Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 34
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
Biometals ; 2022 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-35167013

RESUMO

The mammalian multicopper ferroxidases (MCFs) ceruloplasmin (CP), hephaestin (HEPH) and zyklopen (ZP) comprise a family of conserved enzymes that are essential for body iron homeostasis. Each of these enzymes contains six biosynthetically incorporated copper atoms which act as intermediate electron acceptors, and the oxidation of iron is associated with the four electron reduction of dioxygen to generate two water molecules. CP occurs in both a secreted and GPI-linked (membrane-bound) form, while HEPH and ZP each contain a single C-terminal transmembrane domain. These enzymes function to ensure the efficient oxidation of iron so that it can be effectively released from tissues via the iron export protein ferroportin and subsequently bound to the iron carrier protein transferrin in the blood. CP is particularly important in facilitating iron release from the liver and central nervous system, HEPH is the major MCF in the small intestine and is critical for dietary iron absorption, and ZP is important for normal hair development. CP and HEPH (and possibly ZP) function in multiple tissues. These proteins also play other (non-iron-related) physiological roles, but many of these are ill-defined. In addition to disrupting iron homeostasis, MCF dysfunction perturbs neurological and immune function, alters cancer susceptibility, and causes hair loss, but, despite their importance, how MCFs co-ordinately maintain body iron homeostasis and perform other functions remains incompletely understood.

2.
Mol Cancer Ther ; 21(1): 184-192, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34667112

RESUMO

Pancreatic cancer is the third most common cause of cancer-related deaths in the United States. Although gemcitabine is the standard of care for most patients with pancreatic cancer, its efficacy is limited by the development of resistance. This resistance may be attributable to the evasion of apoptosis caused by the overexpression of BCL-2 family antiapoptotic proteins. In this study, we investigated the role of BCL-XL in gemcitabine resistance to identify a combination therapy to more effectively treat pancreatic cancer. We used CRISPR-Cas9 screening to identify the key genes involved in gemcitabine resistance in pancreatic cancer. Pancreatic cancer cell dependencies on different BCL-2 family proteins and the efficacy of the combination of gemcitabine and DT2216 (a BCL-XL proteolysis targeting chimera or PROTAC) were determined by MTS, Annexin-V/PI, colony formation, and 3D tumor spheroid assays. The therapeutic efficacy of the combination was investigated in several patient-derived xenograft (PDX) mouse models of pancreatic cancer. We identified BCL-XL as a key mediator of gemcitabine resistance. The combination of gemcitabine and DT2216 synergistically induced cell death in multiple pancreatic cancer cell lines in vitro In vivo, the combination significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice compared with the individual agents in pancreatic cancer PDX models. Their synergistic antitumor activity is attributable to DT2216-induced degradation of BCL-XL and concomitant suppression of MCL-1 by gemcitabine. Our results suggest that DT2216-mediated BCL-XL degradation augments the antitumor activity of gemcitabine and their combination could be more effective for pancreatic cancer treatment.


Assuntos
Antineoplásicos/uso terapêutico , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Piperazinas/uso terapêutico , Proteína bcl-X/metabolismo , Animais , Antineoplásicos/farmacologia , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Humanos , Camundongos , Camundongos Endogâmicos NOD , Neoplasias Pancreáticas/patologia , Piperazinas/farmacologia
3.
Cancer Res ; 81(17): 4581-4593, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34158378

RESUMO

The HIV-protease inhibitor nelfinavir has shown broad anticancer activity in various preclinical and clinical contexts. In patients with advanced, proteasome inhibitor (PI)-refractory multiple myeloma, nelfinavir-based therapy resulted in 65% partial response or better, suggesting that this may be a highly active chemotherapeutic option in this setting. The broad anticancer mechanism of action of nelfinavir implies that it interferes with fundamental aspects of cancer cell biology. We combined proteome-wide affinity-purification of nelfinavir-interacting proteins with genome-wide CRISPR/Cas9-based screening to identify protein partners that interact with nelfinavir in an activity-dependent manner alongside candidate genetic contributors affecting nelfinavir cytotoxicity. Nelfinavir had multiple activity-specific binding partners embedded in lipid bilayers of mitochondria and the endoplasmic reticulum. Nelfinavir affected the fluidity and composition of lipid-rich membranes, disrupted mitochondrial respiration, blocked vesicular transport, and affected the function of membrane-embedded drug efflux transporter ABCB1, triggering the integrated stress response. Sensitivity to nelfinavir was dependent on ADIPOR2, which maintains membrane fluidity by promoting fatty acid desaturation and incorporation into phospholipids. Supplementation with fatty acids prevented the nelfinavir-induced effect on mitochondrial metabolism, drug-efflux transporters, and stress-response activation. Conversely, depletion of fatty acids/cholesterol pools by the FDA-approved drug ezetimibe showed a synergistic anticancer activity with nelfinavir in vitro. These results identify the modification of lipid-rich membranes by nelfinavir as a novel mechanism of action to achieve broad anticancer activity, which may be suitable for the treatment of PI-refractory multiple myeloma. SIGNIFICANCE: Nelfinavir induces lipid bilayer stress in cellular organelles that disrupts mitochondrial respiration and transmembrane protein transport, resulting in broad anticancer activity via metabolic rewiring and activation of the unfolded protein response.


Assuntos
Inibidores da Protease de HIV/farmacologia , Lipídeos de Membrana , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Nelfinavir/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Genoma , Glucose/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Lipidômica , Lipídeos/química , Fosfolipídeos/química , Fosforilação , Receptores de Adiponectina/metabolismo , Transdução de Sinais
4.
J Nutr ; 151(9): 2541-2550, 2021 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-34114013

RESUMO

BACKGROUND: The ferroxidase zyklopen (Zp) has been implicated in the placental transfer of iron to the fetus. However, the evidence for this is largely circumstantial. OBJECTIVES: This study aimed to determine whether Zp is essential for placental iron transfer. METHODS: A model was established using 8- to 12-wk-old pregnant C57BL/6 mice on standard rodent chow in which Zp was knocked out in the fetus and fetal components of the placenta. Zp was also disrupted in the entire placenta using global Zp knockout mice. Inductively coupled plasma MS was used to measure total fetal iron, an indicator of the amount of iron transferred by the placenta to the fetus, at embryonic day 18.5 of gestation. Iron transporter expression in the placenta was measured by Western blotting, and the expression of Hamp1, the gene encoding the iron regulatory hormone hepcidin, was determined in fetal liver by real-time PCR. RESULTS: There was no change in the amount of iron transferred to the fetus when Zp was disrupted in either the fetal component of the placenta or the entire placenta. No compensatory changes in the expression of the iron transport proteins transferrin receptor 1 or ferroportin were observed, nor was there any change in fetal liver Hamp1 mRNA. Hephl1, the gene encoding Zp, was expressed mainly in the maternal decidua of the placenta and not in the nutrient-transporting syncytiotrophoblast. Disruption of Zp in the whole placenta resulted in a 26% increase in placental size (P < 0.01). CONCLUSIONS: Our data indicate that Zp is not essential for the efficient transfer of iron to the fetus in mice and is localized predominantly in the maternal decidua. The increase in placental size observed when Zp is knocked out in the entire placenta suggests that this protein may play a role in placental development.


Assuntos
Ceruloplasmina , Placenta , Animais , Ceruloplasmina/genética , Feminino , Feto/metabolismo , Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Placenta/metabolismo , Placentação , Gravidez
5.
Curr Protoc ; 1(5): e136, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34043288

RESUMO

The use of genome editing tools is expanding our understanding of various human diseases by providing insight into gene-disease interactions. Despite the recognized role of toxicants in the development of human health issues and conditions, there is currently limited characterization of their mechanisms of action, and the application of CRISPR-based genome editing to the study of toxicants could help in the identification of novel gene-environment interactions. CRISPR-based functional screens enable identification of cellular mechanisms fundamental for response and susceptibility to a given toxicant. The aim of this review is to inform future directions in the application of CRISPR technologies in toxicological studies. We review and compare different types of CRISPR-based methods including pooled, anchored, combinatorial, and perturb-sequencing screens in vitro, in addition to pooled screenings in model organisms. © 2021 Wiley Periodicals LLC.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , Interação Gene-Ambiente , Humanos , Análise de Sequência
6.
Artigo em Inglês | MEDLINE | ID: mdl-33684654

RESUMO

To evaluate the impact of environmental contaminants on aquatic health, extensive surveys of fish populations have been conducted using bioaccumulation as an indicator of impairment. While these studies have reported mixtures of chemicals in fish tissues, the relationship between specific contaminants and observed adverse impacts remains poorly understood. The present study aimed to characterize the toxicological responses induced by persistent organic pollutants in wild-caught hornyhead turbot (P. verticalis). To do so, hornyhead turbot were interperitoneally injected with a single dose of PCB or PBDE congeners prepared using environmentally realistic mixture proportions. After 96-hour exposure, the livers were excised and analyzed using transcriptomic approaches and analytical chemistry. Concentrations of PCBs and PBDEs measured in the livers indicated clear differences across treatments, and congener profiles closely mirrored our expectations. Distinct gene profiles were characterized for PCB and PBDE exposed fish, with significant differences observed in the expression of genes associated with immune responses, endocrine-related functions, and lipid metabolism. Our findings highlight the key role that transcriptomics can play in monitoring programs to assess chemical-induced toxicity in heterogeneous group of fish (mixed gender and life stage) as is typically found during field surveys. Altogether, the present study provides further evidence of the potential of transcriptomic tools to improve aquatic health assessment and identify causative agents.


Assuntos
Linguado/genética , Éteres Difenil Halogenados/toxicidade , Bifenilos Policlorados/toxicidade , Transcriptoma/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Monitoramento Ambiental , Proteínas de Peixes/genética
7.
Am J Hematol ; 95(9): 1085-1098, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32510613

RESUMO

Transferrin-bound iron (TBI), the physiological circulating iron form, is acquired by cells through the transferrin receptor (TfR1) by endocytosis. In erythroid cells, most of the acquired iron is incorporated into heme in the mitochondria. Cellular trafficking of heme is indispensable for erythropoiesis and many other essential biological processes. Comprehensive elucidation of molecular pathways governing and regulating cellular iron acquisition and heme trafficking is required to better understand physiological and pathological processes affecting erythropoiesis. Here, we report the first genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens in human erythroid cells to identify determinants of iron and heme uptake, as well as heme-mediated erythroid differentiation. We identified several candidate modulators of TBI acquisition including TfR1, indicating that our approach effectively revealed players mechanistically relevant to the process. Interestingly, components of the endocytic pathway were also revealed as potential determinants of transferrin acquisition. We deciphered a role for the vacuolar-type H+ - ATPase (V- ATPase) assembly factor coiled-coil domain containing 115 (CCDC115) in TBI uptake and validated this role in CCDC115 deficient K562 cells. Our screen in hemin-treated cells revealed perturbations leading to cellular adaptation to heme, including those corresponding to trafficking mechanisms and transcription factors potentiating erythroid differentiation. Pathway analysis indicated that endocytosis and vesicle acidification are key processes for heme trafficking in erythroid precursors. Furthermore, we provided evidence that CCDC115, which we identified as required for TBI uptake, is also involved in cellular heme distribution. This work demonstrates a previously unappreciated common intersection in trafficking of transferrin iron and heme in the endocytic pathway of erythroid cells.


Assuntos
Células Eritroides/metabolismo , Heme/metabolismo , Ferro/metabolismo , Proteínas do Tecido Nervoso , Transporte Biológico Ativo , Sistemas CRISPR-Cas , Células Eritroides/citologia , Testes Genéticos , Células HEK293 , Heme/genética , Humanos , Células K562 , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo
8.
Sci Rep ; 9(1): 9437, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31263155

RESUMO

Accumulation of iron has been associated with the pathobiology of various disorders of the central nervous system. Our previous work has shown that hephaestin (Heph) and ceruloplasmin (Cp) double knockout (KO) mice induced iron accumulation in multiple brain regions and that this was paralleled by increased oxidative damage and deficits in cognition and memory. In this study, we enriched astrocytes and oligodendrocytes from the cerebral cortex of neonatal wild-type (WT), Heph KO and Cp KO mice. We demonstrated that Heph is highly expressed in oligodendrocytes, while Cp is mainly expressed in astrocytes. Iron efflux was impaired in Cp KO astrocytes and Heph KO oligodendrocytes and was associated with increased oxidative stress. The expression of Heph, Cp, and other iron-related genes was examined in astrocytes and oligodendrocytes both with and without iron treatment. Interestingly, we found that the expression of the mRNA encoding ferroportin 1, a transmembrane protein that cooperates with CP and HEPH to export iron from cells, was positively correlated with Cp expression in astrocytes, and with Heph expression in oligodendrocytes. Our findings collectively demonstrate that HEPH and CP are important for the prevention of glial iron accumulation and thus may be protective against oxidative damage.


Assuntos
Ceruloplasmina/genética , Ferro/metabolismo , Proteínas de Membrana/genética , Estresse Oxidativo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Ceruloplasmina/deficiência , Proteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Estresse Oxidativo/genética
9.
PLoS Genet ; 15(5): e1008143, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31125343

RESUMO

Maintenance of the correct redox status of iron is functionally important for critical biological processes. Multicopper ferroxidases play an important role in oxidizing ferrous iron, released from the cells, into ferric iron, which is subsequently distributed by transferrin. Two well-characterized ferroxidases, ceruloplasmin (CP) and hephaestin (HEPH) facilitate this reaction in different tissues. Recently, a novel ferroxidase, Hephaestin like 1 (HEPHL1), also known as zyklopen, was identified. Here we report a child with compound heterozygous mutations in HEPHL1 (NM_001098672) who presented with abnormal hair (pili torti and trichorrhexis nodosa) and cognitive dysfunction. The maternal missense mutation affected mRNA splicing, leading to skipping of exon 5 and causing an in-frame deletion of 85 amino acids (c.809_1063del; p.Leu271_ala355del). The paternal mutation (c.3176T>C; p.Met1059Thr) changed a highly conserved methionine that is part of a typical type I copper binding site in HEPHL1. We demonstrated that HEPHL1 has ferroxidase activity and that the patient's two mutations exhibited loss of this ferroxidase activity. Consistent with these findings, the patient's fibroblasts accumulated intracellular iron and exhibited reduced activity of the copper-dependent enzyme, lysyl oxidase. These results suggest that the patient's biallelic variants are loss-of-function mutations. Hence, we generated a Hephl1 knockout mouse model that was viable and had curly whiskers, consistent with the hair phenotype in our patient. These results enhance our understanding of the function of HEPHL1 and implicate altered ferroxidase activity in hair growth and hair disorders.


Assuntos
Oxirredutases/genética , Oxirredutases/metabolismo , Adulto , Alelos , Animais , Sítios de Ligação , Ceruloplasmina/metabolismo , Pré-Escolar , Cobre/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Variação Genética/genética , Células HEK293 , Cabelo , Humanos , Ferro/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Oxirredução , Fenótipo
10.
J Vis Exp ; (145)2019 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-30985763

RESUMO

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genes Homeobox , Animais , Sítios de Ligação , Cromatina , DNA , Regulação da Expressão Gênica , Biblioteca Gênica , Humanos , RNA Guia
11.
BMC Genomics ; 19(1): 877, 2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518325

RESUMO

BACKGROUND: The health and resilience of species in natural environments is increasingly challenged by complex anthropogenic stressor combinations including climate change, habitat encroachment, and chemical contamination. To better understand impacts of these stressors we examined the individual- and combined-stressor impacts of malaria infection, food limitation, and 2,4,6-trinitrotoluene (TNT) exposures on gene expression in livers of Western fence lizards (WFL, Sceloporus occidentalis) using custom WFL transcriptome-based microarrays. RESULTS: Computational analysis including annotation enrichment and correlation analysis identified putative functional mechanisms linking transcript expression and toxicological phenotypes. TNT exposure increased transcript expression for genes involved in erythropoiesis, potentially in response to TNT-induced anemia and/or methemoglobinemia and caused dose-specific effects on genes involved in lipid and overall energy metabolism consistent with a hormesis response of growth stimulation at low doses and adverse decreases in lizard growth at high doses. Functional enrichment results were indicative of inhibited potential for lipid mobilization and catabolism in TNT exposures which corresponded with increased inguinal fat weights and was suggestive of a decreased overall energy budget. Malaria infection elicited enriched expression of multiple immune-related functions likely corresponding to increased white blood cell (WBC) counts. Food limitation alone enriched functions related to cellular energy production and decreased expression of immune responses consistent with a decrease in WBC levels. CONCLUSIONS: Despite these findings, the lizards demonstrated immune resilience to malaria infection under food limitation with transcriptional results indicating a fully competent immune response to malaria, even under bio-energetic constraints. Interestingly, both TNT and malaria individually increased transcriptional expression of immune-related genes and increased overall WBC concentrations in blood; responses that were retained in the TNT x malaria combined exposure. The results demonstrate complex and sometimes unexpected responses to multiple stressors where the lizards displayed remarkable resiliency to the stressor combinations investigated.


Assuntos
Poluentes Ambientais/toxicidade , Lagartos/metabolismo , Transcriptoma/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Mudança Climática , Análise por Conglomerados , Ecossistema , Metabolismo Energético/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lagartos/genética , Lagartos/parasitologia , Linfócitos/citologia , Linfócitos/imunologia , Linfócitos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Plasmodium/patogenicidade , RNA/química , RNA/isolamento & purificação , RNA/metabolismo , Análise de Sequência de RNA , Baço/parasitologia , Baço/fisiologia , Trinitrotolueno/toxicidade
12.
Genes Nutr ; 10(4): 468, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25981695

RESUMO

Iron-deficient anemia is a prevalent disease among humans. We searched for genes regulated by iron deficiency and its regulated mechanism. cDNA microarrays were performed using Hepa1c1c7 cells treated with 100 µM desferrioxamine (DFO), an iron chelator. Early growth response 1 (Egr1) was upregulated with at least 20-fold increase within 4 h and lasted for 24 h, which was confirmed by qRT-PCR. This activation was not seen by ferric ammonium citrate (FAC). DFO increased the transcriptional activity of Egr1-luc (-604 to +160) and serum response element (SRE)-luc reporters by 2.7-folds. In addition, cycloheximide lowered DFO-induced Egr1 mRNA levels. The upregulation of Egr1 by DFO was accompanied by sustained ERK signals along with phosphorylation of Elk-1. The ERK inhibitor (PD98059) prevented the DFO-induced Egr1 mRNAs. Overexpression of Elk-1 mutant (pElk-1S383A) decreased Egr1 reporter activity. DFO lowered reactive oxygen species (ROS) production and increased caspase 3/7 activity and cell death. DFO-induced iron deficiency upregulates Egr1 in part through transcriptional activation via ERK and Elk-1 signals, which may be important in the regulation of cell death in hepatoma cells. Our study demonstrated that iron depletion controlled the expression of Egr1, which might contribute to decisions about cellular fate in response to iron deficiency.

13.
Genes Nutr ; 10(1): 443, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25427953

RESUMO

Hereditary hemochromatosis is an iron overload disorder most commonly caused by a defect in the HFE gene. While the genetic defect is highly prevalent, the majority of individuals do not develop clinically significant iron overload, suggesting the importance of genetic modifiers. Murine hfe knockout models have demonstrated that strain background has a strong effect on the severity of iron loading. We noted that hepatic iron loading in hfe-/- mice occurs primarily over the first postnatal weeks (loading phase) followed by a timeframe of relatively static iron concentrations (plateau phase). We thus evaluated the effects of background strain and of age on hepatic gene expression in Hfe knockout mice (hfe-/-). Hepatic gene expression profiles were examined using cDNA microarrays in 4- and 8-week-old hfe-/- and wild-type mice on two different genetic backgrounds, C57BL/6J (C57) and AKR/J (AKR). Genes differentially regulated in all hfe-/- mice groups, compared with wild-type mice, including those involved in cell survival, stress and damage responses and lipid metabolism. AKR strain-specific changes in lipid metabolism genes and C57 strain-specific changes in cell adhesion and extracellular matrix protein genes were detected in hfe-/- mice. Mouse strain and age are each significantly associated with hepatic gene expression profiles in hfe-/- mice. These affects may underlie or reflect differences in iron loading in these mice.

14.
Aquat Toxicol ; 152: 186-94, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24794047

RESUMO

Cadmium is a heavy metal that can accumulate to toxic levels in the environment leading to detrimental effects in animals and humans including kidney, liver and lung injuries. Using a transcriptomics approach, genes and cellular pathways affected by a low dose of cadmium were investigated. Adult largemouth bass were intraperitoneally injected with 20µg/kg of cadmium chloride (mean exposure level - 2.6µg of cadmium per fish) and microarray analyses were conducted in the liver and testis 48h after injection. Transcriptomic profiles identified in response to cadmium exposure were tissue-specific with the most differential expression changes found in the liver tissues, which also contained much higher levels of cadmium than the testis. Acute exposure to a low dose of cadmium induced oxidative stress response and oxidative damage pathways in the liver. The mRNA levels of antioxidants such as catalase increased and numerous transcripts related to DNA damage and DNA repair were significantly altered. Hepatic mRNA levels of metallothionein, a molecular marker of metal exposure, did not increase significantly after 48h exposure. Carbohydrate metabolic pathways were also disrupted with hepatic transcripts such as UDP-glucose, pyrophosphorylase 2, and sorbitol dehydrogenase highly induced. Both tissues exhibited a disruption of steroid signaling pathways. In the testis, estrogen receptor beta and transcripts linked to cholesterol metabolism were suppressed. On the contrary, genes involved in cholesterol metabolism were highly increased in the liver including genes encoding for the rate limiting steroidogenic acute regulatory protein and the catalytic enzyme 7-dehydrocholesterol reductase. Integration of the transcriptomic data using functional enrichment analyses revealed a number of enriched gene networks associated with previously reported adverse outcomes of cadmium exposure such as liver toxicity and impaired reproduction.


Assuntos
Bass/genética , Bass/metabolismo , Cádmio/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Cádmio/metabolismo , Reparo do DNA/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Estresse Oxidativo/efeitos dos fármacos , Análise Serial de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/metabolismo , Poluentes Químicos da Água/metabolismo
15.
Environ Health Perspect ; 121(6): 683-90, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23649538

RESUMO

BACKGROUND: Differences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity. OBJECTIVES: Grantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability. METHODS: Consortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1ß (IL-1ß) release] using only THP-1 cells. RESULTS: The overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 µg/mL, but did not induce IL-1ß. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1ß production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1ß production in THP-1 cells, with the original MWCNT producing the most IL-1ß. CONCLUSIONS: The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.


Assuntos
Inflamação/induzido quimicamente , Nanopartículas/toxicidade , Nanotubos de Carbono/toxicidade , Titânio/toxicidade , Óxido de Zinco/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Interleucina-1beta/biossíntese , Nanopartículas/química , Nanotubos de Carbono/química , National Institute of Environmental Health Sciences (U.S.) , Ratos , Titânio/química , Estados Unidos
16.
J Trace Elem Med Biol ; 26(2-3): 115-9, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22575541

RESUMO

Intestinal iron absorption is a critical process for maintaining body iron levels within the optimal physiological range. Iron in the diet is found in a wide variety of forms, but the absorption of non-heme iron is best understood. Most of this iron is moved across the enterocyte brush border membrane by the iron transporter divalent metal-ion transporter 1, a process enhanced by the prior reduction of the iron by duodenal cytochrome B and possibly other reductases. Enterocyte iron is exported to the blood via ferroportin 1 on the basolateral membrane. This transporter acts in partnership with the ferroxidase hephaestin that oxidizes exported ferrous iron to facilitate its binding to plasma transferrin. Iron absorption is controlled by a complex network of systemic and local influences. The liver-derived peptide hepcidin binds to ferroportin, leading to its internalization and a reduction in absorption. Hepcidin expression in turn responds to body iron demands and the BMP-SMAD signaling pathway plays a key role in this process. The levels of iron and oxygen in the enterocyte also exert important influences on iron absorption. Disturbances in the regulation of iron absorption are responsible for both iron loading and iron deficiency disorders in humans.


Assuntos
Absorção Intestinal/fisiologia , Ferro/metabolismo , Animais , Transporte Biológico/fisiologia , Enterócitos/metabolismo , Humanos
17.
Environ Health Perspect ; 119(6): 771-7, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21193388

RESUMO

BACKGROUND: In humans, inorganic arsenic (iAs) is metabolized to methylated arsenical species in a multistep process mainly mediated by arsenic (+3 oxidation state) methyltransferase (AS3MT). Among these metabolites is monomethylarsonous acid (MMAIII), the most toxic arsenic species. A recent study in As3mt-knockout mice suggests that unidentified methyltransferases could be involved in alternative iAs methylation pathways. We found that yeast deletion mutants lacking MTQ2 were highly resistant to iAs exposure. The human ortholog of the yeast MTQ2 is N-6 adenine-specific DNA methyltransferase 1 (N6AMT1), encoding a putative methyltransferase. OBJECTIVE: We investigated the potential role of N6AMT1 in arsenic-induced toxicity. METHODS: We measured and compared the cytotoxicity induced by arsenicals and their metabolic profiles using inductively coupled plasma-mass spectrometry in UROtsa human urothelial cells with enhanced N6AMT1 expression and UROtsa vector control cells treated with different concentrations of either iAsIII or MMAIII. RESULTS: N6AMT1 was able to convert MMAIII to the less toxic dimethylarsonic acid (DMA) when overexpressed in UROtsa cells. The enhanced expression of N6AMT1 in UROtsa cells decreased cytotoxicity of both iAsIII and MMAIII. Moreover, N6AMT1 is expressed in many human tissues at variable levels, although at levels lower than those of AS3MT, supporting a potential participation in arsenic metabolism in vivo. CONCLUSIONS: Considering that MMAIII is the most toxic arsenical, our data suggest that N6AMT1 has a significant role in determining susceptibility to arsenic toxicity and carcinogenicity because of its specific activity in methylating MMAIII to DMA and other unknown mechanisms.


Assuntos
Ácido Cacodílico/toxicidade , Compostos Organometálicos/toxicidade , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Urotélio/efeitos dos fármacos , Ácido Cacodílico/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Espectrometria de Massas , Metilação , Compostos Organometálicos/metabolismo , Análise de Sequência de DNA , Urotélio/metabolismo , Leveduras/efeitos dos fármacos , Leveduras/metabolismo
18.
Environ Toxicol Chem ; 30(2): 290-300, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21072851

RESUMO

The delta smelt (Hypomesus transpacificus) is an endangered pelagic fish species endemic to the Sacramento-San Joaquin estuary (CA, USA), and considered an indicator of ecosystem health. Copper is a contaminant of concern in Californian waterways that may affect the development and survival of this endangered species. The experimental combination of molecular biomarkers with higher level effects may allow for interpretation of responses in a functional context that can be used to predict detrimental outcomes caused by exposure. A delta smelt microarray was developed and applied to screen for candidate molecular biomarkers that may be used in monitoring programs. Functional classifications of microarray responses were used along with quantitative polymerase chain reaction determining effects upon neuromuscular, digestive, and immune responses in Cu-exposed delta smelt. Differences in sensitivity were measured between juveniles and larvae (median lethal concentration = 25.2 and 80.4 µg/L Cu(2+), respectively). Swimming velocity declined with higher exposure concentrations in a dose-dependent manner (r = -0.911, p < 0.05), though was not statistically significant to controls. Genes encoding for aspartoacylase, hemopexin, α-actin, and calcium regulation proteins were significantly affected by exposure and were functionally interpreted with measured swimming responses. Effects on digestion were measured by upregulation of chitinase and downregulation of amylase, whereas downregulation of tumor necrosis factor indicated a probable compromised immune system. Results from this study, and many others, support the use of functionally characterized molecular biomarkers to assess effects of contaminants in field scenarios. We thus propose that to attribute environmental relevance to molecular biomarkers, research should concentrate on their application in field studies with the aim of assisting monitoring programs.


Assuntos
Cobre/efeitos adversos , Espécies em Perigo de Extinção , Regulação da Expressão Gênica , Osmeriformes/fisiologia , Poluentes Químicos da Água/efeitos adversos , Animais , California , Cobre/toxicidade , Marcadores Genéticos , Larva/genética , Larva/imunologia , Larva/fisiologia , Análise em Microsséries , Osmeriformes/genética , Osmeriformes/imunologia , Reação em Cadeia da Polimerase , Natação , Água/análise , Poluentes Químicos da Água/toxicidade
19.
Environ Sci Technol ; 44(5): 1841-6, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-20131897

RESUMO

The relative transparency of Daphnia magna (daphnia) and the unique optical properties of quantum dots (QDs) were paired to study the accumulation potential and surface coating effects on uptake of amphiphilic polymer coated CdSe/ZnS QDs. Fluorescence confocal laser scanning microscopy was used to visualize and spectrally distinguish QDs from competing autofluorescent signals arising from the daphnia themselves and their food sources. QDs were found to accumulate within the digestive tracts of daphnia, as well as, in some cases, adhere to the carapace, antennae, and thoracic appendages. After 48 h of gut clearance with and without feeding, QD fluorescence signal was still apparent in the digestive tracts of daphnia, and inductively coupled plasma mass spectrometry (ICP-MS) measurements confirmed that 36-53% of the initial uptake was retained. As surface charge and pegylation can influence the uptake of nanoparticles, uptake of QDs coated with two different amphililic polymers and their polyethylene glycol (PEG) coated counterparts was also examined. Fluorescence microscopy and ICP-MS measurements revealed differences in uptake after 24 h of exposure which were attributed to particle surface coating and stability.


Assuntos
Compostos de Cádmio/análise , Daphnia/metabolismo , Compostos de Selênio/análise , Água/química , Animais , Daphnia/crescimento & desenvolvimento , Polietilenoglicóis/química , Solubilidade , Espectrometria de Fluorescência , Spirulina/metabolismo , Sulfetos/análise , Propriedades de Superfície , Água/parasitologia , Compostos de Zinco/análise
20.
BMC Genomics ; 10: 608, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20003521

RESUMO

BACKGROUND: The delta smelt (Hypomesus transpacificus) is a pelagic fish species listed as endangered under both the USA Federal and Californian State Endangered Species Acts and considered an indicator of ecosystem health in its habitat range, which is limited to the Sacramento-San Joaquin estuary in California, USA. Anthropogenic contaminants are one of multiple stressors affecting this system, and among them, current-use insecticides are of major concern. Interrogative tools are required to successfully monitor effects of contaminants on the delta smelt, and to research potential causes of population decline in this species. We have created a microarray to investigate genome-wide effects of potentially causative stressors, and applied this tool to assess effects of the pyrethroid insecticide esfenvalerate on larval delta smelt. Selected genes were further investigated as molecular biomarkers using quantitative PCR analyses. RESULTS: Exposure to esfenvalerate affected swimming behavior of larval delta smelt at concentrations as low as 0.0625 mug.L-1, and significant differences in expression were measured in genes involved in neuromuscular activity. Alterations in the expression of genes associated with immune responses, along with apoptosis, redox, osmotic stress, detoxification, and growth and development appear to have been invoked by esfenvalerate exposure. Swimming impairment correlated significantly with expression of aspartoacylase (ASPA), an enzyme involved in brain cell function and associated with numerous human diseases. Selected genes were investigated for their use as molecular biomarkers, and strong links were determined between measured downregulation in ASPA and observed behavioral responses in fish exposed to environmentally relevant pyrethroid concentrations. CONCLUSIONS: The results of this study show that microarray technology is a useful approach in screening for, and generation of molecular biomarkers in endangered, non-model organisms, identifying specific genes that can be directly linked with sublethal toxicological endpoints; such as changes in expression levels of neuromuscular genes resulting in measurable swimming impairments. The developed microarrays were successfully applied on larval fish exposed to esfenvalerate, a known contaminant of the Sacramento-San Joaquin estuary, and has permitted the identification of specific biomarkers which could provide insight into the factors contributing to delta smelt population decline.


Assuntos
Comportamento Animal/efeitos dos fármacos , Nitrilas/toxicidade , Osmeriformes/genética , Piretrinas/toxicidade , Natação , Amidoidrolases/genética , Animais , Biomarcadores , Regulação para Baixo , Espécies em Perigo de Extinção , Etiquetas de Sequências Expressas , Expressão Gênica/efeitos dos fármacos , Biblioteca Gênica , Inseticidas/toxicidade , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA , Poluentes Químicos da Água/toxicidade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...