from the hydrolysed cuticle samples were measured after eight hours of incubation. The FFA profiles of the cuticle of adults, and the wings, thoraces and oothecae of both species were established using GC-MS and the results were correlated with the effectiveness of fungal proteases, chitinases and lipases in the hydrolyzation of cuticle samples. Positive correlations would suggest the existence of compounds used by the fungus as nutrients, whereas negative correlations may indicate that these compounds could be engaged in insect defence.
Project description:Discrimination between self and non-self is a prerequisite for any defence mechanism; in innate defence, this discrimination is often mediated by lectins recognizing non-self carbohydrate structures and so relies on an arsenal of host lectins with different specificities towards target organism carbohydrate structures. Recently, cytoplasmic lectins isolated from fungal fruiting bodies have been shown to play a role in the defence of multicellular fungi against predators and parasites. Here, we present a novel fruiting body lectin, CCL2, from the ink cap mushroom Coprinopsis cinerea. We demonstrate the toxicity of the lectin towards Caenorhabditis elegans and Drosophila melanogaster and present its NMR solution structure in complex with the trisaccharide, GlcNAc?1,4[Fuc?1,3]GlcNAc, to which it binds with high specificity and affinity in vitro. The structure reveals that the monomeric CCL2 adopts a ?-trefoil fold and recognizes the trisaccharide by a single, topologically novel carbohydrate-binding site. Site-directed mutagenesis of CCL2 and identification of C. elegans mutants resistant to this lectin show that its nematotoxicity is mediated by binding to ?1,3-fucosylated N-glycan core structures of nematode glycoproteins; feeding with fluorescently labeled CCL2 demonstrates that these target glycoproteins localize to the C. elegans intestine. Since the identified glycoepitope is characteristic for invertebrates but absent from fungi, our data show that the defence function of fruiting body lectins is based on the specific recognition of non-self carbohydrate structures. The trisaccharide specifically recognized by CCL2 is a key carbohydrate determinant of pollen and insect venom allergens implying this particular glycoepitope is targeted by both fungal defence and mammalian immune systems. In summary, our results demonstrate how the plasticity of a common protein fold can contribute to the recognition and control of antagonists by an innate defence mechanism, whereby the monovalency of the lectin for its ligand implies a novel mechanism of lectin-mediated toxicity.
Project description:Protein-RNA cross-linking by UV irradiation at 254 nm wavelength has been established as an unbiased method to identify proteins in direct contact with RNA, and has been successfully applied to investigate the spatial arrangement of protein and RNA in large macromolecular assemblies, e.g. ribonucleoprotein-complex particles (RNPs). The mass spectrometric analysis of such peptide-RNA cross-links provides high resolution structural data to the point of mapping protein-RNA interactions to specific peptides or even amino acids. However, the approach suffers from the low yield of cross-linking products, which can be addressed by improving enrichment and analysis methods. In the present article, we introduce dithiothreitol (DTT) as a potent protein-RNA cross-linker. In order to evaluate the efficiency and specificity of DTT, we used two systems, a small synthetic peptide from smB protein incubated with U1 snRNA oligonucleotide and native ribonucleoprotein complexes from S. cerevisiae. Our results unambiguously show that DTT covalently participates in cysteine-uracil crosslinks, which is observable as a mass increment of 151.9966 Da (C(4)H(8)S(2)O(2)) upon mass spectrometric analysis. DTT presents advantages for cross-linking of cysteine containing regions of proteins. This is evidenced by comparison to experiments where (tris(2-carboxyethyl)phosphine) is used as reducing agent, and significantly less cross-links encompassing cysteine residues are found. We further propose insertion of DTT between the cysteine and uracil reactive sites as the most probable structure of the cross-linking products.
Project description:Particulate matter (PM) air pollution has a significant impact on human morbidity and mortality; however, the mechanisms of PM-induced toxicity are poorly defined. A leading hypothesis states that airborne PM induces harm by generating reactive oxygen species in and around human tissues, leading to oxidative stress. We report here a system employing a microfluidic electrochemical sensor coupled directly to a particle-into-liquid sampler (PILS) system to measure aerosol oxidative activity in an on-line format. The oxidative activity measurement is based on the dithiothreitol (DTT) assay, where, after being oxidized by PM, the remaining reduced DTT is analyzed by the microfluidic sensor. The sensor consists of an array of working, reference, and auxiliary electrodes fabricated in a poly(dimethylsiloxane)-based microfluidic device. Cobalt(II) phthalocyanine-modified carbon paste was used as the working electrode material, allowing selective detection of reduced DTT. The electrochemical sensor was validated off-line against the traditional DTT assay using filter samples taken from urban environments and biomass burning events. After off-line characterization, the sensor was coupled to a PILS to enable on-line sampling/analysis of aerosol oxidative activity. Urban dust and industrial incinerator ash samples were aerosolized in an aerosol chamber and analyzed for their oxidative activity. The on-line sensor reported DTT consumption rates (oxidative activity) in good correlation with aerosol concentration (R(2) from 0.86 to 0.97) with a time resolution of approximately 3 min.
Project description:Transcriptional profiling of ER stress condition comparing control (untreated), dithiothreitol (DTT) treated, or tunicamycin (TM) treated conditions on P. angusta DL1 strain. Six-condition experiment: normal condition vs. ER stress condition (TM 30, 60, 120min; DTT 30, 60, 120min) in P. angusta DL1 strain. Independently grown and harvested. Two replicates per array.
Project description:For fifty years, dithiothreitol (DTT) has been the preferred reagent for the reduction of disulfide bonds in proteins and other biomolecules. Herein we report on the synthesis and characterization of 2,3-bis(mercaptomethyl)pyrazine (BMMP), a readily accessible disulfide-reducing agent with reactivity under biological conditions that is markedly superior to DTT and other known reagents.
Project description:This study examines the associations between the oxidative potential of ambient PM2.5 and PM0.18, measured by means of the dithiothreitol (DTT) assay, and their chemical constituents and modeled sources. Particulate matter (PM) samples were collected from 2012-2013 in Central Los Angeles (LA) and 2013-2014 in Anaheim, California, USA. Detailed chemical analyses of the PM samples, including carbonaceous species, inorganic elements and water-soluble ions, were conducted. Univariate analysis indicated a high correlation (R > 0.60) between the DTT activity and the concentrations of carbonaceous species at both sites. The strongest correlations were observed between DTT and organic tracers of primary vehicle tailpipe emissions including polycyclic aromatic hydrocarbons (PAHs) and hopanes as well as EC, with higher correlations for PM0.18versus PM2.5 components. Moreover, metals and trace elements (e.g., Ba, Cu, Fe, Mn, Pb and Sb) in both size ranges were also associated with DTT activity. Multiple linear regression (MLR) analysis was performed on DTT activity and PM sources identified by a Molecular Marker-Chemical Mass Balance (MM-CMB) model (i.e. major carbonaceous sources: vehicle tailpipe emissions, wood smoke, primary biogenic and secondary organic carbon) together with other typical sources of ambient PM (i.e. crustal material, vehicular abrasion, secondary ions and sea salt). Overall, our findings illustrate the relative importance of different traffic sources on the oxidative potential of ambient PM. Despite major reductions of tailpipe emissions, the lack of similar reductions (and possibly an increase) in non-tailpipe emissions makes them an important source of traffic-related PM in Los Angeles and their increasing role in the overall PM toxicity raises concerns for public health.
Project description:We induced ER stress in Jurkat T-cells with dithiothreitol (DTT), which reduces the protein disulfide bonds and causes the accumulation of misfolded proteins in the ER lumen. For assessment of DTT effects on cells, we performed Affymetrix whole transcriptome gene expression analysis. The cells were treated for 6 hours with 2.5 mM Dithiothreitol (Sigma-Aldrich). Total RNA was isolated from DTT-treated (2 replicates) and control (2 replicates) Jurkat cells and hybridized to a gene expression arrays (GeneChip Gene 1.0 ST Array System; Affymetrix, Santa Clara, CA).
Project description:Dithiothreitol (DTT) is the standard reagent for reducing disulfide bonds between and within biological molecules. At neutral pH, however, >99% of DTT thiol groups are protonated and thus unreactive. Herein, we report on (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), a dithiol that can be synthesized from l-aspartic acid in a few high-yielding steps that are amenable to a large-scale process. DTBA has thiol pK(a) values that are ~1 unit lower than those of DTT and forms a disulfide with a similar E°' value. DTBA reduces disulfide bonds in both small molecules and proteins faster than does DTT. The amino group of DTBA enables its isolation by cation-exchange and facilitates its conjugation. These attributes indicate that DTBA is a superior reagent for reducing disulfide bonds in aqueous solution.