RESUMO
Long-term exposure to fine particulate matter (PM2.5) posed injury for gastrointestinal and respiratory systems, ascribing with the lung-gut axis. However, the cross-talk mechanisms remain unclear. Here, we attempted to establish the response networks of lung-gut axis in mice exposed to PM2.5 at environmental levels. Male Balb/c mice were exposed to PM2.5 (dose of 0.1, 0.5, and 1.0 mg/kg) collected from Chengdu, China for 10 weeks, through intratracheally instillation, and examined the effect of PM2.5 on lung functions of mice. The changes of lung and gut microbiota and metabolic profiles of mice in different groups were determined. Furthermore, the results of multi-omics were conjointly analyzed to elucidate the primary microbes and the associated metabolites in lung and gut responsible for PM2.5 exposure. Accordingly, the cross-talk network and key pathways between lung-gut axis were established. The results indicated that exposed to PM2.5 0.1 mg/kg induced obvious inflammations in mice lung, while emphysema was observed at 1.0 mg/kg. The levels of metabolites guanosine, hypoxanthine, and hepoxilin B3 increased in the lung might contribute to lung inflammations in exposure groups. For microbiotas in lung, PM2.5 exposure significantly declined the proportions of Halomonas and Lactobacillus. Meanwhile, the metabolites in gut including L-tryptophan, serotonin, and spermidine were up-regulated in exposure groups, which were linked to the decreasing of Oscillospira and Helicobacter in gut. Via lung-gut axis, the activations of pathways including Tryptophan metabolism, ABC transporters, Serotonergic synapse, and Linoleic acid metabolism contributed to the cross-talk between lung and gut tissues of mice mediated by PM2.5. In summary, the microbes including Lactobacillus, Oscillospira, and Parabacteroides, and metabolites including hepoxilin B3, guanosine, hypoxanthine, L-tryptophan, and spermidine were the main drivers. In this lung-gut axis study, we elucidated some pro- and pre-biotics in lung and gut microenvironments contributed to the adverse effects on lung functions induced by PM2.5 exposure.
Assuntos
Poluentes Atmosféricos , Lesão Pulmonar , Masculino , Camundongos , Animais , Lesão Pulmonar/induzido quimicamente , Poluentes Atmosféricos/toxicidade , Poluentes Atmosféricos/metabolismo , Triptofano , Multiômica , Espermidina/metabolismo , Espermidina/farmacologia , Pulmão , Material Particulado/toxicidade , Material Particulado/metabolismo , Guanosina/metabolismo , Guanosina/farmacologia , Hipoxantinas/metabolismo , Hipoxantinas/farmacologiaRESUMO
Breast cancer is one of the leading causes of death in females, mainly because of metastasis. Oncometabolites, produced via metabolic reprogramming, can influence metastatic signaling cascades. Accordingly, and based on our previous results, we propose that metabolites from highly metastatic breast cancer cells behave differently from less-metastatic cells and may play a significant role in metastasis. For instance, we aim to identify these metabolites and their role in breast cancer metastasis. Less metastatic cells (MCF-7) were treated with metabolites secreted from highly metastatic cells (MDA-MB-231) and the gene expression of three epithelial-to-mesenchymal transition (EMT) markers including E-cadherin, N-cadherin and vimentin were examined. Some metabolites secreted from MDA-MB-231 cells significantly induced EMT activity. Specifically, hypoxanthine demonstrated a significant EMT effect and increased the migration and invasion effects of MCF-7 cells through a hypoxia-associated mechanism. Hypoxanthine exhibited pro-angiogenic effects via increasing the VEGF and PDGF gene expression and affected lipid metabolism by increasing the gene expression of PCSK-9. Notably, knockdown of purine nucleoside phosphorylase, a gene encoding for an important enzyme in the biosynthesis of hypoxanthine, and inhibition of hypoxanthine uptake caused a significant decrease in hypoxanthine-associated EMT effects. Collectively for the first time, hypoxanthine was identified as a novel metastasis-associated metabolite in breast cancer cells and represents a promising target for diagnosis and therapy.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/patologia , Espectroscopia de Prótons por Ressonância Magnética , Células MCF-7 , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Movimento Celular , Hipoxantinas/farmacologiaRESUMO
OBJECTIVES: To investigate the effect of the L-arginine metabolism on arthritis and inflammation-mediated bone loss. METHODS: L-arginine was applied to three arthritis models (collagen-induced arthritis, serum-induced arthritis and human TNF transgenic mice). Inflammation was assessed clinically and histologically, while bone changes were quantified by µCT and histomorphometry. In vitro, effects of L-arginine on osteoclast differentiation were analysed by RNA-seq and mass spectrometry (MS). Seahorse, Single Cell ENergetIc metabolism by profilIng Translation inHibition and transmission electron microscopy were used for detecting metabolic changes in osteoclasts. Moreover, arginine-associated metabolites were measured in the serum of rheumatoid arthritis (RA) and pre-RA patients. RESULTS: L-arginine inhibited arthritis and bone loss in all three models and directly blocked TNFα-induced murine and human osteoclastogenesis. RNA-seq and MS analyses indicated that L-arginine switched glycolysis to oxidative phosphorylation in inflammatory osteoclasts leading to increased ATP production, purine metabolism and elevated inosine and hypoxanthine levels. Adenosine deaminase inhibitors blocking inosine and hypoxanthine production abolished the inhibition of L-arginine on osteoclastogenesis in vitro and in vivo. Altered arginine levels were also found in RA and pre-RA patients. CONCLUSION: Our study demonstrated that L-arginine ameliorates arthritis and bone erosion through metabolic reprogramming and perturbation of purine metabolism in osteoclasts.
Assuntos
Artrite Experimental , Artrite Reumatoide , Reabsorção Óssea , Humanos , Camundongos , Animais , Osteoclastos , Artrite Reumatoide/patologia , Artrite Experimental/patologia , Inflamação/metabolismo , Camundongos Transgênicos , Arginina/farmacologia , Inosina/metabolismo , Inosina/farmacologia , Hipoxantinas/metabolismo , Hipoxantinas/farmacologia , Purinas/farmacologiaRESUMO
BACKGROUND: Lowered nicotinamide adenine dinucleotide (NAD+) levels in tumor cells drive tumor hyperprogression during immunotherapy, and its restoration activates immune cells. However, the effect of lenvatinib, a first-line treatment for unresectable hepatocellular carcinoma (HCC), on NAD+ metabolism in HCC cells, and the metabolite crosstalk between HCC and immune cells after targeting NAD+ metabolism of HCC cells remain unelucidated. METHODS: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and ultra-high-performance liquid chromatography multiple reaction monitoring-mass spectrometry (UHPLC-MRM-MS) were used to detect and validate differential metabolites. RNA sequencing was used to explore mRNA expression in macrophages and HCC cells. HCC mouse models were used to validate the effects of lenvatinib on immune cells and NAD+ metabolism. The macrophage properties were elucidated using cell proliferation, apoptosis, and co-culture assays. In silico structural analysis and interaction assays were used to determine whether lenvatinib targets tet methylcytosine dioxygenase 2 (TET2). Flow cytometry was performed to assess changes in immune cells. RESULTS: Lenvatinib targeted TET2 to synthesize and increase NAD+ levels, thereby inhibiting decomposition in HCC cells. NAD+ salvage increased lenvatinib-induced apoptosis of HCC cells. Lenvatinib also induced CD8+ T cells and M1 macrophages infiltration in vivo. And lenvatinib suppressed niacinamide, 5-Hydroxy-L-tryptophan and quinoline secretion of HCC cells, and increased hypoxanthine secretion, which contributed to proliferation, migration and polarization function of macrophages. Consequently, lenvatinib targeted NAD+ metabolism and elevated HCC-derived hypoxanthine to enhance the macrophages polarization from M2 to M1. Glycosaminoglycan binding disorder and positive regulation of cytosolic calcium ion concentration were characteristic features of the reverse polarization. CONCLUSIONS: Targeting HCC cells NAD+ metabolism by lenvatinib-TET2 pathway drives metabolite crosstalk, leading to M2 macrophages reverse polarization, thereby suppressing HCC progression. Collectively, these novel insights highlight the role of lenvatinib or its combination therapies as promising therapeutic alternatives for HCC patients with low NAD+ levels or high TET2 levels.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Quinolinas , Animais , Camundongos , Humanos , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , NAD/metabolismo , NAD/farmacologia , NAD/uso terapêutico , Linfócitos T CD8-Positivos , Cromatografia Líquida , Linhagem Celular Tumoral , Espectrometria de Massas em Tandem , Macrófagos/metabolismo , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Hipoxantinas/metabolismo , Hipoxantinas/farmacologia , Hipoxantinas/uso terapêuticoRESUMO
INTRODUCTION: With the mounting number of cancer survivors, the complications following cancer treatment become novel conundrums and starve for countermeasures. Intravenous immunoglobulin (IVIg) is a purified preparation for immune-deficient and autoimmune conditions. OBJECTIVES: Here, we investigated whether IVIg could be employed to fight against radiation injuries and explored the underlying mechanism. METHODS: Hematopoietic or gastrointestinal (GI) tract toxicity was induced by total body or abdominal local irradiation. High-throughput sequencing was performed to analyze the gut microbiota configurations and gene expression profile of small intestine. The untargeted metabolomics of gut microbiome was assessed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses. Hydrodynamic-based gene delivery was used to knockdown the target genes in vivo. RESULTS: Intravenous injection of IVIg protected against radiation-induced hematopoietic and GI tract toxicity in female mice but not in males. IVIg structured sex-characteristic gut microbiota configurations in abdominal irradiated mice. The irradiation enriched gut Lachnospiraceae in female mice but reduced those in males. IVIg injection combined with oral gavage of Lachnospiraceae or its metabolite hypoxanthine, alleviated radiation toxicity in male mice however, Lachnospiraceae or hypoxanthine alone failed to ameliorate the injuries. Abdominal local irradiation drove sex-distinct gene expression signatures in small intestine. Mechanistic investigation showed that replenishment of Lachnospiraceae or hypoxanthine offset abdominal radiation-reduced PLD1 expression in male mice. In females, irradiation elevated PLD1 expression. Deletion of PLD1 in GI tract of female mice erased the radioprotective effects of IVIg. CONCLUSION: IVIg battles against radiation injuries in a sex-specific, gut microbiome-dependent way through Lachnospiraceae/hypoxanthine/PLD1 axis. Our findings provide a sex-precise therapeutic avenue to improve the prognosis of cancer patients with radiotherapy in pre-clinical settings.
Assuntos
Gastroenteropatias , Microbioma Gastrointestinal , Lesões por Radiação , Camundongos , Masculino , Feminino , Animais , Imunoglobulinas Intravenosas/farmacologia , Caracteres Sexuais , Cromatografia Líquida , Espectrometria de Massas em Tandem , Lesões por Radiação/tratamento farmacológico , Hipoxantinas/farmacologiaRESUMO
OBJECTIVE: To investigate the protective effect and mechanism of polydatin (PD) against gouty nephropathy (GN) in mice. METHODS: Twenty-four mice were randomly divided into three groups: the control group (no treatment), the GN group (300 mg/kg hypoxanthine + 150 mg/kg potassium oxonate), and the GN + PD group (300 mg/kg hypoxanthine + 150 mg/kg potassium oxonate + 50 mg/kg PD). Histological changes in the kidneys and the levels of uric acid (UA), blood urea nitrogen (BUN), and serum creatinine (SCr) in the sera were measured. In addition, the expression of gasdermin D (GSDMD) protein in renal tubular epithelial cells, and the expression of NOD-like receptor protein 3 (NLRP3), GSDMD, and caspase-1 proteins in the kidney tissues were determined by immunohistochemistry, immunofluorescence, and Western blot. RESULTS: In vitro, PD inhibited the expression of NLRP3, caspase-1, and GSDMD and protected the renal tubular epithelial cells from pyroptosis. In vivo, PD treatment significantly ameliorated the pathological changes in kidney tissue, and reversed the decrease of serum UA and BUN in GN model mice. The expression of NLRP3, GSDMD, and caspase-1 proteins was also decreased in the PD-treated GN mice. CONCLUSION: The results suggest that PD has a protective effect on mice with GN, which may be related to the downregulation of NLRP3, GSDMD, and caspase-1 proteins and the inhibition of renal tubular epithelial cells pyroptosis.
Assuntos
Gota , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Camundongos , Caspases , Células Epiteliais/metabolismo , Gota/metabolismo , Hipoxantinas/metabolismo , Hipoxantinas/farmacologia , Rim/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/fisiologiaRESUMO
This study aims to investigate the anti-hyperuricemia effect and mechanism of anserine in hyperuricemic rats. Hyperuricemic rats were induced with a combination of 750 mg per kg bw d potassium oxazinate (PO) and 200 mg per kg bw d hypoxanthine for a week, and the rats were separately orally administered anserine (20, 40, 80 mg kg-1) and allopurinol (10 mg kg-1) for three weeks. The results show that the content of serum uric acid (SUA) decreased by approximately 40% and 60% after the intervention of anserine and allopurinol, respectively. The activity of superoxide dismutase (SOD) was increased and the levels of malondialdehyde (MDA), alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were significantly decreased in the anserine groups. After the administration of anserine, the contents of blood urea nitrogen (BUN) and creatinine (Cr) were reduced in the kidney, and the levels of the proinflammatory cytokines IL-1ß, IL-6ß, TNF-α and TGF-ß and inflammatory cell infiltration were reduced in both the liver and kidney. Moreover, the gene expressions of xanthine oxidase (XOD), renal urate transporter 1 (URAT1) and glucose transporter type 9 (GLUT9) were downregulated by anserine administration, and the gene expressions of ATP-binding cassette transporter G2 (ABCG2), organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3) were upregulated at the same time. These findings suggest that hepatorenal injury was repaired by anserine, which further regulated the expression of hepatic XOD and renal URAT1, GLUT9, ABCG2, OAT1 and OAT3 to relieve hyperuricemia in rats.
Assuntos
Hiperuricemia , Transportadores de Ânions Orgânicos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Alanina Transaminase/metabolismo , Fosfatase Alcalina/metabolismo , Alopurinol/metabolismo , Alopurinol/farmacologia , Animais , Anserina/metabolismo , Anserina/farmacologia , Creatinina , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Hiperuricemia/metabolismo , Hipoxantinas/metabolismo , Hipoxantinas/farmacologia , Rim , Fígado/metabolismo , Malondialdeído/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Potássio/metabolismo , Potássio/farmacologia , Ratos , Superóxido Dismutase/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ácido Úrico , Xantina Oxidase/metabolismoRESUMO
Helicobacter pylori (Hp) is a human pathogen that lives in the gastric mucosa of approximately 50% of the world's population causing gastritis, peptic ulcers, and gastric cancer. An increase in resistance to current drugs has sparked the search for new Hp drug targets and therapeutics. One target is the disruption of nucleic acid production, which can be achieved by impeding the synthesis of 6-oxopurine nucleoside monophosphates, the precursors of DNA and RNA. These metabolites are synthesized by Hp xanthine-guanine-hypoxanthine phosphoribosyltransferase (XGHPRT). Here, nucleoside phosphonates have been evaluated, which inhibit the activity of this enzyme with Ki values as low as 200 nM. The prodrugs of these compounds arrest the growth of Hp at a concentration of 50 µM in cell-based assays. The kinetic properties of HpXGHPRT have been determined together with its X-ray crystal structure in the absence and presence of 9-[(N-3-phosphonopropyl)-aminomethyl-9-deazahypoxanthine, providing a basis for new antibiotic development.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/metabolismo , Pentosiltransferases/metabolismo , Sequência de Aminoácidos , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/química , Sítios de Ligação , Cristalografia por Raios X , Gastroenteropatias/tratamento farmacológico , Gastroenteropatias/microbiologia , Gastroenteropatias/patologia , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/patologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/enzimologia , Humanos , Hipoxantina Fosforribosiltransferase/química , Hipoxantina Fosforribosiltransferase/metabolismo , Hipoxantinas/química , Hipoxantinas/metabolismo , Hipoxantinas/farmacologia , Hipoxantinas/uso terapêutico , Cinética , Simulação de Dinâmica Molecular , Organofosfonatos/química , Organofosfonatos/metabolismo , Organofosfonatos/farmacologia , Organofosfonatos/uso terapêutico , Pentosiltransferases/química , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
Multipronged approach was used to synthesize a library of diverse C-8 cyclopentyl hypoxanthine analogs from a common intermediate III. Several potent and selective compounds were identified and evaluated for pharmacokinetic (PK) properties in Wistar rats. One of the compounds 14 with acceptable PK parameters was selected for testing in in vivo primary acute diuresis model. The compound demonstrated significant diuretic activity in this model.
Assuntos
Antagonistas do Receptor A1 de Adenosina/química , Antagonistas do Receptor A1 de Adenosina/farmacologia , Hipoxantinas/química , Hipoxantinas/farmacologia , Antagonistas do Receptor A1 de Adenosina/síntese química , Antagonistas do Receptor A1 de Adenosina/farmacocinética , Animais , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Cromatografia Líquida , Desenho de Fármacos , Células HEK293 , Humanos , Hipoxantinas/síntese química , Hipoxantinas/farmacocinética , Masculino , Espectrometria de Massas , Espectroscopia de Prótons por Ressonância Magnética , Ensaio Radioligante , Ratos , Ratos WistarRESUMO
Hepatocellular carcinoma (HCC) is a highly vascular tumor, and treatment options for patients of advanced-stage are limited. Nitric oxide (NO), which is derived from endothelial nitric oxide synthase (eNOS), provides crucial signals for angiogenesis in the tumor microenvironment. Tetrahydrobiopterin (BH4) is an essential cofactor eNOS and represents a critical determinant of NO production. To examine whether treatment of 2,4-diamino-6-hydroxypyrimidine (DAHP) inhibits angiogenesis of HCC, BALB/c-nu mice were injected with HepG-2 cells with DAHP. Supplemental DAHP treatment decreased K-ras mRNA transcripts, inhibition of phosphorylation of eNOS and Akt, inhibition of guanosine triphosphate cyclohydrolase (GTPCH), and decreased significantly NO synthesis, and then inhibited angiogenesis, compared with the results observed in the saline group. Histopathology demonstrated angiogenesis and tumor formation were significantly inhibited in HCC. DAHP downregulates GTPCH protein expression, corresponding to decreased levels of BH4 and the contents of NO. In addition, DAHP downregulates eNOS and Akt protein expression, corresponding to decreased eNOS phosphorylation at Ser1177 and Akt phosphorylation, compared with the saline control. We suggest that DAHP, recognized as a specific competitive inhibitor of GTPCH, can decrease tumor BH4 and NO by the inhibition of the wild-type Ras-PI3K/Akt pathway, and then inhibiting angiogenesis, and may provide a novel and promising way to target BH4 synthetic pathways to inhibit angiogenesis and to control potential progression of HCC. Whether DAHP has a therapeutic potential will require more direct testing in humans.
Assuntos
Biopterinas/análogos & derivados , Carcinoma Hepatocelular/genética , Regulação para Baixo/genética , Neoplasias Hepáticas/genética , Neovascularização Patológica/genética , Animais , Biopterinas/genética , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Genes ras/genética , Células Hep G2 , Humanos , Hipoxantinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/patologia , Óxido Nítrico/genética , Óxido Nítrico Sintase Tipo III/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genéticaRESUMO
The gold(I) mixed-ligand complexes involving O-substituted derivatives of 9-deazahypoxanthine (HLn) and triphenylphosphine (PPh3) with the general formula [Au(Ln)(PPh3)] (1-5) were prepared and thoroughly characterized by elemental analysis, FT-IR and multinuclear NMR spectroscopy, ESI+ mass spectrometry, single crystal X-ray (HL5 and complex 2) and TG/DTA analyses. Complexes 1-5 were evaluated for their in vitro antitumor activity against nine human cancer lines, i.e. MCF7 (breast carcinoma), HOS (osteosarcoma), A549 (adenocarcinoma), G361 (melanoma), HeLa (cervical cancer), A2780 (ovarian carcinoma), A2780R (ovarian carcinoma resistant to cisplatin), 22Rv1 (prostate cancer) and THP-1 (monocytic leukaemia), for their in vitro anti-inflammatory activity using a model of LPS-activated macrophages, and for their in vivo antiedematous activity by λ-carrageenan-induced hind paw edema model on rats. The results showed that the complexes 1-5 exhibit selective in vitro cytotoxicity against MCF7, HOS, 22Rv1, A2780 and A2780R, with submicromolar IC50 values for 2 against the MCF7 (0.6 µM) and HOS (0.9 µM). The results of in vitro cytotoxicity screening on primary culture of human hepatocytes (HEP220) revealed up to 30-times lower toxicity of compounds against healthy cells as compared with cancer cells. Additionally, the complexes 1-5 significantly influence the secretion and expression of pro-inflammatory cytokines TNF-α and IL-1ß by a similar manner as a commercially used anti-arthritic drug Auranofin. The tested complexes also significantly influence the rate and overall volume of the edema, caused by the intraplantar application of λ-carrageenan polysaccharide to rats. Based on these promising results, the presented compounds could qualify to become feasible candidates for advanced testing as potential antitumor and anti-inflammatory drug-like compounds.
Assuntos
Anti-Inflamatórios/uso terapêutico , Antineoplásicos/uso terapêutico , Edema/tratamento farmacológico , Ouro/química , Hipoxantinas/química , Inflamação/tratamento farmacológico , Neoplasias/tratamento farmacológico , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ouro/farmacologia , Ouro/uso terapêutico , Hepatócitos/efeitos dos fármacos , Humanos , Hipoxantinas/farmacologia , Hipoxantinas/uso terapêutico , Ratos , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
We herein investigated the in vitro effect of hypoxanthine on the activities of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) in erythrocytes, as well as on thiobarbituric acid-reactive substances (TBA-RS), in the plasma of rats. Results showed that hypoxanthine, when added to the incubation medium, enhanced CAT (10.0 µM), GSH-Px and SOD (8.5 µM and 10.0 µM) activities in erythrocytes of 15-day-old rats, reduced CAT activity (10.0 µM) and enhanced GSH-Px activity (10.0 µM) in erythrocytes of 30-day-old rats, reduced CAT activity (10.0 µM) and enhanced GSH-Px activity (8.5 µM and 10.0 µM) in erythrocytes of 60-day-old rats, as compared to controls. In addition, hypoxanthine (10.0 µM) enhanced TBA-RS levels in the plasma of 30- and 60-day old rats. Furthermore, we also tested the influence of allopurinol, trolox, and ascorbic acid on the effects elicited by hypoxanthine on the antioxidant enzymes and TBA-RS. Allopurinol and/or administration of antioxidants prevented most alterations caused by hypoxanthine in the oxidative stress parameters evaluated. Findings suggest that hypoxanthine alters antioxidant defenses and induces lipid peroxidation in the blood of rats; however, in the presence of allopurinol and antioxidants, some of these alterations in oxidative stress caused are prevented. Data indicate that, in humans, antioxidant administration might serve as a potential adjuvant therapy for ameliorating the damage caused by hypoxanthine.
Assuntos
Alopurinol/farmacologia , Ácido Ascórbico/farmacologia , Eritrócitos/enzimologia , Hipoxantinas/fisiologia , Estresse Oxidativo , Vitamina E/farmacologia , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Catalase/metabolismo , Cromanos/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Glutationa Peroxidase/metabolismo , Hipoxantinas/farmacologia , Malondialdeído/metabolismo , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
2,4-Diamino-6-hydroxypyrimidine (DAHP) is considered a specific inhibitor of BH(4) biosynthesis and is widely used in order to elucidate the possible biological function of BH(4) in various cells. In the present study, we found that both the synthesis of tetrahydrobiopterin (BH(4)) and expression of vascular cell adhesion molecule 1 (VCAM-1) were increased in human umbilical vein endothelial cells (HUVEC) treated with proinflammatory cytokines. Thus we examined the effects of DAHP to clarify whether BH(4) might be involved in the expression of VCAM-1 in HUVEC. DAHP reduced the levels of both BH(4) and VCAM-1 induced by TNF-alpha and IFN-gamma. However, the dose-response curves of DAHP for the suppression of the VCAM-1 level and that of BH(4) level were markedly different. Supplementation with sepiapterin failed to restore the depressed VCAM-1 level, although it completely restored the BH(4) level. Furthermore, DAHP significantly reduced the VCAM-1 level under the experimental conditions using TNF-alpha alone, which failed to induce BH(4) production. Taken together, these results indicate that DAHP inhibited the expression of VCAM-1 in a BH(4)-independent manner in HUVEC. In the present study, we also found that DAHP significantly suppressed the accumulation of cytokine-induced NF-kappaB (p65) in the nucleus as well as the mRNA levels of VCAM-1 and GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH(4) synthesis. The data obtained in this study suggest that DAHP reduced VCAM-1 and GTPCH protein synthesis at least partially via suppressing the NF-kappaB level in the nucleus of HUVEC.
Assuntos
Biopterinas/análogos & derivados , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Hipoxantinas/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Biopterinas/análise , Biopterinas/biossíntese , Células Cultivadas , Citocinas/farmacologia , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , GTP Cicloidrolase/análise , GTP Cicloidrolase/biossíntese , Humanos , Interferon gama/farmacologia , Cinética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais/citologiaRESUMO
The cofactor tetrahydrobiopterin plays critical roles in the modulation of the signaling molecules dopamine, serotonin, and nitric oxide. Deficits in cofactor synthesis have been associated with several human hereditary diseases. Responsibility for the regulation of cofactor pools resides with the first enzyme in its biosynthetic pathway, GTP cyclohydrolase I. Because organisms must be able to rapidly respond to environmental and developmental cues to adjust output of these signaling molecules, complex regulatory mechanisms are vital for signal modulation. Mammalian GTP cyclohydrolase is subject to end-product inhibition via an associated regulatory protein and to positive regulation via phosphorylation, although target residues are unknown. GTP cyclohydrolase is composed of a highly conserved homodecameric catalytic core and non-conserved N-terminal domains proposed to be regulatory sites. We demonstrate for the first time in any organism that the N-terminal arms of the protein serve regulatory functions. We identify two different modes of regulation of the enzyme mediated through the N-terminal domains. The first is end-product feedback inhibition, catalytically similar to that of the mammalian enzyme, except that feedback inhibition by the cofactor requires sequences in the N-terminal arms rather than a separate regulatory protein. The second is a novel inhibitory interaction between the N-terminal arms and the active sites, which can be alleviated through the phosphorylation of serine residues within the N termini. Both mechanisms allow for acute and highly responsive regulation of cofactor production as required by downstream signaling pathways.
Assuntos
Drosophila melanogaster/enzimologia , GTP Cicloidrolase/metabolismo , Animais , Boroidretos/farmacologia , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , GTP Cicloidrolase/genética , Hipoxantinas/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The folate antagonist methotrexate (MTX) inhibits synthesis of tetrahydrofolate (THF), pyrimidines and purines, and induces differentiation in several cell types. At 1 microM, MTX reduced proliferation and induced differentiation in HT29 colon cancer cells; the latter effect was augmented (P < 0.001) by thymidine (100 microM) but was reversed (P < 0.001) by the purines, hypoxanthine (Hx; 100 microM) and adenosine (100 microM). In contrast 5-fluoro-uracil (5-FU), a specific thymidylate synthase (TS) inhibitor, had no effect on differentiation, suggesting that MTX-induced differentiation is not due to a reduction in thymidine but to the inhibition of purine biosynthesis. Inhibition of cyclic AMP (cAMP) by RpcAMP (25 microM) further enhanced (P < 0.001) MTX induced differentiation, whereas the cAMP activator forskolin (10 microM) reversed (P < 0.001) MTX induced differentiation. These observations implicate a central role of adenosine and cAMP in MTX induced differentiation. By combining Western blot analysis with liquid chromatography-mass spectrometry (LC-MS)and HPLC analyses we also reveal both the expression and activity of key enzymes (i.e. methionine synthase (MS), s-adenosylhomocysteinase, cystathionine beta-synthase and ornithine decarboxylase) regulating methyl cycle, transsulfuration and polyamine pathways in HT29 colon cancer cells. At 1 microM, MTX induced differentiation was associated with a marked reduction in the intracellular concentrations of adenosine and, consequently, S-adenosylmethionine (SAM), S-adenosylhomocysteine, polyamines and glutathione (GSH). Importantly, the marked reduction in methionine that accompanied MS inhibition following MTX treatment was non-limiting with respect to SAM synthesis. Collectively, these findings indicate that the effects of MTX on cellular differentiation and single carbon metabolism are primarily due to the intracellular depletion of purines.
Assuntos
Adenocarcinoma/patologia , Diferenciação Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Metotrexato/farmacologia , Purinas/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenosina/metabolismo , Adenosina/farmacologia , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HT29 , Humanos , Hipoxantinas/farmacologia , MAP Quinase Quinase Quinases/efeitos dos fármacos , MAP Quinase Quinase Quinases/metabolismo , Metionina/metabolismo , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Poliaminas/metabolismo , Transdução de Sinais , Tetra-Hidrofolatos/farmacologia , Timidina/farmacologiaRESUMO
BACKGROUND AND PURPOSE: Purinergic nucleoside inosine elicits protection and regeneration during various injuries. The purpose of this study was to examine the protective effects of inosine against cerebral ischemia. METHODS: Adult Sprague-Dawley rats were anesthetized. Inosine, hypoxathine, or vehicle was administered intracerebroventricularly before transient right middle cerebral artery occlusion (MCAo). Animals were placed in behavioral chambers 2 days to 2 weeks after MCAo and then euthanized for tri-phenyl-tetrazolium chloride staining. Glutamate release was measured by microdialysis/high-performance liquid chromatography, and single-unit action potentials were recorded from neurons in the parietal cortex. RESULTS: Stroke animals receiving inosine pretreatment demonstrated a higher level of locomotor activity and less cerebral infarction. Intracerebroventricular administration of the same dose of hypoxanthine did not confer protection. Coadministration of selective A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191) significantly reduced inosine-mediated protection. Inosine did not alter basal glutamate release, nor did it reduce ischemia-evoked glutamate overflow from cerebral cortex. However, inosine antagonized glutamate-induced electrophysiological excitation in cerebral cortical neurons. CONCLUSIONS: Inosine inhibits glutamate postsynaptic responses and reduces cerebral infarction. Its protective effect against ischemia/reperfusion-related insults may involve activation of adenosine A3 receptors.
Assuntos
Isquemia Encefálica/prevenção & controle , Inosina/farmacologia , Animais , Isquemia Encefálica/etiologia , Hipoxantinas/administração & dosagem , Hipoxantinas/farmacologia , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/prevenção & controle , Injeções Intraventriculares , Inosina/administração & dosagem , Masculino , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Fibroproliferative response of rat heart and lung fibroblasts to the lanthanide cerium was examined, as the element has been implicated in the causation of cardiac and pulmonary fibrosis. Fibroblasts from both of the organs were morphologically identical, and the response to fetal bovine serum, a nonspecific mitogen, was also comparable. The oxygen radical generator (hypoxanthine + xanthine oxidase [Hyp. + XO]) induced a proliferative response that was neutralized in both cardiac and lung fibroblasts by free-radical scavengers. Superoxide dismutase was more effective than catalase in reducing the mitogenic effect of Hyp. + XO. The free-radical scavenger N-acetyl-L-cysteine neutralized the free-radical-mediated changes in pulmonary fibroblasts but had a negative effect in cardiac fibroblasts, indicating a tissue-dependent variation. Reactive oxygen species are known to act as biological mediators of tissue fibrosis induced by metallic compounds. Exposure to low levels of cerium (0.5 microM) stimulated a mitogenic response in cardiac fibroblasts, but the pulmonary fibroblasts were not sensitized by the element. Tissue-dependent variation in proliferative response to cerium shows a positive association with intracellular generation of reactive oxygen species. Fibrotic changes in cerium pneumoconiosis may either be replacement fibrosis following tissue damage or mediated by nonfibroblastic cells. The study confirms that cardiac and pulmonary fibroblasts are dissimilar cellular subtypes.
Assuntos
Divisão Celular/efeitos dos fármacos , Cério/farmacologia , Fibroblastos/efeitos dos fármacos , Radicais Livres/metabolismo , Pulmão/citologia , Miocárdio/citologia , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Animais , Catalase/farmacologia , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Sequestradores de Radicais Livres/metabolismo , Hipoxantinas/farmacologia , Especificidade de Órgãos , Ratos , Ratos Wistar , Superóxido Dismutase/farmacologia , Xantina Oxidase/farmacologiaRESUMO
AIM: It has recently been shown that nitric oxide synthase in the presence of suboptimal levels of tetrahydrobiopterin (BH(4)), an essential cofactor of nitric oxide synthase, may favor increased production of oxygen free radicals. This study was designed to define the role of BH(4) in myocardial ischemia-reperfusion injury. METHODS: Isolated perfused rat hearts were subjected to 37 degrees C ischemia and reperfusion. Hearts were received with BH(4) or vehicle for 5 min just before ischemia and during the first 5 min of the reperfusion period. The effects of BH(4) on left ventricular function, myocardial contents of lipid peroxidation and high energy phosphates, and levels of lactate dehydrogenase and nitrite plus nitrate in perfusate before ischemia and after reperfusion were estimated. Moreover, the effect of BH(4) given with 2,4-diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of BH(4) production, intraperitoneally 24 h before the experiments were estimated. RESULTS: BH(4) improved contractile and metabolic abnormalities in reperfused hearts. Furthermore, BH(4) significantly alleviated ischemic contracture during ischemia, and restored diminished perfusate levels of nitrite plus nitrate after reperfusion. On the other hand, DAHP-treatment aggravated ischemia-reperfusion induced functional and metabolic abnormalities. Administration of BH(4) improved DAHP-induced functional and metabolic abnormalities. CONCLUSION: Results demonstrated that BH(4) lessens ischemia-reperfusion injury in isolated perfused rat hearts. Conversely, deficiency of BH(4) seems to accelerate endothelial dysfunction and myocardial ischemia-reperfusion injury. Present data may be compatible with the hypothesis that nitric oxide synthase in the presence of insufficiency of BH(4) serve as the cause of oxidative injury.
Assuntos
Antioxidantes/farmacologia , Biopterinas/análogos & derivados , Biopterinas/uso terapêutico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Antioxidantes/uso terapêutico , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Coração/fisiopatologia , Hipoxantinas/farmacologia , Técnicas In Vitro , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Masculino , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Óxido Nítrico/análise , Óxido Nítrico/metabolismo , Ratos , Ratos Sprague-Dawley , Função Ventricular Esquerda/efeitos dos fármacos , Função Ventricular Esquerda/fisiologiaRESUMO
OBJECTIVE: Guanosine triphosphate-cyclohydrolase I (GTP-CHI) is the first and rate-limiting enzyme for the de novo biosynthesis of biopterin. The objective of present study was to observe the effect of 2,4-diamino-6-hydroxy-pyrimidine (DAHP), an inhibitor of GTP-CHI, on the development of postburn Staphylococcus aureus sepsis. DESIGN: A prospective, controlled animal study. SETTING: A research laboratory in a hospital. SUBJECTS: Male Wistar rats. INTERVENTIONS: Fifty-six male Wistar rats were randomly divided into four groups as follows: normal control group (n = 10), scald control group (n = 10), postburn sepsis group (n = 20), and DAHP treatment group (n = 16). In the scald control group, rats were subjected to a 20% total body surface area third-degree scald injury and then were killed at 24 hrs. In the postburn sepsis group (n = 20), rats were inflicted with 20% total body surface area third-degree scald followed by Staphylococcus aureus challenge, and they were further divided into 2- and 6-hr groups. In the DAHP treatment group (n = 16), animals were intraperitoneally injected with a dose of 1 g/kg DAHP before Staphylococcus aureus challenge and then were further divided into 2- and 6-hr groups. Tissue samples from liver, kidneys, lungs, and heart were collected to determine GTP-CHI, inducible nitric oxide synthase, and tumor necrosis factor-alpha messenger RNA expression. Meanwhile, biopterin and nitric oxide concentrations in these tissues were also measured. MEASUREMENTS AND MAIN RESULTS: After the scald injury followed by Staphylococcus aureus challenge, GTP-CHI messenger RNA expression and biopterin concentrations were significantly elevated in various tissues such as liver, heart, kidneys, and lungs, as were the values of inducible nitric oxide synthase messenger RNA expression and nitric oxide formation (p <.01). Pretreatment with DAHP significantly reduced GTP-CHI/biopterin induction (p <.05-.01), and the up-regulation of inducible nitric oxide synthase/nitric oxide was also suppressed. Furthermore, DAHP administration inhibited the gene expression of tumor necrosis factor-alpha. Two hours after septic challenge, tumor necrosis factor-alpha messenger RNA expression in liver, kidneys, and lungs in the DAHP-treated group was 35.7%, 37.3%, and 33.0% of that in the postburn septic group, respectively. Additionally, in animals without DAHP treatment, the 6-hr mortality rate was 55.6% (20 of 36), whereas it was only 25.0% in DAHP-treated animals (4 of 16, p =.08). CONCLUSIONS: Early treatment with DAHP might be a potential strategy to prevent the development of postburn Staphylococcal sepsis, which appears to be associated with down-regulation of biopterin and nitric oxide formation by DAHP.
Assuntos
Biopterinas/análogos & derivados , Queimaduras/microbiologia , Inibidores Enzimáticos/uso terapêutico , GTP Cicloidrolase/antagonistas & inibidores , Hipoxantinas/uso terapêutico , Sepse/prevenção & controle , Infecções Estafilocócicas/prevenção & controle , Análise de Variância , Animais , Biopterinas/biossíntese , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hipoxantinas/farmacologia , Masculino , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/biossíntese , Estudos Prospectivos , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Sepse/etiologia , Sepse/microbiologia , Infecções Estafilocócicas/etiologiaRESUMO
Tetrahydrobiopterin (BH4) is an essential co-factor for nitric oxide synthases (NOS). The aim of the present work was to study whether BH4 deficiency affects the vulnerability of neurones in primary culture to hypoxia. Intracellular BH4 levels were depleted by pre-incubating neurones with 5 mm 2,4-diamino-6-hydroxypyrimidine (DAHP) for 18 h, after which cells were exposed for 1 h to normoxic or hypoxic conditions. Our results showed that whereas neurones were resistant to hypoxia-induced cellular damage, BH4 deficiency in neurones led to oxidative stress, mitochondrial depolarization, ATP depletion and necrosis after 1 h of hypoxia. Indeed, hypoxia specifically inhibited mitochondrial complex IV activity in BH4-deficient neurones. All these effects were counteracted when neuronal BH4 levels were restored by incubating cells with exogenous BH4 during the hypoxic period. Moreover, hypoxia-induced damage in BH4-deficient neurones was prevented when Nomega-nitro-l-arginine monomethyl ester (NAME), haemoglobin or superoxide dismutase plus catalase were present during the hypoxic period, suggesting that peroxynitrite might be involved in the process. In fact, BH4 deficiency elicited neuronal NO dysfunction, resulting in an increase in peroxynitrite generation by cells, as shown by the enhancement in tyrosine nitration; this was prevented by supplements of BH4, NAME, haemoglobin or superoxide dismutase plus catalase during hypoxia. Our results suggest that BH4 deficiency converts neuronal NOS into an efficient peroxynitrite synthase, which is responsible for the increase in neuronal vulnerability to hypoxia-induced mitochondrial damage and necrosis.