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Doxorubicin (Dox) is among the most widely used cancer chemotherapeutic drugs. The clinical use of Dox is, however, limited due to its cardiotoxicity. Studies over the past several decades have suggested various mechanisms of Dox-induced cardiotoxicity (DIC). Among them are oxidative stress, topoisomerase inhibition, and mitochondrial damage. Several novel molecular targets and signaling pathways underlying DIC have emerged over the past few years. The most notable advances include discovery of ferroptosis as a major form of cell death in Dox cytotoxicity, and elucidation of the involvement of cardiogenetics and regulatory RNAs as well as multiple other targets in DIC. In this review, we discuss these advances, focusing on latest cutting-edge research discoveries from mechanistic studies reported in influential journals rather than surveying all research studies available in the literature.
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Introduction: Leydig cells isolated from the testis are able to sustain high levels of testosterone production in vitro, but only for up to 3 days. Such cells are valuable for addressing the acute effects of chemicals on steroidogenic function, but not for repeated or chronic effects. Methodology is now available by which adult Leydig cells can be derived in vitro from seminiferous tubule-associated stem cells. In contrast to isolated Leydig cells, the Leydig cells derived in this way can synthesize and secrete high levels of testosterone for months. Herein, we asked whether this system might be used to address the effect of mono-(2-ethylhexyl) phthalate (MEHP) exposure on the formation of Leydig cells from tubule-associated stem cells, and on the Leydig cells after their formation. Methods: Adult Brown Norway rats received an intraperitoneal injection of ethane dimethanesulfonate (EDS) to eliminate the existing Leydig cells. Seminiferous tubules then were isolated and cultured in medium containing Insulin-Transferrin- Selenium (ITS), Smoothened Agonist (SAG), and luteinizing hormone (LH). Results: Culture of the tubules for 8 weeks resulted in the formation of cells on the surfaces of the tubules that stained for CYP11A1 and STAR and produced high levels of testosterone. When the tubules were cultured in medium containing increasing concentrations of MEHP, concentration-dependent effects on Leydig cell formation occurred. To determine the effect of MEHP on newly produced Leydig cells, tubules were cultured for 8 weeks in the absence of MEHP, resulting in the formation of adult Leydig cells, and then in medium containing increasing concentrations of MEHP. Concentration-dependent decreases in testosterone production by the adult Leydig cells were seen, and these decreases proved to be reversible. Discussion: The use of this new system should make it possible to determine the mechanisms by which acute, repeated, or chronic exposures to increasing concentrations of MEHP and/or exposure to other chemicals affect the formation of Leydig cells from stem cells, as well as the steroidogenic function of adult Leydig cells.
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Dyslipidemia is a disorder where abnormally lipid concentrations circulate in the bloodstream. The disorder is common in type 2 diabetics (T2D) and is linked with T2D comorbidities, particularly cardiovascular disease. Dyslipidemia in T2D is typically characterized by elevated plasma triglyceride and low high-density lipoprotein cholesterol (HDL-C) levels. There is a significant gap in the literature regarding dyslipidemia in rural parts of Africa, where lipid profiles may not be captured through routine surveillance. This study aimed to characterize the prevalence and demo-graphic profile of dyslipidemia in T2D in the rural community of Ganadougou, Mali. We performed a cross-sectional study of 104 subjects with T2D in Ganadougou between November 2021 and March 2022. Demographic and lipid profiles were collected through cross-sectional surveys and serological analyses. The overall prevalence of dyslipidemia in T2D patients was 87.5% (91/104), which did not differ by sex (P = .368). High low-density lipoprotein cholesterol (LDL-C) was the most common lipid abnormality (78.9%, [82/104]). Dyslipidemia was associated with age and hypertension status (P = .013 and.036, respectively). High total and high LDL-C parameters were significantly associated with hypertension (P = .029 and .006, respectively). In low-resource settings such as rural Mali, there is a critical need to improve infrastructure for routine dyslipidemia screening to guide its prevention and intervention approaches. The high rates of dyslipidemia observed in Gandadougou, consistent with concomitant increases in cardiovascular diseases in Africa suggest that lipid profile assessments should be incorporated into routine medical care for T2D patients in African rural settings.
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Diabetes is currently a crisis in sub-Saharan West Africa (SSWA) with dramatic implications for public health and national budgets prioritizing infectious diseases. There is limited recent literature about the prevalence, awareness, and risk factors for type 2 diabetes (T2D) in rural parts of SSWA. This study characterized T2D prevalence and risk factors for the rural Malian community of Nièna, which is situated in Mali's second-largest province of Sikasso. Between December 2020 and July 2021, a cross-sectional study of 412 participants was conducted in the Nièna community using clinical questionnaires and rapid diagnostic tests. Among 412 participants, there were 143 (34.7%) and 269 (65.3%) males and females, respectively. The overall prevalence of T2D in Nièna was 7.5% (31/412), and prevalence rates were 8.6% (23/269) and 5.6% (8/143) for females and males, respectively. Age, family history of diabetes, hypertension, waist circumference, and fetal macrosomia were significantly associated with T2D (p = 0.007, p < 0.001, p = 0.003, p = 0.013, and p < 0.001, respectively). Notably, 61.3% (19/31) of T2D subjects were unaware of their diabetic status before the study. Field surveys have considerable utility in driving T2D awareness in rural African settings.
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Diabetes Mellitus Tipo 2 , Femenino , Masculino , Humanos , Diabetes Mellitus Tipo 2/epidemiología , Estudios Transversales , Malí/epidemiología , Prevalencia , Factores de RiesgoRESUMEN
Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is widely used in manufacturing. Previous studies have shown that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, has inhibitory effects on luteinizing hormone (LH)-stimulated steroid biosynthesis by Leydig cells. The molecular mechanisms underlying its effects, however, remain unclear. In the present study, we examined the effects of MEHP on changes in mitochondrial function in relationship to reduced progesterone formation by MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with MEHP (0-300 µM for 24 h) resulted in dose-dependent inhibition of LH-stimulated progesterone biosynthesis. Biochemical analysis data revealed that the levels of the mature steroidogenic acute regulatory protein (STAR), a protein that works at the outer mitochondrial membrane to facilitate the translocation of cholesterol for steroid formation, was significantly reduced in response to MEHP exposures. MEHP also caused reductions in MA-10 cell mitochondrial membrane potential (ΔΨm) and mitochondrial respiration as evidenced by decreases in the ability of the mitochondria to consume molecular oxygen. Additionally, significant increases in the generation of mitochondrial superoxide were observed. Taken together, these results indicate that MEHP inhibits steroid formation in MA-10 cells at least in part by its effects on mitochondrial function.
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Dietilhexil Ftalato/análogos & derivados , Células Intersticiales del Testículo/química , Mitocondrias/efectos de los fármacos , Plastificantes/toxicidad , Animales , Línea Celular Tumoral , Colesterol/metabolismo , Dietilhexil Ftalato/administración & dosificación , Dietilhexil Ftalato/toxicidad , Relación Dosis-Respuesta a Droga , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/fisiología , Oxígeno/metabolismo , Plastificantes/administración & dosificación , Esteroides/biosíntesisRESUMEN
Mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate (DEHP), is a plasticizer with endocrine disruptor activity that has been shown to stimulate basal steroid biosynthesis in Leydig cells. The mechanism by which it does so is unknown. Using MA-10 mouse tumor Leydig cells, we assessed the effects of MEHP on reactive oxygen species (ROS) levels, and on the signal transduction pathways that mobilize cholesterol. Exposure to 0-300 µM MEHP stimulated basal progesterone production in a dose-dependent manner. Progesterone stimulation was correlated with increases in the phosphorylation of hormone-sensitive lipase (HSL; aka cholesteryl ester hydrolase), which is involved in the production of free cholesterol, and of steroidogenic acute regulatory (STAR) protein expression. Co-treating MA-10 cells with MEHP and the ROS scavenger N-acetyl cysteine (NAC) blocked the activation of HSL, blunted MEHP-induced STAR, and reduced basal progesterone formation. These observations suggest that ROS generation by MEHP leads to activation of HSL and increase in STAR which, together, result in increased free-cholesterol bioavailability and progesterone formation.
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Colesterol/metabolismo , Dietilhexil Ftalato/análogos & derivados , Disruptores Endocrinos/toxicidad , Células Intersticiales del Testículo/efectos de los fármacos , Plastificantes/toxicidad , Progesterona/metabolismo , Esterol Esterasa/metabolismo , Acetilcisteína/farmacología , Animales , Línea Celular Tumoral , Dietilhexil Ftalato/toxicidad , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Oxidación-Reducción , Fosfoproteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Extracellular signal-regulated kinase (Erk)1/2 activity signals myeloid cell differentiation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Previously, we reported that Erk1/2 activation (phosphorylation) induced by TPA required reactive oxygen species (ROS) as a second messenger. Here, we hypothesized that ROS generated in response to TPA inhibit Erk1/2-directed phosphatase activity, which leads to an increase phosphorylation of Erk1/2 to signal p21(WAF1/Cip1)-mediated growth arrest in ML-1 cells. Incubation of ML-1 cells with TPA resulted in a marked accumulation of phosphorylated Erk1/2, and is subsequent to H2O2 generation. Interestingly, post-TPA-treatment with N-acetylcysteine (NAC) stimulated a marked and a rapid dephosphorylation of Erk1/2, suggesting a regeneration of Erk1/2-directed phospahatase activity by NAC. ROS generation in ML-1 cells induced by TPA was suggested to occur in the mitochondrial electron transport chain (METC) based on the following observations: (i) undifferentiated ML-1 cells not only lack p67-phox and but also express a low level of p47-phox key components required for NADPH oxidase enzymatic activity, (ii) pretreatment with DPI, an inhibitor of NADH- and NADPH-dependent enzymes, or rhein, an inhibitor of complex I, blocked the ROS generation, and (iii) examination of the microarray analysis data and Western blot analysis data revealed an induction of MnSOD expression at both mRNA and protein levels in response to TPA. MnSOD is a key member of the mitochondrial defense system against mitochondrial-derived superoxide. Together, this study suggested that TPA stimulated ROS generation as a second messenger to activate Erk1/2 via a redox-mediated inhibition of Erk1/2-directed phosphatase in ML-1 cells.
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Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/metabolismo , Carcinógenos/metabolismo , Línea Celular , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Activación Enzimática , Inducción Enzimática , Inhibidores Enzimáticos/metabolismo , Colorantes Fluorescentes/metabolismo , Perfilación de la Expresión Génica , Humanos , Peróxido de Hidrógeno/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidantes/metabolismo , Oxidación-Reducción , Fenantridinas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Sepsis is characterized by an inappropriate host immune-inflammatory response and sustained oxidative damage. Nrf2, a bZIP oxidant-responsive transcription factor, regulates a battery of cytoprotective genes including antioxidants and maintains cellular redox homeostasis. Mouse studies have demonstrated a critical role of Nrf2 in improving survival during sepsis. This preclinical ex vivo study using neutrophils and peripheral blood mononuclear cells (PBMCs) as a surrogate cells evaluates the efficacy of CDDO-Im and CDDO-Me [imidazole and methyl ester derivative of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO)] to activate the Nrf2 pathway and protect from lipopolysaccharide (LPS)-induced inflammatory response in humans. CDDO-Im treatment significantly induced Nrf2-dependent antioxidative genes (HO-1, GCLC, GCLM, and NQO1) in PBMCs isolated from six normal subjects. CDDO-Im increased nuclear accumulation of Nrf2 protein. Pretreatment of PBMC by CDDO-Im significantly attenuated LPS-induced cytokine expression. Similar increases in levels of antioxidant genes and suppression of LPS-induced cytokine expression was observed after CDDO-Me pretreatment. CDDO-Im also greatly inhibited LPS, fMLP, TNF-alpha, and TPA-induced ROS generation in neutrophils. In conclusion, these results demonstrate that activation of the Nrf2-dependent antioxidative pathway by CDDO-Im or CDDO-Me protects against the LPS-induced inflammatory response and suggest that they can be potential therapeutic candidates for intervening sepsis syndrome.
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Citocinas/metabolismo , Imidazoles/farmacología , Leucocitos Mononucleares/inmunología , Factor 2 Relacionado con NF-E2/fisiología , Neutrófilos/inmunología , Ácido Oleanólico/análogos & derivados , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Lipopolisacáridos/toxicidad , Factor 2 Relacionado con NF-E2/genética , Neutrófilos/efectos de los fármacos , Ácido Oleanólico/farmacología , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Formil Péptido/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Molecular dioxygen (O2) is an essential element of aerobic life, yet incomplete reduction or excitation of O2 during aerobic metabolisms generates diverse oxygen-containing reactive species, commonly known as reactive oxygen species (ROS). On the one hand, ROS pose a serious threat to aerobic organisms via inducing oxidative damage to cellular constituents. On the other hand, these reactive species, when their generation is under homeostatic control, also play important physiological roles (e.g., constituting an important component of immunity and participating in redox signaling). This article defines oxygen and the key facts about oxygen, and discusses the relationship between oxygen and the emergence of early animals on Earth. The article then describes the discovery of oxygen by three historical figures and examines the birth of the concepts of oxygen toxicity and the underlying free radical mechanisms. The article ends with a brief introduction to the emerging field of ROS-mediated redox signaling and physiological responses.
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Although exposures to environmental toxicants occur throughout life, little attention has been paid to the possible effects of exposures early in life on later exposure effects. We asked whether DEHP administered in utero (GD14-parturition) affects how male rats respond to later exposures. Neither in utero nor juvenile (PND21-35) exposures to 100mg/kg/day DEHP affected testis weight or histology as assessed on PND35. However, after in utero DEHP, subsequent juvenile exposure resulted in significantly reduced testis weight and altered testicular histology. Both in utero and juvenile exposures resulted in significant reductions in serum testosterone, but there was no effect of earlier on later exposure. Whether or not there had been in utero DEHP exposure, juvenile DEHP exposure had no effect on body, kidney or liver weights. These observations indicate that in utero exposure can, but will not necessarily, alter later exposure effects, with outcomes dependent upon endpoints measured and dose.
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Dietilhexil Ftalato/toxicidad , Disruptores Endocrinos/toxicidad , Contaminantes Ambientales/toxicidad , Plastificantes/toxicidad , Efectos Tardíos de la Exposición Prenatal , Testículo/efectos de los fármacos , Animales , Femenino , Masculino , Tamaño de los Órganos/efectos de los fármacos , Embarazo , Ratas , Ratas Sprague-Dawley , Testículo/patología , Testosterona/sangreRESUMEN
MitoSOX-based assays are widely used to detect mitochondrial reactive oxygen species (ROS), especially superoxide. To this end, 5 µM MitoSOX is commonly used. In this ROS Protocols article, we described the flow cytometric protocol involving the use of various concentrations of MitoSOX (1, 2.5, 5 µM) for detecting mitochondrial ROS in control and mitochondrial DNA-deficient (MD) melanoma B16-F10 cells. We also compared the MitoSOX-based flow cytometry with lucigenin-derived chemiluminometry for their ability to reliably detect the relative differences in mitochondrial ROS formation in the control and MD cells. Our results suggested that 1 µM, rather than the commonly used 5 µM, appeared to be the optimal concentration of MitoSOX for detecting mitochondrial ROS via flow cytometry.
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Human monocytic THP-1 cells can be induced to differentiate to macrophages when treated with phorbol 12-myristate 13-acetate (PMA). It is understood that before initiating cell differentiation, PMA treatment must first induce an inhibition of cell growth. Since the initial biochemical and molecular events that are associated with this growth inhibition have not been characterized, the present study was carried out to elucidate the molecular mechanisms associated with the PMA-induced growth arrest of THP-1 cells. Our results indicate that PMA inhibits THP-1 cells at G1-phase of the cell cycle, via a complex mechanism associated with the modulation of the expression of several cell cycle regulators, initiated by the cellular generation of reactive oxygen species (ROS). Both p21WAF1/CIP1 mRNA and protein were upregulated 24 h post PMA treatment as demonstrated by ribonuclease protection assay and Western blotting, respectively. Because these cells lack functional p53, this effect was independent of p53 activity. Electrophoretic mobility shift assay showed that the PMA-induced activation of the p21WAF1/CIP1 promoter was driven by the specific protein 1 (Sp1) transcription factor through Sp1-binding sites. Additionally, our study demonstrates that PMA-induces the upregulation of p21 through a protein kinase C (PKC)-mediated ROS-dependent signaling mechanism involving MAP kinase activation.
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Proliferación Celular/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Monocitos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Antioxidantes/farmacología , Quinasas CDC2-CDC28/antagonistas & inhibidores , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Flavonoides/farmacología , Fase G1/efectos de los fármacos , Humanos , Fosforilación , Proteína Quinasa C/metabolismo , ARN Mensajero/efectos de los fármacos , Proteína de Retinoblastoma/efectos de los fármacos , Proteína de Retinoblastoma/metabolismo , Fase S/efectos de los fármacos , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Factores de TiempoRESUMEN
Previous studies have shown that phthalate exposure can suppress steroidogenesis. However, the affected components of the steroidogenic pathway, and the mechanisms involved, remain uncertain. We show that incubating MA-10 Leydig cells with mono-(2-ethylhexyl) phthalate (MEHP) resulted in reductions in luteinizing hormone (LH)-stimulated cAMP and progesterone productions. cAMP did not decrease in response to MEHP when the cells were incubated with cholera toxin or forskolin. Incubation of MEHP-treated cells with dibutyryl-cAMP, 22-hydroxycholesterol or pregnenolone inhibited the reductions in progesterone. Increased levels of reactive oxygen species (ROS) occurred in response to MEHP. In cells in which intracellular glutathione was depleted by buthionine sulfoximine pretreatment, the increases in ROS and decreases in progesterone in response to MEHP treatment were exacerbated. These results indicate that MEHP inhibits MA-10 Leydig cell steroidogenesis by targeting LH-stimulated cAMP production and cholesterol transport, and that a likely mechanism by which MEHP acts is through increased oxidative stress.
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Dietilhexil Ftalato/análogos & derivados , Células Intersticiales del Testículo/efectos de los fármacos , Progesterona/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Dietilhexil Ftalato/toxicidad , Regulación hacia Abajo , Glutatión/metabolismo , Células Intersticiales del Testículo/metabolismo , Hormona Luteinizante/farmacología , Masculino , Ratones , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
12-O-tetradecanoylphorbol 13-acetate (TPA) induces the differentiation of human myeloid ML-1 cells to macrophages. In the current study, the expression, responsiveness, and regulation of toll-like receptors (TLRs) in TPA-induced ML-1-derived macrophages were investigated. We have found that TPA-induced differentiation of ML-1 cells was accompanied by the upregulation of TLR1, TLR2, TLR4, and CD14 expression at both the mRNA and protein levels. Interestingly, TLR1 and TLR4 protein expression on ML-1 cells could be blocked by pretreatment with U0126, suggesting the role of an Erk1/2-induced differentiation signal in this process. In addition, the expression of IRAK-2, a key member of the TLR/IRAK-2/NF-κB-dependent signaling cascade was also induced in response to TPA. Accordingly, we demonstrated an increased cellular release of inflammatory cytokines (TNF-α and various interleukins) upon stimulation with LPS and LTA ligands for TLR4 and TLR2, respectively. Furthermore, using luminol-dependent chemiluminescence, addition of LPS and LTA induces a sustained DPI-inhibitable generation of reactive oxygen species (ROS) by the differentiated ML-1 cells. Together, these data suggest that the increase in the responsiveness of TPA-treated ML-1 cells to LPS and LTA occurs in response to the upregulation of their respective receptors as well as an induction of the IRAK-2 gene expression.
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Di-(2-ethylhexyl) phthalate, a widely used plasticizer, and its active metabolite, mono-(2-ethylhexyl) phthalate (MEHP), have been shown to exert adverse effects on the reproductive tract in developing and adult animals. As yet, however, the molecular mechanisms by which they act are uncertain. In the present study, we address the molecular and cellular mechanisms underlying the effects of MEHP on basal and human chorionic gonadotropin (hCG)-stimulated steroid production by MA-10 Leydig cells, using a systems biology approach. MEHP induced dose-dependent decreases in hCG-stimulated steroid formation. Changes in mRNA and protein expression in cells treated with increasing concentrations of MEHP in the presence or absence of hCG were measured by gene microarray and protein high-throughput immunoblotting analyses, respectively. Expression profiling indicated that low concentrations of MEHP induced the expression of a number of genes that also were expressed after hCG stimulation. Cross-comparisons between the hCG and MEHP treatments revealed two genes, Anxa1 and AR1. We suggest that these genes may be involved in a new self-regulatory mechanism of steroidogenesis. The MEHP-induced decreases in hCG-stimulated steroid formation were paralleled by increases in reactive oxygen species generation, with the latter mediated by the Cyp1a1 gene and its network. A model for the mechanism of MEHP action on MA-10 Leydig cell steroidogenesis is proposed.
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Gonadotropina Coriónica/farmacología , Dietilhexil Ftalato/análogos & derivados , Tumor de Células de Leydig/metabolismo , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Esteroides/biosíntesis , Animales , Western Blotting , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Dietilhexil Ftalato/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes/efectos de los fármacos , Redes Reguladoras de Genes/genética , Immunoblotting , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Progesterona/biosíntesis , Ratas , Especies Reactivas de Oxígeno/metabolismo , Testosterona/biosíntesisRESUMEN
Intercellular adhesion molecule 1 (ICAM-1) has been implicated in playing a key role in the mechanism of inflammatory process initiated in response to environmental agents, and during normal hematopoietic cell differentiation. Though induction of ICAM-1 by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in myeloid cells has been reported, the molecular mechanism by which TPA upregulates ICAM-1 expression remains unclear. In the present study, we investigated the signaling mechanism associated with TPA-induced ICAM-1 expression in ML-1 cells. Herein, our microarray, flow cytometry, and Western blot analysis indicated that ICAM-1 was constitutively expressed at a low level in ML-1 cells, but its expression was further upregulated at both the mRNA and protein levels in response to TPA. ICAM-1 expression in response to TPA was inhibited by pretreatment with GF109203X [a specific inhibitor of protein kinase C (PKC)], or with PD98059 and U0126 (specific inhibitors of MEK), suggesting the importance of PKC, and Erk1/2 signaling cascades in this response. Interestingly, ICAM-1 expression in response to TPA-induced PKC activation was linked to the generation of reactive oxygen species (ROS), as pretreatment with NAC (an ROS scavenger) blocked both ErK1/2 activation and ICAM-1 expression induced by TPA. In addition, TPA-induced ICAM-1 expression was blocked by inhibition of nuclear factor-kappaB (NF-kappaB) activation following pretreatment with BAY11-7085 (a specific inhibitor of NF-kappaB activation). TPA-induced NF-kappaB activation was shown by increased degradation of IkB (NF-kappaB specific inhibitory protein). Together, these observations demonstrated that TPA, a potent activator of PKC, induces ICAM-1 expression via a ROS- and ERK1/2-dependent signaling mechanism in ML-1 cells.
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Molécula 1 de Adhesión Intercelular/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Western Blotting , Butadienos/farmacología , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Flavonoides/farmacología , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Indoles/farmacología , Molécula 1 de Adhesión Intercelular/metabolismo , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Maleimidas/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/fisiología , Modelos Biológicos , FN-kappa B/metabolismo , FN-kappa B/fisiología , Nitrilos/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteína Quinasa C/fisiología , Transducción de Señal/efectos de los fármacos , Sulfonas/farmacologíaRESUMEN
BACKGROUND: Alcoholism is a common cause of cirrhosis. Hepatic stellate cells are the main source of collagen that ultimately leads to hepatic fibrosis and cirrhosis. Reactive oxygen species (ROS) enhance stellate cell activation and stimulate fibrogenesis. In this study, the acute effects of ethanol (ET) and acetaldehyde (AC) were determined on the production of ROS in isolated rat hepatic stellate cells. METHODS: Rat stellate cells were isolated in situ by perfusion of the portal vein and cultured. Hydrogen peroxide (H(2)O(2)) was determined by luminol-derived chemiluminescence (CL), while superoxide anion (O(2*-)) production was assessed by the fluorescent probe hydroethidine. RESULTS: AC increased the formation of H(2)O(2) and O(2*-), and these effects were first detectable at AC concentrations of 5 and 10 microM, respectively, reaching a maximum at 50 to 75 microM. Reduction of glutathione (GSH) synthesis by 1-buthionine sulfoximide (BSO) or by GSH conjugation with dimethylmaleate (DEM) further enhanced the effects of AC on H(2)O(2) and O(2*-) formation, while N-acetylcysteine (NAC) decreased H(2)O(2) and eliminated the enhanced generation of O(2*-) caused by AC. Raloxifene, which inhibits O(2*-) production by NAD(P)H oxidase, reduced the effects of AC on H(2)O(2) and O(2*-) production. ET increased H(2)O(2) or O(2*-) only in the presence of BSO or DEM. CONCLUSION: This study shows that concentrations of AC, which occur in vivo after the ingestion of alcoholic beverages, result in the formation of ROS in rat hepatic stellate cells. The increases in ROS are known to activate stellate cells promoting fibrogenesis.
Asunto(s)
Acetaldehído/farmacología , Etanol/administración & dosificación , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Sepsis induced lethality is characterized by amplified host innate immune response. Nrf2, a bZIP transcription factor, regulates a battery of cellular antioxidative genes and maintains cellular redox homeostasis. This study demonstrates that increasing Nrf2 activity by a potent small molecule activator, CDDO-Im (1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole), protects from deregulation of lipopolysaccharide (LPS) induced innate immune response. In response to LPS stimuli, nrf2-deficient (nrf2 -/-) peritoneal neutrophils showed increased NADPH oxidase-dependent ROS generation, proinflammatory cytokines (Tnf-alpha and Il-6) and chemokines (Mip2 and Mcp-1) relative to wild-type (nrf2 +/+) cells. Pretreatment of peritoneal neutrophils with CDDO-Im induced antioxidative genes (Ho-1, Gclc, Gclm, and Nqo1) and attenuated LPS induced ROS generation as well as expression of proinflammatory cytokines exclusively in nrf2 +/+ neutrophils but not in nrf2 -/- cells. In corroboration with in vitro studies, pretreatment with CDDO-Im induced Nrf2-dependent antioxidative genes, attenuated LPS induced proinflammatory cytokine expression, and decreased mortality specifically in the nrf2 +/+ mice. In conclusion, the results suggest that Nrf2 is associated with oxidative regulation of LPS induced innate immune response in neutrophils. Activation of Nrf2-dependent compensatory antioxidative pathways by CDDO-Im protects from LPS induced inflammatory response and mortality.