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1.
Cell Physiol Biochem ; 54(1): 126-141, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32017483

RESUMO

BACKGROUND/AIMS: Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter and hormone with important physiological functions in many organs, including the intestine. We have previously shown that 5-HT activates the aryl hydrocarbon receptor (AhR) in intestinal epithelial cells (IECs) via a serotonin transporter (SERT)-dependent mechanism. AhR is a nuclear receptor that binds a variety of molecules including tryptophan (TRP) metabolites to regulate physiological processes in the intestine including xenobiotic detoxification and immune modulation. We hypothesized that 5-HT activates AhR indirectly by interfering with metabolic clearance of AhR ligands by cytochrome P450 1A1 (CYP1A1). METHODS: Inhibition of CYP1A1 activity by 5-HT was assessed in the human intestinal epithelial cell line Caco-2 and recombinant CYP1A1 microsomes using both luciferase and LC-MS/MS. Degradation of 5-HT by recombinant CYP1A1 was measured by LC-MS/MS. For in vitro studies, CYP1A1 and CYP1B1 mRNA expression levels were measured by RT-PCR and CYP1A1 activity was measured by ethoxyresorufin-O-deethylase (EROD) assays. For in vivo studies, AhR ligands were administered to SERT KO mice and WT littermates and intestinal mucosa CYP1A1 mRNA was measured. RESULTS: We show that 5-HT inhibits metabolism of both the pro-luciferin CYP1A1 substrate Luc-CEE as well as the high affinity AhR ligand 6-formylindolo[3,2-b] carbazole (FICZ). Recombinant CYP1A1 assays revealed that 5-HT is metabolized by CYP1A1 in an NADPH dependent manner. Treatment with 5-HT in TRP-free medium, which is devoid of trace AhR ligands, showed that 5-HT requires the presence of AhR ligands to activate AhR. Cotreatment with 5-HT and FICZ confirmed that 5-HT potentiates induction of AhR target genes by AhR ligands. However, this was only true for ligands which are CYP1A1 substrates such as FICZ. Administration of ß-napthoflavone by gavage or indole-3-carbinol via diet to SERT KO mice revealed that lack of SERT impairs intestinal AhR activation. CONCLUSION: Our studies provide novel evidence of crosstalk between serotonergic and AhR signaling where 5-HT can influence the ability of AhR ligands to activate the receptor in the intestine.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Serotonina/farmacologia , Transcrição Genética/efeitos dos fármacos , Animais , Células CACO-2 , Carbazóis/farmacologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Humanos , Ligantes , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/deficiência , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , beta-Naftoflavona/administração & dosagem
2.
Toxicol Lett ; 320: 37-45, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31778776

RESUMO

As a major toxicant which is abundant in tobacco smoking, benzo(a)pyrene (BaP) is considered as a strong carcinogen of lung cancer. In spite of the intensive research, the role that BaP plays in lung cancer still lacks a comprehensive and precise understanding. Recently, a long non-coding RNA, linc00673, has emerged as a central player in different kinds of malignancies, including non-small cell lung cancer (NSCLC). In the present study, we found that BaP with the concentration of no more than 8 µM did not affect cell proliferation in the NSCLC cell line A549, while it significantly enhanced A549 cell migration and invasion. Further results revealed that BaP promoted mesenchymal biomarkers expression and inhibited the major epithelial biomarker E-cadherin in a time and dose dependent manner, which indicated epithelial-mesenchymal transition (EMT) was induced by BaP in A549 cells. Through quantitative real-time PCR, we observed that BaP significantly elevated the expression level of linc00673. While after the knockdown of aryl hydrocarbon receptor (AHR), the up-regulating effect of BaP on linc00673 was reversed. Furthermore, silencing linc00673 significantly suppressed the BaP-induced migration, invasion, and EMT in A549 cells. In summary, our study demonstrates that BaP promotes A549 cell migration, invasion and EMT through up-regulating the expression of linc00673 in an AHR-dependent manner.


Assuntos
Benzo(a)pireno/toxicidade , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , RNA Longo não Codificante/metabolismo , Células A549 , Antígenos CD/genética , Antígenos CD/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caderinas/genética , Caderinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Invasividade Neoplásica , RNA Longo não Codificante/genética , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Fatores de Tempo , Regulação para Cima
3.
Aquat Toxicol ; 218: 105334, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31743820

RESUMO

Tributyltin (TBT) and dioxin-like polychlorinated biphenyls (PCBs) are environmental contaminants that are highly toxic to fish and co-occur in New Bedford Harbor (NBH), an estuarine Superfund site located in Massachusetts, USA. Atlantic killifish (Fundulus heteroclitus) that reside in NBH (and other highly contaminated sites along the east coast of the United States) have developed resistance to activation of the aryl hydrocarbon receptor (AHR) pathway and the toxicity of dioxin-like chemicals, such as 3,3',4,4',5-pentachlorobiphenyl, PCB126. In many biological systems, TBT disregulates adipose and bone development via the PPARγ-RXR pathway; AHR activation also disrupts adipose and bone homeostasis, potentially through molecular crosstalk between AHR and PPARγ. However, little is known about how co-exposure and the interaction of these pathways modulate the toxicological effects of these contaminants. Here, we tested the hypotheses that TBT would induce teratogenesis in killifish via activation of PPARγ and that PCB126 co-exposure would suppress PPARγ pathway activation in PCB-sensitive killifish from a reference site (Scorton Creek, SC, PCB-sensitive) but not in PCB-tolerant NBH killifish. Killifish embryos from both populations exposed to TBT (50 and 100 nM) displayed caudal fin deformities. TBT did not change the expression of pparg or its target genes related to adipogenesis (fabp11a and fabp1b) in either population. However, expression of osx/sp7, an osteoblast marker gene, and col2a1b, a chondroblast marker gene, was significantly suppressed by TBT only in SC killifish. An RXR-specific agonist, but not a PPARγ-specific agonist, induced caudal fin deformities like those observed in TBT-treated embryos. PCB126 did not induce caudal fin deformities and did not exacerbate TBT-induced fin deformities. Further, PCB126 increased expression of pparg in SC embryos and not NBH embryos, but did not change the expression of fabp1b. Taken together, these results suggest that in killifish embryos the PPARγ pathway is regulated in part by AHR, but is minimally active at least in this early life stage. In killifish, RXR activation, rather than PPARγ activation, appears to be the mechanism by which TBT induces caudal fin teratogenicity, which is not modulated by AHR responsiveness.


Assuntos
Nadadeiras de Animais/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Fundulidae , PPAR gama/metabolismo , Bifenilos Policlorados/toxicidade , Receptores de Hidrocarboneto Arílico/metabolismo , Compostos de Trialquitina/toxicidade , Poluentes Químicos da Água/toxicidade , Nadadeiras de Animais/anormalidades , Animais , Resistência a Medicamentos/efeitos dos fármacos , Sinergismo Farmacológico , Embrião não Mamífero/anormalidades , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Massachusetts , PPAR gama/genética , Receptor Cross-Talk , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais/efeitos dos fármacos
4.
Toxicol Lett ; 319: 85-94, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31730885

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is a chronic hepatic disease associated with the excessive accumulation of lipids in the liver. Premenopausal women are protected from the liver metabolic complications of obesity compared with body mass index (BMI)-matched men. This protection may be related to estrogen's ability to limit liver fat accumulation. Aryl hydrocarbon receptor (AhR), a novel regulator of NAFLD, may be an important target for regulating estrogen homeostasis. In present study, we used benzo[a]pyrene (BaP), a classic and potent ligand of AhR, to activate AhR pathway causes overexpression of the estrogen-metabolizing enzyme cytochrome P450 1A1 (CYP1A1) and affects the expression of important genes involved in hepatic lipid regulation. BaP induces CYP1A1 expression through AhR signaling and inhibits the protective effect of 17ß-estradiol (E2) on hepatic steatosis, characterized by triglyceride accumulation, and markers of liver damage are significantly elevated. The expression of adipogenic genes involved in the hepatic lipid metabolism of sterol regulatory element-binding protein-1c (SREBP-1c) was increased compared with that in the control group. Furthermore, the mRNA and protein levels of peroxisome proliferator-activated receptor alpha (PPARα), which is involved in fatty acid oxidation, were significantly reduced. Taken together, our results revealed that the steatotic effect of AhR is likely due to overexpression of the E2 metabolic enzyme CYP1A1, which affects the estrogen signaling pathway, leading to the suppression of fatty acid oxidation, inhibition of the hepatic export of triglycerides, and an increase in peripheral fat mobilization. The results from this study may help establish AhR as a novel therapeutic and preventive target for fatty liver disease.


Assuntos
Hepatopatia Gordurosa não Alcoólica/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Animais , Benzo(a)pireno/farmacologia , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , Estradiol/farmacologia , Estrogênios/metabolismo , Feminino , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , PPAR alfa/biossíntese , PPAR alfa/genética , Receptores de Hidrocarboneto Arílico/agonistas , Transdução de Sinais/efeitos dos fármacos , Proteína de Ligação a Elemento Regulador de Esterol 1/biossíntese , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Triglicerídeos/metabolismo
5.
Biochim Biophys Acta Rev Cancer ; 1873(1): 188335, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816350

RESUMO

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor (TF) that is a member of the Per-Arnt-Sim family of proteins. AhR regulates diverse processes, including malignant transformation, hematopoietic cell development, and fate determination of immune cell lineages. Moreover, AhR forms a crucial link between innate and adaptive arms of the immune system. Malignant cells frequently evolve multiple mechanisms for suppressing tumor-specific responses, including the induction of suppressive pathways involving AhR and its metabolic byproducts in the tumor microenvironment that promote immune evasion and tumor progression. Thus, interest is high in further defining the role of AhR in carcinogenesis and immune development and regulation, particularly regarding the therapeutic interventions that unleash immune responses to cancer cells. Here, we provide an overview of the role of AhR in the regulation of innate and adaptive immune response and discuss the implications of targeting this pathway to augment the immune response in cancer patients.


Assuntos
Regulação da Expressão Gênica/genética , Sistema Imunitário/metabolismo , Neoplasias/genética , Receptores de Hidrocarboneto Arílico/genética , Microambiente Tumoral/genética , Imunidade Adaptativa/genética , Imunidade Adaptativa/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Tolerância Imunológica/genética , Tolerância Imunológica/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Receptores de Hidrocarboneto Arílico/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologia
6.
Toxicol Lett ; 321: 114-121, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31830554

RESUMO

Despite numerous studies on the toxicities of planar polycyclic aromatic hydrocarbons (PAHs), very little is known about the toxicological profiles of non-planar PAHs. In the present study, the cytotoxicity of corannulene (COR), a typical bowl-shaped PAH with a myriad of applications in the area of material chemistry, and benzo[a]pyrene (BaP), a typical planar PAH with similar molecular weight, were systematically compared in various cell lines. Compared with BaP, exposure to COR resulted in less cytotoxic responses in both human (HepG2) and murine (Hepa1-6) hepatoma cells, which was characterized with a slower cellular accumulation as well as a weaker induction of cytochrome P450 1 (CYP1/Cyp1) isozymes. Knockdown of aryl hydrocarbon receptor (AhR) by siRNA attenuated the inductive effect of COR on CYP1A/Cyp1a mRNA levels in these two cell lines. Further analysis revealed that derivatization greatly influenced the cytotoxicity of COR, which was positively correlated with their binding affinities to the AhR, as demonstrated by in silico molecular docking. Overall, these results suggest that AhR appears to be involved in the cytotoxic responses of COR and its derivatives, providing a fundamental understanding of the biological effects of bowl-like PAHs.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Hepatócitos/efeitos dos fármacos , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Receptores de Hidrocarboneto Arílico/agonistas , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , Indução Enzimática , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Camundongos , Simulação de Acoplamento Molecular , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade
7.
Toxicol Lett ; 318: 86-91, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31669099

RESUMO

Proton pump inhibitors (PPIs) have been used worldwide to treat gastrointestinal disorders. A recent study showed that long-term use of PPIs caused iron deficiency; however, it is unclear whether PPIs affect iron metabolism directly. We investigated the effect of PPIs on the peptide hepcidin, an important iron regulatory hormone. First, we used the FDA Adverse Event Reporting System database and analyzed the influence of PPIs. We found that PPIs, as well as H2 blockers, increased the odds ratio of iron-deficient anemia. Next, HepG2 cells were used to examine the action of PPIs and H2 blockers on hepcidin. PPIs augmented hepcidin expression, while H2 blockers did not. In fact, the PPI omeprazole increased hepcidin secretion, and omeprazole-induced hepcidin upregulation was inhibited by gene silencing or the pharmacological inhibition of the aryl hydrocarbon receptor. In mouse experiments, omeprazole also increased hepatic hepcidin mRNA expression and blood hepcidin levels. In mice treated with omeprazole, protein levels of duodenal and splenic ferroportin decreased. Taken together, PPIs directly affect iron metabolism by suppressing iron absorption through the inhibition of duodenal ferroportin via hepcidin upregulation. These findings provide a new insight into the molecular mechanism of PPI-induced iron deficiency.


Assuntos
Anemia Ferropriva/induzido quimicamente , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Duodeno/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepcidinas/metabolismo , Absorção Intestinal/efeitos dos fármacos , Ferro/sangue , Inibidores da Bomba de Prótons/toxicidade , Receptores de Hidrocarboneto Arílico/metabolismo , Anemia Ferropriva/sangue , Anemia Ferropriva/fisiopatologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Transporte de Cátions/metabolismo , Duodeno/metabolismo , Duodeno/fisiopatologia , Células Hep G2 , Hepatócitos/metabolismo , Antagonistas dos Receptores Histamínicos H2/toxicidade , Humanos , Ferro/deficiência , Masculino , Camundongos Endogâmicos C57BL , Receptores de Hidrocarboneto Arílico/genética
8.
Yonsei Med J ; 61(1): 56-63, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31887800

RESUMO

PURPOSE: Elevated aryl hydrocarbon receptor (AhR) transactivating (AHRT) activity and uremia in chronic kidney disease (CKD) may interact with each other, further complicating the disease course. In this study, we prospectively estimated serum AHRT activity using a highly sensitive cell-based AhR-dependent luciferase activity assay in CKD patients and compared differences therein according to treatment modality. MATERIALS AND METHODS: Patients undergoing peritoneal dialysis (PD) (n=22) and hemodialysis (HD) (n=38) and patients with pre-dialysis CKD stage IV or V (n=28) were included. AHRT activity and intracellular adenosine triphosphate (ATP) levels were measured. We performed a correlation analysis for AHRT activity, ATP levels, and various clinical parameters. RESULTS: AHRT activity and intracellular ATP levels were inversely correlated and differed according to treatment modalities. AHRT activity was higher in non-dialysis CKD patients than in patients undergoing dialysis and was higher in patients undergoing HD, compared to PD. AHRT activity decreased after HD treatment in HD patients. ATP levels were higher in healthy controls than in patients with pre-dialysis CKD and PD and were further decreased in patients with HD. We noted significant correlations between multiple clinical parameters associated with cardiovascular risk factors and AHRT activity. CONCLUSION: AHRT activity was elevated in CKD patients, while dialysis treatment reduced AHRT activity. Further studies are warranted to specify AHRT activity and to evaluate the precise roles thereof in patients with CKD.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Diálise Renal , Insuficiência Renal Crônica/metabolismo , Ativação Transcricional , Trifosfato de Adenosina/metabolismo , Feminino , Humanos , Espaço Intracelular/metabolismo , Testes de Função Renal , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/fisiopatologia
9.
Int J Nanomedicine ; 14: 9525-9534, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824148

RESUMO

Introduction: Advancement of novel anticancer drugs into clinical use is frequently halted by their lack of solubility, reduced stability under physiological conditions, and non-specific uptake by normal tissues, causing systemic toxicity. Their progress to use in the clinic could be accelerated by the development of new formulations employing suitable and complementary drug delivery vehicles. Methods: A robust method for apoferritin (AFt)-encapsulation of antitumour benzothiazoles has been developed for enhanced activity against and drug delivery to benzothiazole-sensitive cancers. Results: More than 70 molecules of benzothiazole 5F 203 were encapsulated per AFt cage. Post-encapsulation, the size and integrity of the protein cages were retained as evidenced by dynamic light scattering. ToF-SIMS depth profiling using an argon cluster beam confirmed 5F 203 exclusively within the AFt cavity. Improved encapsulation of benzothiazole lysyl-amide prodrugs was achieved (~130 molecules of Phortress per AFt cage). Transferrin receptor 1, TfR1, was detected in lysates prepared from most cancer cell lines studied, contributing to enhanced anticancer potency of the AFt-encapsulated benzothiazoles (5F 203, Phortress, GW 610, GW 608-Lys). Nanomolar activity was demonstrated by AFt-formulations in breast, ovarian, renal and gastric carcinoma cell lines, whereas GI50 >50 µM was observed in non-tumourigenic MRC-5 fibroblasts. Intracellular 5F 203, a potent aryl hydrocarbon receptor (AhR) ligand, and inducible expression of cytochrome P450 (CYP) 1A1 were detected following exposure of sensitive cells to AFt-5F 203, confirming that the activity of benzothiazoles was not compromised following encapsulation. Conclusion: Our results show enhanced potency and selectivity of AFt-encapsulated 5F 203 against carcinomas derived from breast, ovarian, renal, colorectal as well as gastric cancer models, and offer realistic prospects for potential refinement of tumour-targeting and treatment, and merit further in vivo investigations.


Assuntos
Antineoplásicos/farmacologia , Apoferritinas/metabolismo , Sistemas de Liberação de Medicamentos , Tiazóis/farmacologia , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Humanos , Receptores de Hidrocarboneto Arílico/metabolismo , Tiazóis/química
10.
Environ Pollut ; 255(Pt 3): 113357, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31671369

RESUMO

Gut microbiota is of critical importance to host health. Aryl hydrocarbon receptor (AhR) is found to be closely involved in the regulation of gut microbial dynamics. However, it is still not clear how AhR signaling shapes the gut microbiota. In the present study, adult zebrafish were acutely exposed to an AhR antagonist (CH223191), an AhR agonist (polychlorinated biphenyl 126; PCB126) or their combination for 7 d. Overall intestinal health and gut microbial community were temporally monitored (1 d, 3 d and 7 d) and inter-compared among different groups. The results showed that single exposure to PCB126 significantly disrupted the overall health of intestines (i.e., neural signaling, inflammation, epithelial barrier integrity, oxidative stress). However, CH223191 failed to inhibit but enhanced the physiological toxicities of PCB126, implying the involvement of extra mechanisms rather than AhR in the regulation of intestinal physiological activities. Dysbiosis of gut microbiota was also caused by PCB126 over time as a function of sex. It is intriguing that CH223191 successfully abolished the holistic effects of dioxin on gut microbiota, which inferred that growth of gut microbes was directly controlled by AhR activation without the involvement of host feedback modulation. When coming to detailed alterations at certain taxon, both antagonistic and synergistic interactions existed between CH223191 and dioxin, depending on fish sex, exposure duration and bacterial species. Correlation analysis found that gut inflammation was positively associated with pathogenic Legionella bacteria, but was negatively associated with epithelial barrier integrity, suggesting that integral intestinal epithelial barrier can prevent the influx of pathogenic bacteria to induce inflammatory response. Overall, this study has deciphered, for the first time, the direct regulative effects of AhR activity on gut microbiota. Future research is warranted to elucidate the specific mechanisms of AhR action on certain bacterial population.


Assuntos
Dioxinas/toxicidade , Microbioma Gastrointestinal/efeitos dos fármacos , Animais , Bactérias/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Disbiose , Intestinos , Microbiota , Bifenilos Policlorados , Dibenzodioxinas Policloradas , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Peixe-Zebra/metabolismo
11.
J Toxicol Sci ; 44(10): 711-720, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31588062

RESUMO

Recent studies have demonstrated a relationship between the disruption of zinc homeostasis and the onset of diseases. However, little is known about the factors that disrupt zinc homeostasis. Here, we investigated the effects of ß-naphthoflavone, an exogenous ligand of aryl hydrocarbon receptor (AHR), on intracellular zinc levels. Human hepatoma HepG2 cells were treated with ß-naphthoflavone for 3 days, and intracellular labile and total zinc levels were assessed through flow cytometry and inductively coupled plasma atom emission spectroscopy, respectively. The mRNA levels of zinc transporters were determined by real-time PCR. Treatment of cells with ß-naphthoflavone induced a decrease in intracellular labile zinc in a dose-dependent manner, with significantly decreased levels observed at 1 µM compared with controls. Additionally, intracellular total zinc levels demonstrated a decreasing trend with 10 µM ß-naphthoflavone. Zinc pyrithione recovered the decrease in intracellular labile zinc levels induced by ß-naphthoflavone, while zinc sulfate had no effect. Moreover, significant decreases in the mRNA levels of zinc transporters ZnT10 and ZIP5 were observed in response to 10 µM ß-naphthoflavone. These results demonstrated that ß-naphthoflavone has the potential to disrupt zinc homeostasis in hepatocytes. Although the underlying mechanism remains to be determined, suppression of zinc transporter transcription through AHR activation may be involved in the ß-naphthoflavone-induced disruption of intracellular zinc levels.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Zinco/metabolismo , beta-Naftoflavona/toxicidade , Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte de Cátions/genética , Citocromo P-450 CYP1A1/genética , Células Hep G2 , Hepatócitos/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Ligantes , Neoplasias Hepáticas/metabolismo
12.
Environ Pollut ; 255(Pt 2): 113329, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31600704

RESUMO

Transcription factors including pregnane X receptor (Pxr) and nuclear factor-erythroid 2-related factor-2 (Nrf2) are important modulators of Adenosine triphosphate-binding cassette (ABC) transporters in mammalian cells. However, whether such modulation is conserved in zebrafish embryos remains largely unknown. In this manuscript, pxr- and nrf2-deficient models were constructed with CRISPR/Cas9 system, to evaluate the individual function of Pxr and Nrf2 in the regulation of ABC transporters and detoxification of heavy metal ions like Cd2+ and Ag+. As a result, both Cd2+ and Ag+ conferred extensive interactions with ABC transporters in wild type (WT) embryos: their accumulation and toxicity were affected by the activity of ABC transporters, and they significantly induced the mRNA expressions of ABC transporters. These induction effects were reduced by the mutation of pxr and nrf2, but elevations in the basal expression of ABC transporters compensated for the loss of their inducibility. This could be an explanation for remaining transporter function in both mutant models as well as the unaltered toxicity of metal ions in pxr-deficient embryos. However, mutation of nrf2 disrupted the production of glutathione (GSH), resulting in the enhanced toxicity of Cd2+/Ag+ in zebrafish embryos. In addition, elevated expressions of other transcription factors like aryl hydrocarbon receptor (ahr) 1b, peroxisome proliferator-activated receptor (ppar)-ß, and nrf2 were found in pxr-deficient models without any treatment, while enhanced induction of ahr1b, ppar-ß and pxr could only be seen in nrf2-deficient embryos after the treatment of metal ions, indicating different compensation phenomena for the absence of transcription factors. After all, pxr-deficient and nrf2-deficient zebrafish embryos are useful tools in the functional investigation of Pxr and Nrf2 in the early life stages of aquatic organisms. However, the compensatory mechanisms should be taken into consideration when interpreting the results and need in-depth investigations.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Metais Pesados/toxicidade , Fator 2 Relacionado a NF-E2/genética , Receptor de Pregnano X/genética , Peixe-Zebra/embriologia , Animais , Glutationa/metabolismo , Inativação Metabólica , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Esteroides/genética , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo
13.
Nat Commun ; 10(1): 4579, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31594926

RESUMO

Rheumatoid arthritis (RA) is a chronic inflammatory disorder characterized by progressive bone erosion. Leflunomide is originally developed to suppress inflammation via its metabolite A77 1726 to attenuate bone erosion. However, distinctive responsiveness to Leflunomide is observed among RA individuals. Here we show that Leflunomide exerts immunosuppression but limited efficacy in RA individuals distinguished by higher serum C-reactive protein (CRPHigher, CRPH), whereas the others with satisfactory responsiveness to Leflunomide show lower CRP (CRPLower, CRPL). CRP inhibition decreases bone erosion in arthritic rats. Besides the immunomodulation via A77 1726, Leflunomide itself induces AHR-ARNT interaction to inhibit hepatic CRP production and attenuate bone erosion in CRPL arthritic rats. Nevertheless, high CRP in CRPH rats upregulates HIF1α, which competes with AHR for ARNT association and interferes Leflunomide-AHR-CRP signaling. Hepatocyte-specific HIF1α deletion or a HIF1α inhibitor Acriflavine re-activates Leflunomide-AHR-CRP signaling to inhibit bone erosion. This study presents a precision medicine-based therapeutic strategy for RA.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Reabsorção Óssea/prevenção & controle , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Imunossupressores/farmacologia , Leflunomida/farmacologia , Acriflavina/farmacologia , Acriflavina/uso terapêutico , Adulto , Animais , Artrite Experimental/sangue , Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Reabsorção Óssea/sangue , Reabsorção Óssea/imunologia , Proteína C-Reativa/análise , Proteína C-Reativa/imunologia , Proteína C-Reativa/metabolismo , Células Cultivadas , Colágeno/imunologia , Feminino , Hepatócitos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Imunossupressores/uso terapêutico , Leflunomida/uso terapêutico , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Ratos , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Resultado do Tratamento
14.
Food Chem Toxicol ; 134: 110803, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31563530

RESUMO

Polychlorinated biphenyls (PCBs) would do serious damage to multiple systems, while coplanar polychlorinated biphenyls, the most toxic member of the family, has been widely taken into consideration. In this study, ICR mice were fed with different doses of PCB126 to explore the underlying molecular mechanisms on immunotoxicity. The results showed that PCB126 caused immunosuppression as evidenced by inhibiting the ratios of thymus and spleen weights, changing the organizational structure and decreasing levels and mRNA expression of TNF-α, IFN-γ and IL-2. PCB126 inhibited the SOD activity and spurred the accumulation of MDA in spleen and thymus. Meanwhile, it also disturbed the Nrf2 signaling pathway as evidenced by up-regulating the mRNA expression of Nrf2 and Keap1. Additionally, a remarkable reduction in the mRNA expression of AhR and enhancement in the mRNA expression of Cyp1 enzymes (Cyp1a1, Cyp1a2 and Cyp1b1) were observed, which increased the ROS levels. PCB126 could increase protein expression of Bax, Caspase-3, Caspase-8 and Caspase-9, while the protein expression of Bcl-2 was decreased. In summary, the results indicated that PCB126 modulated the AhR signaling pathway, which interacted with apoptosis and oxidative stress to induce immunotoxicity, enrich the immunotoxicological mechanisms of PCB126.


Assuntos
Apoptose/efeitos dos fármacos , Dioxinas/toxicidade , Mitocôndrias/metabolismo , Bifenilos Policlorados/toxicidade , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Baço/efeitos dos fármacos , Baço/imunologia , Animais , Peso Corporal/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Feminino , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos ICR , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Baço/citologia , Baço/enzimologia , Superóxido Dismutase-1/metabolismo , Timo/efeitos dos fármacos , Timo/enzimologia , Timo/metabolismo
15.
Environ Pollut ; 255(Pt 1): 113171, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31539851

RESUMO

Environmental contaminants, to which humans are widely exposed, cause or worsen several diseases, like cardiovascular diseases and cancers. Among these molecules, polycyclic aromatic hydrocarbons (PAHs) stand out since they are ubiquitous pollutants found in ambient air and diet. Because of their toxic effects, public Health agencies promote development of research studies aiming at increasing the knowledge about PAHs and the discovery of biomarkers of exposure and/or effects. Extracellular vesicles (EVs), including small extracellular vesicles (S-EVs or exosomes) and large extracellular vesicles (L-EVs or microvesicles), are delivery systems for multimolecular messages related to the nature and status of the originating cells. Because they are produced by all cells and detected within body fluids, EV releases could act as cell responses and thereby serve as biomarkers. To test whether EVs can serve as biomarkers of PAHs exposure, we evaluate the effects of these pollutants on EV production using an in vitro approach (human endothelial cell line, HMEC-1) and an in vivo approach (urine samples from PAHs-exposed rats). Our study indicates that, i) PAH exposure increases in vitro the EV production by endothelial cells and in vivo the release of EVs in urine, and that the stimulating effects of PAHs concern both S-EVs and L-EVs; ii) PAH exposure and more particularly exposure to B[a]P, can influence the composition of exosomes produced by endothelial cells; iii) the aryl hydrocarbon receptor, a cytosolic receptor associated to most deleterious effects of PAHs, would be involved in the PAH effects on the release of S-EVs, but not L-EVs. These results suggest that EVs may have utility for monitoring exposure to PAHs, and more particularly to B[a]P, considered as reference PAH, and to detect the related early cellular response prior to end-organ damages.


Assuntos
Células Endoteliais/metabolismo , Poluentes Ambientais/toxicidade , Vesículas Extracelulares/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Urina/química , Animais , Biomarcadores/metabolismo , Líquidos Corporais/química , Linhagem Celular , Exposição Ambiental/efeitos adversos , Monitoramento Ambiental/métodos , Exossomos , Feminino , Humanos , Ratos , Receptores de Hidrocarboneto Arílico/metabolismo
16.
Int J Mol Sci ; 20(18)2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31540101

RESUMO

Sesquiterpenes, the main components of plant essential oils, are bioactive compounds with numerous health-beneficial activities. Sesquiterpenes can interact with concomitantly administered drugs due to the modulation of drug-metabolizing enzymes (DMEs). The aim of this study was to evaluate the modulatory effects of six sesquiterpenes (farnesol, cis-nerolidol, trans-nerolidol, α-humulene, ß-caryophyllene, and caryophyllene oxide) on the expression of four phase I DMEs (cytochrome P450 3A4 and 2C, carbonyl reductase 1, and aldo-keto reductase 1C) at both the mRNA and protein levels. For this purpose, human precision-cut liver slices (PCLS) prepared from 10 patients and transfected HepG2 cells were used. Western blotting, quantitative real-time PCR and reporter gene assays were employed in the analyses. In the reporter gene assays, all sesquiterpenes significantly induced cytochrome P450 3A4 expression via pregnane X receptor interaction. However in PCLS, their effects on the expression of all the tested DMEs at the mRNA and protein levels were mild or none. High inter-individual variabilities in the basal levels as well as in modulatory efficacy of the tested sesquiterpenes were observed, indicating a high probability of marked differences in the effects of these compounds among the general population. Nevertheless, it seems unlikely that the studied sesquiterpenes would remarkably influence the bioavailability and efficacy of concomitantly administered drugs.


Assuntos
Aldo-Ceto Redutases/metabolismo , Carbonil Redutase (NADPH)/metabolismo , Citocromo P-450 CYP3A/metabolismo , Família 2 do Citocromo P450/metabolismo , Receptor de Pregnano X/agonistas , Sesquiterpenos/farmacologia , Idoso , Idoso de 80 Anos ou mais , Sistema Enzimático do Citocromo P-450/metabolismo , Farneseno Álcool/farmacologia , Feminino , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Fígado/enzimologia , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , /farmacologia , Receptor de Pregnano X/metabolismo , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/metabolismo
17.
Int J Mol Sci ; 20(18)2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31547315

RESUMO

Nicotine is one of the most toxic secondary plant metabolites in nature and it is highly toxic to herbivorous insects. The overexpression of CYP6CY3 and its homologous isozyme CYP6CY4 in Myzus persicae nicotianae is correlated with nicotine tolerance. The expanded (AC)n repeat in promoter is the cis element for CYP6CY3 transcription. These repeat sequences are conserved in the CYP6CY3 gene from Aphis gossypii and the homologous P450 genes in Acyrthosiphon pisum. The potential transcriptional factors that may regulate CYP6CY3 were isolated by DNA pulldown and sequenced in order to investigate the underlying transcriptional regulation mechanism of CYP6CY3. These identified transcriptional factors, AhR and ARNT, whose abundance was highly correlated with an abundance of the CYP6CY3 gene, were validated. RNAi and co-transfection results further confirm that AhR and ARNT play a major role in the transcriptional regulation of the CYP6CY3 gene. When the CYP6CY3 transcript is destabilized by AhR/ARNT RNAi, the transcription of the CYP6CY4 is dramatically up-regulated, indicating a compensatory mechanism between the CYP6CY3 and CYP6CY4 genes. Our present study sheds light on the CYP6CY3 and CYP6CY4 mediated nicotine adaption of M. persicae nicotianae to tobacco. The current studies shed light on the molecular mechanisms that underlie the genotypic and phenotypic changes that are involved in insect host shifts and we conclude that AhR/ARNT regulate the expression of CYP6CY3 and CYP6CY4 cooperatively, conferring the nicotine adaption of M. persicae nicotianae to tobacco.


Assuntos
Afídeos/fisiologia , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Família 6 do Citocromo P450/metabolismo , Proteínas de Insetos/metabolismo , Nicotina/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Adaptação Fisiológica , Animais , Afídeos/genética , Família 6 do Citocromo P450/genética , Interações Hospedeiro-Parasita , Proteínas de Insetos/genética , Tabaco/metabolismo , Tabaco/parasitologia , Ativação Transcricional
18.
Ecotoxicol Environ Saf ; 183: 109505, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31394372

RESUMO

Biotests like the fish embryo toxicity test have become increasingly popular in risk assessment and evaluation of chemicals found in the environment. The large range of possible endpoints is a big advantage when researching on the mode of action of a certain substance. Here, we utilized the frequently used model organism zebrafish (Danio rerio) to examine regulative mechanisms in the pathway of the aryl-hydrocarbon receptor (AHR) in early development. We exposed embryos to representatives of two chemical classes known to elicit dioxin-like activity: benzo[a]pyrene for polycyclic aromatic hydrocarbons (PAHs) and 2,3-benzofuran for polar O-substituted heterocycles as a member of heterocyclic compounds in general (N-, S-, O-heterocycles; NSO-hets). We measured gene transcription of the induced P450 cytochromes (cyp1), their formation of protein and biotransformation activity throughout the whole embryonic development until 5 days after fertilization. The results show a very specific time course of transcription depending on the chemical properties (e.g. halogenation, planarity, Kow), the physical decay and the biodegradability of the tested compound. However, although this temporal pattern was not precisely transferable onto the protein level, significant regulation in enzymatic activity over time could be detected. We conclude, that a careful choice of time and end point as well as consideration of the chemical properties of a substance are fairly important when planning, conducting and especially evaluating biotests.


Assuntos
Benzo(a)pireno/toxicidade , Benzofuranos/toxicidade , Receptores de Hidrocarboneto Arílico/metabolismo , Poluentes Químicos da Água/toxicidade , Animais , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Testes de Toxicidade/métodos , Testes de Toxicidade/normas , Transcrição Genética/efeitos dos fármacos , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
19.
J Exp Clin Cancer Res ; 38(1): 335, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370872

RESUMO

BACKGROUND: The chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity. METHODS: Molecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA. RESULTS: We first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC. CONCLUSIONS: In the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Metilcolantreno/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metilcolantreno/química , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Transporte Proteico , Receptores de Hidrocarboneto Arílico/química , Receptores Estrogênicos/química , Receptores Acoplados a Proteínas-G/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
20.
In Vitro Cell Dev Biol Anim ; 55(8): 633-640, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31385165

RESUMO

The immunosuppressive function of mesenchymal stem cells (MSCs) is well known. Aryl hydrocarbon receptor (AhR), a transcription factor of the bHLH/PAS family, is widely expressed in several cells and is involved in various physiological and pathological processes. Previously, we found that the expression of AhR was downregulated in MSCs isolated from mice with neutrophilic asthma and that the activation of AhR enhanced the function of MSCs to alleviate neutrophilic asthma. We hypothesized that AhR activation enhanced MSCs for their immunosuppressive function. We aimed to investigate whether AhR activation can augment the suppressive function of MSCs against splenocyte proliferation. We co-cultured MSCs or AhR-activated MSCs with splenocytes at different ratios. The results showed that AhR activation in MSCs upregulated the expression of inducible nitric oxide (iNOS), which promoted the production of nitric oxide (NO), thus enhancing the inhibitory effect on splenocyte proliferation. The NO donor S-nitroso-N-acetylpenicillamine also inhibited the proliferation of splenocytes, and the iNOS inhibitor N(G)-nitro L-arginine methyl ester and NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide partially reversed the immunosuppressive function. Our study indicates that the AhR activation of MSCs might have an important role in the regulation of splenocyte proliferation and might serve as a potential strategy for treating immune-related diseases.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/metabolismo , Baço/patologia , Animais , Comunicação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Óxidos N-Cíclicos/farmacologia , Feminino , Imidazóis/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mitomicina/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , S-Nitroso-N-Acetilpenicilamina/farmacologia , Regulação para Cima/efeitos dos fármacos
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