RESUMEN
Neuropeptides constitute a vast and functionally diverse family of neurochemical signaling molecules and are widely involved in the regulation of various physiological processes. The nematode Caenorhabditis elegans is well-suited for the study of neuropeptide biochemistry and function, as neuropeptide biosynthesis enzymes are not essential for C. elegans viability. This permits the study of neuropeptide biosynthesis in mutants lacking certain neuropeptide-processing enzymes. Mass spectrometry has been used to study the effects of proprotein convertase and carboxypeptidase mutations on proteolytic processing of neuropeptide precursors and on the peptidome in C. elegans However, the enzymes required for the last step in the production of many bioactive peptides, the carboxyl-terminal amidation reaction, have not been characterized in this manner. Here, we describe three genes that encode homologs of neuropeptide amidation enzymes in C. elegans and used tandem LC-MS to compare neuropeptides in WT animals with those in newly generated mutants for these putative amidation enzymes. We report that mutants lacking both a functional peptidylglycine α-hydroxylating monooxygenase and a peptidylglycine α-amidating monooxygenase had a severely altered neuropeptide profile and also a decreased number of offspring. Interestingly, single mutants of the amidation enzymes still expressed some fully processed amidated neuropeptides, indicating the existence of a redundant amidation mechanism in C. elegans All MS data are available via ProteomeXchange with the identifier PXD008942. In summary, the key steps in neuropeptide processing in C. elegans seem to be executed by redundant enzymes, and loss of these enzymes severely affects brood size, supporting the need of amidated peptides for C. elegans reproduction.
Asunto(s)
Amidina-Liasas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Oxigenasas de Función Mixta/metabolismo , Complejos Multienzimáticos/metabolismo , Neuropéptidos/metabolismo , Amidina-Liasas/química , Amidina-Liasas/genética , Secuencia de Aminoácidos , Animales , Vías Biosintéticas , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Cobre/metabolismo , Eliminación de Gen , Humanos , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mutación , Neuropéptidos/genética , Alineación de Secuencia , Espectrometría de Masas en TándemRESUMEN
Using the well-documented model organism Caenorhabditis elegans, a combined analysis of metal speciation in the exposure medium and body burdens of metals (Zn, Cu and Cd) was performed, and factors that are predictive of toxicological endpoints in single metal and mixed metal exposures were identified. Cu, and to a lesser extent Cd, is found to associate with Escherichia coli in the exposure medium (the food source for C. elegans) as evidenced by the observed decrease in both their dissolved and free metal ion concentrations. Together with a critical analysis of literature data, our results suggest that free metal ion concentrations and thus aqueous uptake routes are the best predictor of internal concentrations under all conditions considered, and of metal toxicity in single metal exposures. Additional factors are involved in determining the toxicity of metal mixtures. In general, the eventual adverse effects of metals on biota are expected to be a consequence of the interplay between chemical speciation in the exposure medium, timescale of exposure, exposure route as well as the nature and timescale of the biotic handling pathways.
Asunto(s)
Bioacumulación , Cadmio/toxicidad , Caenorhabditis elegans/efectos de los fármacos , Cobre/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Carga Corporal (Radioterapia) , Cadmio/metabolismo , Caenorhabditis elegans/metabolismo , Cobre/metabolismo , Exposición Dietética/efectos adversos , Escherichia coli/metabolismo , Contaminantes Químicos del Agua/metabolismoRESUMEN
Tris(2-chloroethyl) phosphate (TCEP) is a pervasive flame retardant that has been identified as a chemical of concern given its health effects and therefore its use has since been tightly regulated. Tris(2-chloroisopropyl) phosphate (TCIPP), an analogue of TCEP, is believed to be its replacement. However, compared to TCEP, little is known of the toxicological impacts of TCIPP. We used RNA sequencing as unbiased and sensitive tool to identify and compare effects on a transcriptome level of TCEP and TCIPP in the human hepatocellular carcinoma cell line, HepG2. We identified that compared to other flame retardants, TCEP and TCIPP had little cytotoxicity. Treatment with sub-cytotoxic concentrations of the two compounds revealed that both chemicals elicited similar effects; both compounds were found to affect genes involved in immune responses and steroid hormone biosynthesis, while also affecting xenobiotic metabolism pathways in a similar manner. Specifically for effects on immune responses, both compounds were shown to alter the expression of the receptor of the potent and pleiotropic complement component, C5a. Additionally, expression of genes encoding for effector proteins involved in the complement cascade along with other potent inflammatory regulators were found altered in response to TCEP and TCIPP, further emphasizing their potential effects on immune function. Taken together, given that TCIPP elicited similar effects compared to TCEP, and at lower concentrations, the potential health effects of TCIPP need to be further studied for a complete risk assessment of the compound.
Asunto(s)
Retardadores de Llama/toxicidad , Organofosfatos/toxicidad , Citotoxinas/toxicidad , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Células Hep G2/efectos de los fármacos , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARN , Transcriptoma/efectos de los fármacosRESUMEN
The free-living nematode Caenorhabditis elegans is one of the most studied models in a wide variety of research fields with applications in agro- or pharmaceutical industries. It has been used for the development of new anthelminthic drugs and was proven to yield key insights in neurodegenerative diseases and metabolic syndromes. Due to its suitability for high-throughput genetic screens, efficiency for RNA interference approaches and the availability of thousands of mutants, most studies were carried out at the genetic level. However, determining the cellular function of each gene product remains an unfinished goal in this post-genomic era. A systems biology approach focusing on the actual gene products (i.e. proteins) can help unraveling this puzzle. A fundamental pillar in this research is mass spectrometry-based proteomics. We here provide an in-depth overview of proteomics-related studies in C. elegans research, with special emphasis on the methodologies and biological applications.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Perfilación de la Expresión Génica/métodos , Proteoma/metabolismo , Proteómica/métodos , Transducción de Señal/fisiología , AnimalesRESUMEN
Polymodal nociceptors sense and integrate information on injurious mechanical, thermal, and chemical stimuli. Chemical signals either activate nociceptors or modulate their responses to other stimuli. One chemical known to activate or modulate responses of nociceptors is acetylcholine (ACh). Across evolution nociceptors express subunits of the nicotinic acetylcholine receptor (nAChR) family, a family of ACh-gated ion channels. The roles of ACh and nAChRs in nociceptor function are, however, poorly understood. Caenorhabditis elegans polymodal nociceptors, PVD, express nAChR subunits on their sensory arbor. Here we show that mutations reducing ACh synthesis and mutations in nAChR subunits lead to defects in PVD function and morphology. A likely cause for these defects is a reduction in cytosolic calcium measured in ACh and nAChR mutants. Indeed, overexpression of a calcium pump in PVD mimics defects in PVD function and morphology found in nAChR mutants. Our results demonstrate, for the first time, a central role for nAChRs and ACh in nociceptor function and suggest that calcium permeating via nAChRs facilitates activity of several signaling pathways within this neuron.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Nocicepción , Receptores Nicotínicos/metabolismo , Acetilcolina/metabolismo , Animales , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Mutación , Nociceptores/metabolismo , Receptores Nicotínicos/genéticaRESUMEN
The ability to optically excite or silence specific cells using optogenetics has become a powerful tool to interrogate the nervous system. Optogenetic experiments in small organisms have mostly been performed using whole-field illumination and genetic targeting, but these strategies do not always provide adequate cellular specificity. Targeted illumination can be a valuable alternative but it has only been shown in motionless animals without the ability to observe behavior output. We present a real-time, multimodal illumination technology that allows both tracking and recording the behavior of freely moving C. elegans while stimulating specific cells that express channelrhodopsin-2 or MAC. We used this system to optically manipulate nodes in the C. elegans touch circuit and study the roles of sensory and command neurons and the ultimate behavioral output. This technology enhances our ability to control, alter, observe and investigate how neurons, muscles and circuits ultimately produce behavior in animals using optogenetics.
Asunto(s)
Conducta Animal , Caenorhabditis elegans/fisiología , Músculos/fisiología , Neuronas/fisiología , Fenómenos Ópticos , Fotobiología/métodos , Percepción Visual , Animales , Factores de TiempoRESUMEN
The emerging field of optogenetics allows for optical activation or inhibition of excitable cells. In 2005, optogenetic proteins were expressed in the nematode Caenorhabditis elegans for the first time. Since then, C. elegans has served as a powerful platform upon which to conduct optogenetic investigations of synaptic function, circuit dynamics and the neuronal basis of behaviour. The C. elegans nervous system, consisting of 302 neurons, whose connectivity and morphology has been mapped completely, drives a rich repertoire of behaviours that are quantifiable by video microscopy. This model organism's compact nervous system, quantifiable behaviour, genetic tractability and optical accessibility make it especially amenable to optogenetic interrogation. Channelrhodopsin-2 (ChR2), halorhodopsin (NpHR/Halo) and other common optogenetic proteins have all been expressed in C. elegans. Moreover, recent advances leveraging molecular genetics and patterned light illumination have now made it possible to target photoactivation and inhibition to single cells and to do so in worms as they behave freely. Here, we describe techniques and methods for optogenetic manipulation in C. elegans. We review recent work using optogenetics and C. elegans for neuroscience investigations at the level of synapses, circuits and behaviour.
Asunto(s)
Caenorhabditis elegans/genética , Neuronas/fisiología , Optogenética/métodos , Sinapsis/genética , Animales , Caenorhabditis elegans/fisiología , Caenorhabditis elegans/efectos de la radiación , Femenino , Luz , Masculino , Neuronas/efectos de la radiación , Sinapsis/fisiología , Sinapsis/efectos de la radiaciónRESUMEN
In mammals, hypothalamic gonadotropin-releasing hormone (GnRH) is a neuropeptide that stimulates the release of gonadotropins from the anterior pituitary. The existence of a putative functional equivalent of this reproduction axis in protostomian invertebrates has been a matter of debate. In this study, the ligand for the GnRH receptor in the nematode Caenorhabditis elegans (Ce-GnRHR) was found using a bioinformatics approach. The peptide and its precursor are reminiscent of both insect adipokinetic hormones and GnRH-preprohormone precursors from tunicates and higher vertebrates. We cloned the AKH-GnRH-like preprohormone and the Ce-GnRHR and expressed the GPCR in HEK293T cells. The GnRHR was activated by the C. elegans AKH-GnRH-like peptide (EC(50) = 150 nM) and by Drosophila AKH and other nematode AKH-GnRHs that we found in EST databases. Analogous to both insect AKH receptor and vertebrate GnRH receptor signaling, Ce-AKH-GnRH activated its receptor through a Galpha(q) protein with Ca(2+) as a second messenger. Gene silencing of Ce-GnRHR, Ce-AKH-GnRH, or both resulted in a delay in the egg-laying process, comparable to a delay in puberty in mammals lacking a normal dose of GnRH peptide or with a mutated GnRH precursor or receptor gene. The present data support the view that the AKH-GnRH signaling system probably arose very early in metazoan evolution and that its role in reproduction might have been developed before the divergence of protostomians and deuterostomians.
Asunto(s)
Caenorhabditis elegans/fisiología , Hormonas de Insectos/metabolismo , Oligopéptidos/metabolismo , Óvulo , Ácido Pirrolidona Carboxílico/análogos & derivados , Receptores LHRH/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/metabolismo , Metabolismo de los Hidratos de Carbono , Línea Celular , Clonación Molecular , Cartilla de ADN , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Hormonas de Insectos/química , Hormonas de Insectos/genética , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/genética , Ácido Pirrolidona Carboxílico/química , Ácido Pirrolidona Carboxílico/metabolismo , Receptores LHRH/química , Receptores LHRH/genética , Homología de Secuencia de AminoácidoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0200080.].
RESUMEN
Neuropeptides are small messenger molecules that can be found in all metazoans, where they govern a diverse array of physiological processes. Because neuropeptides seem to be conserved among pest species, selected peptides can be considered as attractive targets for drug discovery. Much can be learned from the model system Caenorhabditis elegans because of the availability of a sequenced genome and state-of-the-art postgenomic technologies that enable characterization of endogenous peptides derived from neuropeptide-like protein (NLP) precursors. Here, we provide an overview of the NLP peptide family in C. elegans and discuss their resemblance with arthropod neuropeptides and their relevance for anthelmintic discovery.
Asunto(s)
Artrópodos/genética , Caenorhabditis elegans/genética , Neuropéptidos/genética , Secuencia de Aminoácidos , Animales , Antihelmínticos/uso terapéutico , Secuencia Conservada , Descubrimiento de Drogas , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
The neuropeptides pigment dispersing factor (PDF) and vasoactive intestinal peptide (VIP) are known as key players in the circadian clock system of insects and mammals, respectively. In this study, we report the discovery and characterization of a widely conserved PDF-like neuropeptide precursor pathway in nematodes. Using a combinatorial approach of biochemistry and peptidomics, we have biochemically isolated, identified and characterized three PDF-like neuropeptides in the free-living nematode Caenorhabditis elegans. The two PDF encoding genes, which were designated pdf-1 and pdf-2, display a very strong conservation within the phylum of nematodes. Many of the PDF expressing cells in C. elegans play a role in the control of locomotion and the integration of environmental stimuli, among which light. Our real-time PCR analysis indicates that both PDF genes are consistently expressed during the day and do not affect each other's expression. The transcription of both PDF genes seems to be regulated by atf-2 and ces-2, which encode bZIP transcription factors homologous to Drosophila vrille and par domain protein 1 (Pdp1epsilon), respectively. Together, our data suggest that the PDF neuropeptide pathway, which seems to be conserved throughout the protostomian evolutionary lineage, might be more complex than previously assumed.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Neuropéptidos/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Clonación Molecular , Secuencia Conservada , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Datos de Secuencia Molecular , Alineación de Secuencia , Transducción de Señal/fisiología , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Neuromedin U (NMU) in vertebrates is a structurally highly conserved neuropeptide of which highest levels are found in the pituitary and gastrointestinal tract. In Drosophila, two neuropeptide genes encoding pyrokinins (PKs), capability (capa) and hugin, are possible insect homologs of vertebrate NMU. Here, the ligand for an orphan G protein-coupled receptor in the nematode Caenorhabditis elegans (Ce-PK-R) was found using a bioinformatics approach. After cloning and expressing Ce-PK-R in HEK293T cells, we found that it was activated by a neuropeptide from the C. elegans NLP-44 precursor (EC(50)=18nM). This neuropeptide precursor is reminiscent of insect CAPA precursors since it encodes a PK-like peptide and two periviscerokinin-like peptides (PVKs). Analogous to CAPA peptides in insects and NMUs in vertebrates, whole mount immunostaining in C. elegans revealed that the CAPA precursor is expressed in the nervous system. The present data also suggest that the ancestral CAPA precursor was already present in the common ancestor of Protostomians and Deuterostomians and that it might have been duplicated into CAPA and HUGIN in insects. In vertebrates, NMU is the putative homolog of a protostomian CAPA-PK.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Neuropéptidos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/agonistas , Proteínas de Caenorhabditis elegans/genética , Línea Celular , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Transducción de SeñalRESUMEN
Neuropeptides are important signaling molecules that function in cell-cell communication as neurotransmitters or hormones to orchestrate a wide variety of physiological conditions and behaviors. These endogenous peptides can be monitored by high throughput peptidomics technologies from virtually any tissue or organism. The neuropeptide complement of the soil nematode Caenorhabditis elegans has been characterized by on-line two-dimensional liquid chromatography and quadrupole time-of-flight tandem mass spectrometry (2D-nanoLC Q-TOF MS/MS). Here, we use an alternative peptidomics approach combining liquid chromatography (LC) with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to map the peptide content of C. elegans and another Caenorhabditis species, Caenorhabditis briggsae. This study allows a better annotation of neuropeptide-encoding genes from the C. briggsae genome and provides a promising basis for further evolutionary comparisons.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis/genética , Neuropéptidos/genética , Proteómica , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Neuropéptidos/análisis , Precursores de Proteínas/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The nematode Caenorhabditis elegans joins the menagerie of behavioral model systems next to the fruit fly Drosophila melanogaster, the marine snail Aplysia californica and the mouse. In contrast to Aplysia, which contains 20,000 neurons having cell bodies of hundreds of microns in diameter, C. elegans harbors only 302 tiny neurons from which the cell lineage is completely described, as is the case for all the other somatic cells. As such, this nervous system appears at first sight incommensurable with those of higher organisms, although genome-wide comparison of predicted C. elegans genes with their counterparts in vertebrates revealed many parallels. Together with its short lifespan and ease of cultivation, suitability for high-throughput genetic screenings and genome-wide RNA interference approaches, access to an advanced genetic toolkit and cell-ablation techniques, it seems that this tiny transparent organism of only 1mm in length has nothing to hide. Recently, highly exciting developments have occurred within the field of neuropeptidergic signaling in C. elegans, not only because of the availability of a sequenced genome since 1998, but especially because of state of the art post genomic technologies, that allow for molecular characterization of the signaling molecules. Here, we will focus on endogenous, bioactive (neuro)peptides and mainly discuss biosynthesis, peptide sequence information, localization and G-protein coupled receptors of the three major peptide families in C. elegans.
Asunto(s)
Caenorhabditis elegans/fisiología , Neuropéptidos/fisiología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Expresión Génica , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Members of the cholecystokinin (CCK)/gastrin family of peptides, including the arthropod sulfakinins, and their cognate receptors, play an important role in the regulation of feeding behavior and energy homeostasis. Despite many efforts after the discovery of CCK/gastrin immunoreactivity in nematodes 23 yr ago, the identity of these nematode CCK/gastrin-related peptides has remained a mystery ever since. The Caenorhabditis elegans genome contains two genes with high identity to the mammalian CCK receptors and their invertebrate counterparts, the sulfakinin receptors. By using the potential C. elegans CCK receptors as a fishing hook, we have isolated and identified two CCK-like neuropeptides encoded by neuropeptide-like protein-12 (nlp-12) as the endogenous ligands of these receptors. The neuropeptide-like protein-12 peptides have a very limited neuronal expression pattern, seem to occur in vivo in the unsulfated form, and react specifically with a human CCK-8 antibody. Both receptors and ligands share a high degree of structural similarity with their vertebrate and arthropod counterparts, and also display similar biological activities with respect to digestive enzyme secretion and fat storage. Our data indicate that the gastrin-CCK signaling system was already well established before the divergence of protostomes and deuterostomes.
Asunto(s)
Caenorhabditis elegans/fisiología , Colecistoquinina/fisiología , Gastrinas/fisiología , Transducción de Señal/fisiología , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Cromatografía Líquida de Alta Presión , Clonación Molecular/métodos , Sistemas de Lectura Abierta , Receptores CCR2/genética , Receptores de Colecistoquinina/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The toxicity effects of zinc (Zn), copper (Cu), and cadmium (Cd), both as single metals and in combination, were examined in the nematode Caenorhabditis elegans. Metal effects on lethality were analyzed in a time-dependent manner using different concentrations in K-medium. To investigate the effects on locomotion and chemosensation, lethal concentration at 20% (LC20) values were used. The results showed that Cu toxicity was higher compared with Cd and Zn, resulting in higher mortality rates and a more reduced locomotion. Lethality increased over time for all metals. When Cd was added to Cu, and vice versa, significant increases in toxicity were noted. Different interaction effects were observed for the mixtures ZnCd, ZnCu, CuCd, and ZnCuCd. Zinc seemed to have a neutral toxic effect on Cd, while in combination with Cu, a similar additive effect was seen as for the CuCd combination. Binary and tertiary metal mixtures caused a strong decrease in locomotion, except for the ZnCd combination, where Zn seemed to have a neutral effect. After LC2024 h exposure, reduced crawling speed (except for Zn) and reduced thrashing behavior (except for Zn and the ZnCd mixture) were observed. Almost no significant effects were observed on chemosensation. Because the same trend of mixture effects was noted in locomotion and in lethality tests, locomotion can probably be considered a sensitive endpoint for metal toxicities. Environ Toxicol Chem 2018;37:145-159. © 2017 SETAC.
Asunto(s)
Conducta Animal/efectos de los fármacos , Cadmio/toxicidad , Caenorhabditis elegans/efectos de los fármacos , Cobre/toxicidad , Zinc/toxicidad , Animales , Quimiotaxis/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Tiempo de Reacción , Análisis de Regresión , Sensación/efectos de los fármacosRESUMEN
A wide variety of bioactive peptides are present in all metazoan species where they govern diverse functions as small messenger molecules. In the last 15 years, mass spectrometry-based methods have identified endogenous peptides in diverse species. Mass spectrometry enables the precise peptide sequences to be determined, including the potential existence of truncated versions or the presence of post-translational modifications. Because small modifications can have a large effect on biological activity, knowledge of the actual peptide sequences paves the way for further functional studies such as analysis of neuropeptidergic signaling cascades. Zebrafish (Danio rerio) is an important animal model that is commonly used in a wide range of studies. Here we provide a detailed description of the peptide extraction procedure and peptidomics workflow for zebrafish.
Asunto(s)
Encéfalo/metabolismo , Espectrometría de Masas/métodos , Neuropéptidos/análisis , Proteómica/métodos , Pez Cebra/metabolismo , Animales , Neuropéptidos/aislamiento & purificación , Neuropéptidos/metabolismoRESUMEN
The nematode Caenorhabditis elegans lends itself as an excellent model organism for peptidomics studies. Its ease of cultivation and quick generation time make it suitable for high-throughput studies. Adult hermaphrodites contain 959 somatic nuclei that are ordered in defined, differentiated tissues. The nervous system, with its 302 neurons, is probably the most known and studied endocrine tissue. Moreover, its neuropeptidergic signaling pathways display a large number of similarities with those observed in other metazoans. However, various other tissues have also been shown to express several neuropeptides. This includes the hypodermis, gonad, gut, and even muscle. Hence, whole mount peptidomics of C. elegans cultures provides an integral overview of peptidergic signaling between the different tissues of the entire organism. Here, we describe a peptidomics approach used for the identification of endogenous (neuro)peptides in C. elegans. Starting from a detailed peptide extraction procedure, we will outline the setup for an online liquid chromatography-mass spectrometry (LC-MS) analysis and describe subsequent data analysis approaches.
Asunto(s)
Caenorhabditis elegans/metabolismo , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Neuropéptidos/análisis , Neuropéptidos/metabolismo , Proteómica/métodos , AnimalesRESUMEN
The flame retardant, 9,10-dihydro-9-oxa-10-phosphaphenanthrene 10-oxide (DOPO), has been receiving great interest given its superior fire protection properties, and its predicted low level of persistence, bioaccumulation, and toxicity. However, empirical toxicological data that are essential for a complete hazard assessment are severely lacking. In this study, we attempted to identify the potential toxicological modes of action by transcriptome (RNA-seq) profiling of the human liver hepatocellular carcinoma cell line, HepG2. Such insight may help in identifying compounds of concern and potential toxicological phenotypes. DOPO was found to have little cytotoxic potential, with lower effective concentrations compared to other flame retardants studied in the same cell line. Differentially expressed genes revealed a wide range of molecular effects including changes in protein, energy, DNA, and lipid metabolism, along with changes in cellular stress response pathways. In response to 250 µM DOPO, the most perturbed biological processes were fatty acid metabolism, androgen metabolism, glucose transport, and renal function and development, which is in agreement with other studies that observed similar effects of other flame retardants in other species. However, treatment with 2.5 µM DOPO resulted in very few differentially expressed genes and failed to indicate any potential effects on biology, despite such concentrations likely being orders of magnitude greater than would be encountered in the environment. This, together with the low levels of cytotoxicity, supports the potential replacement of the current flame retardants by DOPO, although further studies are needed to establish the nephrotoxicity and endocrine disruption of DOPO.
RESUMEN
Tris (2-butoxyethyl) phosphate (TBOEP) is a compound produced at high volume that is used as both a flame retardant and a plasticizer. It is persistent and bioaccumulative, yet little is known of its toxicological modes of action. Such insight may aid risk assessment in a weight-of-evidence approach supplementing current testing strategies. We used an RNA sequencing approach as an unbiased and sensitive tool to explore potential negative health effects of sub-cytotoxic concentrations of TBOEP on the transcriptome of the human liver hepatocellular carcinoma cell line, HepG2, with the lowest concentration used potentially holding relevance to human physiological levels. Over-representation and gene set enrichment analysis corresponded well and revealed that TBOEP treatments resulted in an upregulation of genes involved in protein and energy metabolism, along with DNA replication. Such increases in cell and macromolecule metabolism could explain the increase in mitochondrial activity at lower TBOEP concentrations. In addition, TBOEP affected a wide variety of biological processes, the most notable one being the general stress response, wound healing. Finally, TBOEP showed effects on steroid hormone biosynthesis and activation, regulation, and potentiation of immune responses, in agreement with other studies. As such, this study is the first study investigating genome-wide changes in gene transcription in response to TBOEP in human cells.