RESUMO
Three-dimensional quantitative phase imaging is an emerging method, which provides the 3D distribution of the refractive index (RI) and the dry mass in live and fixed cells as well as in tissues. However, an insufficiently answered question is the influence of chemical cell fixation procedures on the results of RI reconstructions. Therefore, this work is devoted to systematic investigations on the RI in cellular organelles of live and fixed cells including nucleus, nucleolus, nucleoplasm, and cytoplasm. The research was carried out on four different cell lines using a common paraformaldehyde (PFA)-based fixation protocol. The selected cell types represent the diversity of mammalian cells and therefore the results presented provide a picture of fixation caused RI changes in a broader context. A commercial Tomocube HT-1S device was used for 3D RI acquisition. The changes in the RI values after the fixation process are detected in the reconstructed phase distributions and amount to the order of 10-3 . The RI values decrease and the observed RI changes are found to be different between various cell lines; however, all of them show the most significant loss in the nucleolus. In conclusion, our study demonstrates the evident need for standardized preparation procedures in phase tomographic measurements. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.
Assuntos
Microscopia , Refratometria , Formaldeído , Polímeros , TomografiaRESUMO
The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their unique properties, can interfere with biochemical test methods, so multiple tests are required to fully assess their cellular effects. For a more reliable and comprehensive assessment, it is therefore imperative to include methods in nanoparticle testing routines that are not affected by particles and allow for the efficient integration of additional molecular techniques into the workflow. Digital holographic microscopy (DHM), an interferometric variant of quantitative phase imaging (QPI), has been demonstrated as a promising method for the label-free assessment of the cytotoxic potential of nanoparticles. Due to minimal interactions with the sample, DHM allows for further downstream analyses. In this study, we investigated the capabilities of DHM in a multimodal approach to assess cytotoxicity by directly comparing DHM-detected effects on the same cell population with two downstream biochemical assays. Therefore, the dry mass increase in RAW 264.7 macrophages and NIH-3T3 fibroblast populations measured by quantitative DHM phase contrast after incubation with poly(alkyl cyanoacrylate) nanoparticles for 24 h was compared to the cytotoxic control digitonin, and cell culture medium control. Viability was then determined using a metabolic activity assay (WST-8). Moreover, to determine cell death, supernatants were analyzed for the release of the enzyme lactate dehydrogenase (LDH assay). In a comparative analysis, in which the average half-maximal effective concentration (EC50) of the nanocarriers on the cells was determined, DHM was more sensitive to the effect of the nanoparticles on the used cell lines compared to the biochemical assays.
Assuntos
Nanopartículas , Animais , Camundongos , Células NIH 3T3 , Nanopartículas/toxicidade , Nanopartículas/química , Células RAW 264.7 , Sobrevivência Celular/efeitos dos fármacos , Holografia/métodos , Imageamento Quantitativo de FaseRESUMO
Reducing side effects in non-cancerous tissue is a key aim of modern radiotherapy. Here, we assessed whether the use of the antioxidants hydroxytyrosol (HT) and thioredoxin-mimetic peptide CB3 (TMP) attenuated radiation-induced normal tissue toxicity in vitro. We used primary human umbilical vein endothelial cells (HUVECs) and human epidermal keratinocytes (HaCaT) as normal tissue models. Cells were treated with HT and TMP 24 h or immediately prior to irradiation. Reactive oxygen species (ROS) were assessed via luminescent- and fluorescence-based assays, migration was investigated using digital holographic microscopy, and clonogenic survival was quantified by colony formation assays. Angiogenesis and wound healing were evaluated via time-dependent microscopy. Secreted cytokines were validated in quantitative polymerase chain reaction (qPCR) studies. Treatment with HT or TMP was well tolerated by cells. The application of either antioxidant before irradiation resulted in reduced ROS formation and a distinct decrease in cytokines compared to similarly irradiated, but otherwise untreated, controls. Antioxidant treatment also increased post-radiogenic migration and angiogenesis while accelerating wound healing. HT or TMP treatment immediately before radiotherapy increased clonogenic survival after radiotherapy, while treatment 24 h before radiotherapy enhanced baseline proliferation. Both antioxidants may decrease radiation-induced normal tissue toxicity and deserve further pre-clinical investigation.
RESUMO
BACKGROUND AND AIM: While preliminary evidence points to pro-tumorigenic roles for the Musashi (MSI) RNA-binding proteins Musashi-1 (MSI1) and Musashi-2 (MSI2) in some breast cancer subtypes, no data exist for inflammatory breast cancer (IBC). METHODS: MSI gene expression was quantified in IBC SUM149PT cells. We then used small interfering RNA-based MSI1 and MSI2 double knockdown (DKD) to understand gene expression and functional changes upon MSI depletion. We characterized cancer stem cell characteristics, cell apoptosis and cell cycle progression via flow cytometry, mammospheres via spheroid assays, migration and proliferation via digital holographic microscopy, and cell viability using BrdU assays. Chemoresistance was determined for paclitaxel and cisplatin with MTT assays and radioresistance was assessed with clonogenic analyses. In parallel, we supported our in vitro data by analyzing publicly available patient IBC gene expression datasets. RESULTS: MSI1 and MSI2 are upregulated in breast cancer generally and IBC specifically. MSI2 is more commonly expressed compared to MSI1. MSI DKD attenuated proliferation, cell cycle progression, migration, and cell viability while increasing apoptosis. Stem cell characteristics CD44(+)/CD24(-), TERT and Oct4 were associated with MSI expression in vivo and were decreased in vitro after MSI DKD as was ALDH expression and mammosphere formation. In vivo, chemoresistant tumors were characterized by MSI upregulation upon chemotherapy application. In vitro, MSI DKD was able to alleviate chemo- and radioresistance. CONCLUSIONS: The Musashi RNA binding proteins are dysregulated in IBC and associated with tumor proliferation, cancer stem cell phenotype, chemo- and radioresistance. MSI downregulation alleviates therapy resistance and attenuates tumor proliferation in vitro.
Assuntos
Neoplasias Inflamatórias Mamárias , Neoplasias , Humanos , Neoplasias Inflamatórias Mamárias/tratamento farmacológico , Neoplasias Inflamatórias Mamárias/genética , Neoplasias Inflamatórias Mamárias/metabolismo , Neoplasias/patologia , Células-Tronco Neoplásicas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proliferação de Células , Proteínas de Ligação a RNA/genéticaRESUMO
Cytotoxicity quantification of nanoparticles is commonly performed by biochemical assays to evaluate their biocompatibility and safety. We explored quantitative phase imaging (QPI) with digital holographic microscopy (DHM) as a time-resolved in vitro assay to quantify effects caused by three different types of organic nanoparticles in development for medical use. Label-free proliferation quantification of native cell populations facilitates cytotoxicity testing in biomedical nanotechnology. Therefore, DHM quantitative phase images from measurements on nanomaterial and control agent incubated cells were acquired over 24 h, from which the temporal course of the cellular dry mass was calculated within the observed field of view. The impact of LipImage™ 815 lipidots® nanoparticles, as well as empty and cabazitaxel-loaded poly(alkyl cyanoacrylate) nanoparticles on the dry mass development of four different cell lines (RAW 264.7, NIH-3T3, NRK-52E, and RLE-6TN), was observed vs. digitonin as cytotoxicity control and cells in culture medium. The acquired QPI data were compared to a colorimetric cell viability assay (WST-8) to explore the use of the DHM assay with standard biochemical analysis methods downstream. Our results show that QPI with DHM is highly suitable to identify harmful or low-toxic nanomaterials. The presented DHM assay can be implemented with commercial microscopes. The capability for imaging of native cells and the compatibility with common 96-well plates allows high-throughput systems and future embedding into existing experimental routines for in vitro cytotoxicity assessment.
Assuntos
Holografia , Nanopartículas , Bioensaio , Linhagem Celular , Holografia/métodos , Microscopia/métodosRESUMO
State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial interference. Digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI), facilitates reliable proliferation quantification of native cell populations and the extraction of morphological features in a fast and label- and interference-free manner by biophysical parameters. DHM therefore has been identified as versatile tool for cytotoxicity testing in biomedical nanotechnology. In a comparative study performed at two collaborating laboratories, we investigated the interlaboratory variability and performance of DHM in nanomaterial toxicity testing, utilizing complementary standard operating procedures (SOPs). Two identical custom-built off-axis DHM systems, developed for usage in biomedical laboratories, equipped with stage-top incubation chambers were applied at different locations in Europe. Temporal dry mass development, 12-h dry mass increments and morphology changes of A549 human lung epithelial cell populations upon incubation with two variants of poly(alkyl cyanoacrylate) (PACA) nanoparticles were observed in comparison to digitonin and cell culture medium controls. Digitonin as cytotoxicity control, as well as empty and cabazitaxel-loaded PACA nanocarriers, similarly impacted 12-h dry mass development and increments as well as morphology of A549 cells at both participating laboratories. The obtained DHM data reflected the cytotoxic potential of the tested nanomaterials and are in agreement with corresponding literature on biophysical and chemical assays. Our results confirm DHM as label-free cytotoxicity assay for polymeric nanocarriers as well as the repeatability and reproducibility of the technology. In summary, the evaluated DHM assay could be efficiently implemented at different locations and facilitates interlaboratory in vitro toxicity testing of nanoparticles with prospects for application in regulatory science.
Assuntos
Holografia , Microscopia , Digitonina , Holografia/métodos , Humanos , Técnicas In Vitro , Microscopia/métodos , Reprodutibilidade dos TestesRESUMO
Nanotechnologies such as nanoparticles are established components of new medical devices and pharmaceuticals. The use and distribution of these materials increases the requirement for standardized evaluation of possible adverse effects, starting with a general cytotoxicity screening. The Horizon 2020 project "Regulatory Science Framework for Nano(bio)material-based Medical Products and Devices (REFINE)" identified in vitro cytotoxicity quantification as a central task and first step for risk assessment and development for medical nanocarriers. We have performed an interlaboratory comparison on a cell-assay matrix including a kinetic lactate dehydrogenase (LDH) release cell death and WST-8 cell viability assay adapted for testing organic nanocarriers in four well-characterized cell lines of different organ origins. Identical experiments were performed by three laboratories, namely the Biomedical Technology Center (BMTZ) of the University of Münster, SINTEF Materials and Chemistry (SINTEF), and the National Institute for Public Health and the Environment (RIVM) of the Netherlands according to new standard operating procedures (SOPs). The experiments confirmed that LipImage™ 815 lipidots® are non-cytotoxic up to a concentration of 128 µg/mL and poly(alkyl cyanoacrylate) (PACA) nanoparticles for drug delivery of cytostatic agents caused dose-dependent cytotoxic effects on the cell lines starting from 8 µg/mL. PACA nanoparticles loaded with the active pharmaceutical ingredient (API) cabazitaxel showed a less pronounced dose-dependent effect with the lowest concentration of 2 µg/mL causing cytotoxic effects. The mean within laboratory standard deviation was 4.9% for the WST-8 cell viability assay and 4.0% for the LDH release cell death assay, while the between laboratory standard deviation was 7.3% and 7.8% for the two assays, respectively. Here, we demonstrated the suitability and reproducibility of a cytotoxicity matrix consisting of two endpoints performed with four cell lines across three partner laboratories. The experimental procedures described here can facilitate a robust cytotoxicity screening for the development of organic nanomaterials used in medicine.
Assuntos
Nanopartículas , Linhagem Celular , Sobrevivência Celular , L-Lactato Desidrogenase/metabolismo , Nanopartículas/toxicidade , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Engineered nanomaterials have been found to induce oxidative stress. Cellular oxidative stress, in turn, can result in the induction of antioxidant and detoxification enzymes which are controlled by the nuclear erythroid 2-related factor 2 (NRF2) transcription factor. Here, we present the results of a pre-validation study which was conducted within the frame of BIORIMA ("biomaterial risk management") an EU-funded research and innovation project. For this we used an NRF2 specific chemically activated luciferase expression reporter gene assay derived from the human U2OS osteosarcoma cell line to screen for the induction of the NRF2 mediated gene expression following exposure to biomedically relevant nanobiomaterials. Specifically, we investigated Fe3O4-PEG-PLGA nanomaterials while Ag and TiO2 "benchmark" nanomaterials from the Joint Research Center were used as reference materials. The viability of the cells was determined by using the Alamar blue assay. We performed an interlaboratory study involving seven different laboratories to assess the applicability of the NRF2 reporter gene assay for the screening of nanobiomaterials. The latter work was preceded by online tutorials to ensure that the procedures were harmonized across the different participating laboratories. Fe3O4-PEG-PLGA nanomaterials were found to induce very limited NRF2 mediated gene expression, whereas exposure to Ag nanomaterials induced NRF2 mediated gene expression. TiO2 nanomaterials did not induce NRF2 mediated gene expression. The variability in the results obtained by the participating laboratories was small with mean intra-laboratory standard deviation of 0.16 and mean inter laboratory standard deviation of 0.28 across all NRF2 reporter gene assay results. We conclude that the NRF2 reporter gene assay is a suitable assay for the screening of nanobiomaterial-induced oxidative stress responses.
RESUMO
Aquatic ecosystems around the world face serious threats from anthropogenic contaminants. Results from 8 years of field and laboratory investigations indicate that sublethal contaminant exposure is occurring in the early life stages of striped bass in the San Francisco Estuary, a population in continual decline since its initial collapse during the 1970s. Biologically significant levels of polychlorinated biphenyls, polybrominated diphenyl ethers, and current-use/legacy pesticides were found in all egg samples from river-collected fish. Developmental changes previously unseen with standard methods were detected with a technique using the principles of unbiased stereology. Abnormal yolk utilization, brain and liver development, and overall growth were observed in larvae from river-collected fish. Histopathological analyses confirmed and identified developmental alterations. Using this methodology enabled us to present a conclusive line of evidence for the maternal transfer of xenobiotics and their adverse effects on larval striped bass in this estuary.
Assuntos
Bass/crescimento & desenvolvimento , Larva/efeitos dos fármacos , Poluentes da Água/toxicidade , Xenobióticos/toxicidade , Animais , Feminino , Pesqueiros , Éteres Difenil Halogenados/toxicidade , Larva/crescimento & desenvolvimento , Masculino , Oceanos e Mares , Óvulo/efeitos dos fármacos , Praguicidas/toxicidade , Bifenilos Policlorados/toxicidade , Rios , São Francisco , Saco Vitelino/efeitos dos fármacosRESUMO
Acetylcholinesterase (AChE) activity is one of the most common biomarkers of neurotoxicity used in aquatic organisms. However, compared to its extensive use as biomarker, the effects of natural factors on AChE activity remain unclear especially in estuarine fishes. The aim of this study was to evaluate the effects of natural factors on AChE activity of striped bass (Morone saxatilis) juveniles. Brain AChE activity was measured in YOY (Young-Of-Year) individuals collected monthly from August 2007 to January 2008 at 12 different sites in the San Francisco Estuary system. The spatio-temporal variability of AChE was analyzed relative to water temperature and salinity as well as fish size. AChE activity was highly positively correlated with water temperature and to a lesser extent negatively with fish size while no relationship was detected with salinity. Taking into account these natural factors when using AChE as a biomarker will help to determine and understand the effects of neurotoxic contaminants on fish in estuarine systems.
Assuntos
Acetilcolinesterase/análise , Bass/fisiologia , Biomarcadores/metabolismo , Encéfalo/enzimologia , Animais , Tamanho Corporal , Ativação Enzimática/efeitos dos fármacos , Neurotoxinas/toxicidade , Análise de Regressão , Salinidade , Temperatura , Poluentes Químicos da Água/toxicidadeRESUMO
This study investigated sublethal, molecular effects of two current-use insecticides, chlorpyrifos (CP) and esfenvalerate (EV) in juvenile Chinook salmon. Heat-shock protein (hsp60, hsp70, hsp90) expression was quantified by Western blotting in muscle, liver and gill, and transcription of four cytokines (TGF-beta, IL-1beta, IGF-1, Mx-protein) was measured by real-time TaqMan PCR in anterior kidney and spleen. Expression of hsp was increased in muscle and liver at 1.2 and 7.2 microg/L CP, and at 0.01 and 0.1 microg/L EV, respectively. Transcription of IL-1beta and TGF-beta was elevated in kidney at 1.2 microg/L CP, while EV had no effect. No changes in cytokine transcription were observed in the spleen. Our results show that these insecticides cause cellular effects at environmental concentrations, and that hsps are sensitive indicators of sublethal exposure to CP and EV. In addition, CP may exert immunotoxic effects by altering the transcription of important mediators of the fish immune system.
Assuntos
Citocinas/genética , Proteínas de Choque Térmico/genética , Inseticidas/toxicidade , Neurotoxinas/toxicidade , Salmão/genética , Animais , California , Citocinas/efeitos dos fármacos , Primers do DNA , DNA Complementar/genética , Brânquias/efeitos dos fármacos , Brânquias/fisiologia , Proteínas de Choque Térmico/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/fisiologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Reação em Cadeia da Polimerase , Transcrição Gênica/efeitos dos fármacosRESUMO
Changes in the gene transcription of stress response genes in resident fish can be powerful biomarkers for the identification of sublethal impacts of environmental stressors on aquatic ecosystems. In this study, we tested the effects of two reference toxicants, copper (Cu) and the pyrethroid insecticide esfenvalerate [(S)-alpha-cyano-3-phenoxybenzyl-(S)-2-(4-chlorophenyl)-3-methylbutyrate], on lethal (mortality) and sublethal endpoints (growth, swimming behavior, transcription levels of stress response genes) in juvenile (81-90-day-old) striped bass (Morone saxatilis). We established cellular stress response markers for proteotoxicity (HSP70, HSP90), phase I detoxification mechanism (CYP1A1), metal-binding (metallothionein), as well as immune-function and pathogen-defense (TGF-beta, Mx-protein, nRAMP). Quantitative real-time TaqMan PCR was used to examine tissue-specific changes in the transcriptome of liver, spleen, white muscle, anterior kidney and gills after 7-day Cu exposures and 24-h esfenvalerate exposures. On the transcriptome level, exposure to Cu showed strongest effects on the transcription of metallothionein in spleen tissue, causing a 4-fold increase of mRNA at 42ppb total Cu and a 10-fold increase at 160 ppb Cu. Exposure to Cu also caused significant tissue-specific changes in gene transcription for immune-system related genes. Esfenvalerate exposure had tissue-specific effects on the transcription of HSP70, HSP90 and CYP1A1. The most significant effects were detected in liver tissue after exposure to 0.64 microg/L esfenvalerate. Our results show that the stress response at the transcriptome level is a more sensitive indicator for Cu and esfenvalerate exposures at low concentrations than swimming behavior, growth or mortality. The accuracy of studies on quantitative changes in the transcriptome can benefit from an initial evaluation or the inclusion of several different tissues and the use of multiple housekeeping genes.
Assuntos
Bass/fisiologia , Cobre/toxicidade , Inseticidas/toxicidade , Nitrilas/toxicidade , Piretrinas/toxicidade , Transcrição Gênica/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Primers do DNA/química , Proteínas de Peixes/análise , Proteínas de Peixes/genética , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Estresse Fisiológico/genética , Estresse Fisiológico/fisiopatologia , Estresse Fisiológico/veterinária , Análise de Sobrevida , Fatores de TempoRESUMO
Rapid expression of heat shock protein (hsp) families in response to a variety of stressors has been demonstrated in many organisms, including fish. The present 60-d challenge study was designed to compare hsp induction in juvenile Chinook salmon following exposure to individual pesticides, virus, and both stressors combined. Heat shock protein expression patterns over time were monitored and related to the extent of virus infection and mortality. Acetylcholinesterase (AChE) inhibition and recovery in response to applied stressors were measured in brain. High enzyme inhibition levels have been correlated with imminent mortality, and other sublethal physiological effects have been observed in fish concurrent with depressed AChE activity. Mortality was elevated considerably in fish exposed to 0.08 microg/L of the pyrethroid esfenvalerate (EV). Mortality due to infectious hematopoietic necrosis virus (IHNV) was lower in groups previously treated with pesticides; however, these fish died sooner than individuals exposed to virus only. Both pesticides, EV and the organophosphate (OP) chlorpyrifos (CP), as well as virus exposure, induced hsp expression, but highest hsp levels were observed after the combined treatments, suggesting an additive effect between virus and pesticides. Highest virus titers were accompanied by strongest hsp induction, indicating a connection between virus concentration and hsp expression. In conclusion, the measurement of hsp expression appears to be a very sensitive, integrative indicator of stress. Esfenvalerate and IHNV did not affect AChE activity, and exposure to 3.7 microg/L CP led to significantly inhibited AChE for at least 20 d. The time required for complete recovery of AChE activity raises concern about deleterious behavioral effects.
Assuntos
Acetilcolinesterase/efeitos dos fármacos , Inibidores da Colinesterase/toxicidade , Proteínas de Choque Térmico/metabolismo , Vírus da Necrose Hematopoética Infecciosa/patogenicidade , Praguicidas/toxicidade , Salmão/virologia , Poluentes Químicos da Água/toxicidade , Animais , Estresse FisiológicoRESUMO
Acetylcholinesterase (AChE) activity has traditionally been monitored as a biomarker of organophosphate (OP) and/or carbamate exposure. However, AChE activity may not be the most sensitive endpoint for these agrochemicals, because OPs can cause adverse physiological effects at concentrations that do not affect AChE activity. Carboxylesterases are a related family of enzymes that have higher affinity than AChE for some OPs and carbamates and may be more sensitive indicators of environmental exposure to these pesticides. In this study, carboxylesterase and AChE activity, cytochrome P4501A (CYP1A) protein levels, and mortality were measured in individual juvenile Chinook salmon (Oncorhynchus tshawytscha) following exposure to an OP (chlorpyrifos) and a pyrethroid (esfenvalerate). As expected, high doses of chlorpyrifos and esfenvalerate were acutely toxic, with nominal concentrations (100 and 1 microg/l, respectively) causing 100% mortality within 96 h. Exposure to chlorpyrifos at a high dose (7.3 microg/l), but not a low dose (1.2 microg/l), significantly inhibited AChE activity in both brain and muscle tissue (85% and 92% inhibition, respectively), while esfenvalerate exposure had no effect. In contrast, liver carboxylesterase activity was significantly inhibited at both the low and high chlorpyrifos dose exposure (56% and 79% inhibition, respectively), while esfenvalerate exposure still had little effect. The inhibition of carboxylesterase activity at levels of chlorpyrifos that did not affect AChE activity suggests that some salmon carboxylesterase isozymes may be more sensitive than AChE to inhibition by OPs. CYP1A protein levels were approximately 30% suppressed by chlorpyrifos exposure at the high dose, but esfenvalerate had no effect. Three teleost species, Chinook salmon, medaka (Oryzias latipes) and Sacramento splittail (Pogonichthys macrolepidotus), were examined for their ability to hydrolyze a series of pyrethroid surrogate substrates and in all cases hydrolysis activity was undetectable. Together these data suggest that (1) carboxylesterase activity inhibition may be a more sensitive biomarker for OP exposure than AChE activity, (2) neither AChE nor carboxylesterase activity are biomarkers for pyrethroid exposure, (3) CYP1A protein is not a sensitive marker for these agrochemicals and (4) slow hydrolysis rates may be partly responsible for acute pyrethroid toxicity in fish.
Assuntos
Acetilcolinesterase/metabolismo , Carboxilesterase/metabolismo , Clorpirifos/toxicidade , Citocromo P-450 CYP1A1/metabolismo , Inseticidas/toxicidade , Nitrilas/toxicidade , Oncorhynchus mykiss/metabolismo , Piretrinas/toxicidade , Acetilcolinesterase/química , Análise de Variância , Animais , Biomarcadores/metabolismo , Encéfalo/metabolismo , Carboxilesterase/química , Relação Dose-Resposta a Droga , Fígado/metabolismo , Músculo Esquelético/metabolismo , Análise de SobrevidaRESUMO
Sublethal concentrations of pollutants may compromise fish, resulting in increased susceptibility to endemic pathogens. To test this hypothesis, juvenile chinook salmon (Oncorhynchus tshawytscha) were exposed to sublethal levels of esfenvalerate or chlorpyrifos either alone or concurrently with infectious hematopoietic necrosis virus (IHNV). Three trials were performed with fish exposed to concentrations of IHNV between 0.8 x 10(2) and 2.7 x 10(6) plaque-forming units/ml and to 5.0 microg/L of chlorpyrifos or 0.1 microg/L of esfenvalerate. The presence and concentration of IHNV in dead fish were assayed by virus isolation and plaque assay techniques, respectively. Among groups exposed to both esfenvalerate and IHNV, 83% experienced highly significant (p < 0.001) mortality, ranging from 20 to 90% at 3 d post-virus exposure, and cumulatively died from 2.4 to 7.7 d sooner than fish exposed to IHNV alone. This trend was not seen in any other treatment group. Virus assays of dead fish indicate a lethal synergism of esfenvalerate and IHNV. Chlorpyrifos had no observed effect on total mortality or IHNV susceptibility. The present results suggest that accepted levels of pollutants may be seemingly nonlethal to fish but, in fact, be acting synergistically with endemic pathogens to compromise survivorship of wild fish populations through immunologic or physiologic disruption.
Assuntos
Vírus da Necrose Hematopoética Infecciosa/patogenicidade , Inseticidas/toxicidade , Mortalidade , Nitrilas/toxicidade , Piretrinas/toxicidade , Salmão/virologia , Poluentes Químicos da Água/toxicidade , Animais , Linhagem CelularRESUMO
We determined the effects of two pesticides, chlorpyrifos and esfenvalerate in juvenile Chinook salmon. Four to five month old salmon were exposed to a range of pesticide concentrations, and tissue samples of surviving fish were analyzed for stress protein expression, cytokine transcription, and acetylcholinesterase (AChE) activity. At the highest concentrations, both pesticides led to complete mortality, whereas medium and low concentrations resulted in high survival rates. Significant differences in stress protein expression, cytokine transcription and AChE activity were found between control and surviving chlorpyrifos-exposed fish. Elevated stress protein expression was the only detectable response to esfenvalerate.
Assuntos
Clorpirifos/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Piretrinas/toxicidade , RNA Mensageiro/metabolismo , Salmão/metabolismo , Acetilcolinesterase/biossíntese , Análise de Variância , Animais , Western Blotting , Encéfalo/metabolismo , Citocinas/metabolismo , Eletroforese em Gel de Poliacrilamida , Indução Enzimática/efeitos dos fármacos , Proteínas de Choque Térmico/biossíntese , Mortalidade , Músculo Esquelético/metabolismo , Nitrilas , Reação em Cadeia da Polimerase/métodosRESUMO
Impairment of fish immune function as a consequence of polluted aquatic environments can result in changes in susceptibility to disease. In this study, we investigated the effects of two widely used insecticides, chlorpyrifos (CP) and esfenvalerate (EV), and a pathogen, infectious hematopoietic necrosis virus (IHNV), singly and in combination, on survival and cytokine (Mx protein, IL-1beta, TGF-beta and IGF-1) expression in juvenile Chinook salmon. Fish were exposed for 96 h to sublethal concentrations of CP (3.7 microg L(-1)) or EV (0.08 microg L(-1)), allowed to recover in clean water for seven days, then exposed to IHNV (6.4x10(5)TCID(50)mL(-1)) for 1.5h. Mortality was recorded daily, and spleen and anterior kidney samples were collected on day 4 (after CP or EV treatment), day 20 and day 60 (after CP or EV treatment and subsequent IHNV exposure) of the experiment. Significant mortality after 60 days was observed following exposure to EV (17%) or IHNV (20%), and prior insecticide exposure did not synergize the acute effects of pathogen treatment. By day 4, exposure to CP as well as EV led to a significant decrease of Mx protein and IL-1beta expression; by day 20, EV-exposed fish significantly overexpressed IL-1beta. Mx protein transcription was up-regulated in spleen and kidney of all IHNV-exposed fish groups by day 20. All but one treatment (EV) led to significantly decreased IGF-1 transcription in spleen on days 20 and 60, whereas a short-term increase was seen after CP exposure (day 4). In kidney, decreases of IGF-1 transcription were less pronounced. TGF-beta transcription was up-regulated in CP/IHNV and EV/IHNV exposure groups. Our results indicate that CP and EV alter the expression of cytokines, but this did not negatively affect the ability of fish to survive a subsequent exposure to IHNV. Induced TGF-beta transcription indicated that the combined stressors affected fish in a synergistic manner, but the consequences are unknown. Increased transcription of Mx protein was a reliable indicator of virus exposure.