RESUMO
We synthesized a directed library of compounds to explore the structure-activity relationships of peroxisome proliferator-activated receptor δ (PPARδ) activation relative to mesenchymal stem cell (MSC) osteogenesis. Our scaffold used para-substituted cinnamic acids as a polar headgroup, a heteroatom and heterocycle core connecting units, and substituted phenyl groups for the lipophilic tail. Compounds were screened for their ability to increase osteogenesis in MSCs, and the most promising were examined for subunit specificity using a quantitative PPAR transactivation assay. Six compounds were selected for in vivo studies in an ovariectomized mouse model of human postmenopausal osteoporosis. Four compounds improved bone density in vivo, with two (12d and 31a) having activity comparable to that of GW0742, a well-studied PPARδ-selective agonist. 31a (2-methyl-4-[N-methyl-N-[5-methylene-4-methyl-2-[4-(trifluoromethyl)phenyl]thiazole]]aminocinnamic acid) had the highest selectivity for PPARδ compared to other subtypes, its selectivity far exceeding that of GW0742. Our results confirm that PPARδ is a new drug target for possible treatment of osteoporosis via in situ manipulation of MSCs.
Assuntos
Diferenciação Celular , Osteogênese , PPAR delta/agonistas , Tiazóis/química , Animais , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Desenho de Fármacos , Feminino , Fêmur/diagnóstico por imagem , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteogênese/efeitos dos fármacos , Osteoporose/tratamento farmacológico , PPAR delta/metabolismo , Relação Estrutura-Atividade , Tiazóis/metabolismo , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Microtomografia por Raio-XRESUMO
INTRODUCTION: Studies reveal positive interviewer perceptions of multiple mini-interview (MMI) upon MMI completion. No studies evaluate change in interviewer perceptions during MMI implementation. The objective was to evaluate the change in interviewer perceptions during the implementation of the MMI model at the University of Toledo College of Pharmacy and Pharmaceutical Sciences. METHODS: Interviewers (faculty volunteers, preceptors, student pharmacists) were eligible for inclusion in the prospective cohort. Consenting individuals (1) completed a pre-MMI training survey regarding perceptions of MMI, (2) participated in a 90-minute MMI training program (PowerPoint presentation and review of videos demonstrating MMI practices), (3) completed a post-MMI training survey, and (4) after interviews, completed a post-interview survey. The six Likert-scale MMI perception questions were independently analyzed for changes in the rank response across the three survey time points using Friedman's nonparametric repeated-measures analysis. Each question was evaluated for all respondents together, and for nine different respondent subgroups. The overall criteria for significance was α = 0.05 for each question, with Bonferroni correction for the ten overall comparisons made for each question. RESULTS: Thirty-two interviewers participated (20 faculty members, five preceptors, and seven student pharmacists). From the pre-MMI training survey through the post-interview survey, interviewers gained confidence in their ability to explain the rationale behind the MMI model, were more likely to agree that six minutes was adequate time to assess an applicant and believed MMI provides a fair assessment of an applicant's noncognitive attributes. CONCLUSIONS: After interviewers received training and gained experience with MMI, perceptions of MMI improved.
Assuntos
Entrevistas como Assunto/métodos , Percepção , Seleção de Pessoal/normas , Critérios de Admissão Escolar/estatística & dados numéricos , Faculdades de Farmácia/estatística & dados numéricos , Estudos de Coortes , Humanos , Entrevistas como Assunto/estatística & dados numéricos , Seleção de Pessoal/métodos , Seleção de Pessoal/estatística & dados numéricos , Estudos Prospectivos , Pesquisa Qualitativa , Faculdades de Farmácia/organização & administração , Inquéritos e QuestionáriosRESUMO
Activation of lipid-burning pathways in the fat-storing white adipose tissue (WAT) is a promising strategy to improve metabolic health and reduce obesity, insulin resistance, and type II diabetes. For unknown reasons, bilirubin levels are negatively associated with obesity and diabetes. Here, using mice and an array of approaches, including MRI to assess body composition, biochemical assays to measure bilirubin and fatty acids, MitoTracker-based mitochondrial analysis, immunofluorescence, and high-throughput coregulator analysis, we show that bilirubin functions as a molecular switch for the nuclear receptor transcription factor peroxisome proliferator-activated receptor α (PPARα). Bilirubin exerted its effects by recruiting and dissociating specific coregulators in WAT, driving the expression of PPARα target genes such as uncoupling protein 1 (Ucp1) and adrenoreceptor ß 3 (Adrb3). We also found that bilirubin is a selective ligand for PPARα and does not affect the activities of the related proteins PPARγ and PPARδ. We further found that diet-induced obese mice with mild hyperbilirubinemia have reduced WAT size and an increased number of mitochondria, associated with a restructuring of PPARα-binding coregulators. We conclude that bilirubin strongly affects organismal body weight by reshaping the PPARα coregulator profile, remodeling WAT to improve metabolic function, and reducing fat accumulation.
Assuntos
Tecido Adiposo Branco/metabolismo , Bilirrubina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Mitocôndrias/metabolismo , PPAR alfa/metabolismo , Animais , Bilirrubina/metabolismo , Camundongos , Receptores Adrenérgicos beta 3/biossíntese , Proteína Desacopladora 1/biossínteseRESUMO
A new indole based chalcone molecule MOMIPP induced methuosis mediated cell death in gliobastoma and other cancer cell lines. But the drug was insoluble in water and had a very short plasma half-life. The purpose of this work was to develop a formulation that can provide sustained levels of MOMIPP in vivo. Initial studies established drug solubility in various solvents. N-methyl pyrrolidone (NMP) was determined as an excellent solvent for the drug. Subsequently a poloxamer-407 based thermoreversible gel containing NMP was used to develop the formulation. Rheological studies were performed via oscillatory temperature mode, continuous shear analysis, and oscillatory frequency mode experiments. The mechanical properties of the formulations were tested using a texture profile analyzer. The gelation temperature and time of formulations increased with increasing amounts of NMP. However, the viscosity at 20 °C and storage modulus decreased as the amount of NMP increased. Characterization studies helped to identify the gel formulation that was used to administer the drug orally, sub-cutaneously, and intra-peritoneally. When the gel was given intraperitoneally the target plasma and brain levels of over 5 µM was maintained for about 8 h. Thus, a thermoreversible gel formulation that can deliver MOMIPP in animal studies was successfully developed.
Assuntos
Antineoplásicos , Hidrogéis , Animais , Encéfalo/metabolismo , Géis , Indóis , Poloxâmero/metabolismo , Piridinas , Reologia , Temperatura , ViscosidadeRESUMO
BACKGROUND: Synthetic indolyl- pyridinyl- propenones (IPPs) induce methuosis, a form of non-apoptotic cell death, in glioblastoma and other cancer cell lines. Methuosis is characterized by accumulation of cytoplasmic vacuoles derived from macropinosomes and late endosomes, followed by metabolic failure and rupture of the plasma membrane. However, not all IPPs that cause vacuolization are cytotoxic. The main goals of the present study were to identify key signaling pathways that contribute to methuosis induced by cytotoxic IPPs and to evaluate the anti-tumor potential of a prototype IPP in vivo. METHODS: We utilized metabolic flux analysis, glucose uptake, immunoblotting, and selective pharmacological inhibitors to compare the effects of closely related cytotoxic and non-cytotoxic IPPs in cultured glioblastoma cells. To determine whether the use of methuosis-inducing IPPs might be feasible in a therapeutic context, we quantified the distribution of our lead IPP compound, MOMIPP, in mouse plasma and brain, and tested its ability to inhibit tumor growth in an intracerebral glioblastoma xenograft model. RESULTS: The cytotoxic IPP compound, MOMIPP, causes early disruptions of glucose uptake and glycolytic metabolism. Coincident with these metabolic changes, MOMIPP selectively activates the JNK1/2 stress kinase pathway, resulting in phosphorylation of c-Jun, Bcl-2 and Bcl-xL. At the same concentration, the non-cytotoxic analog, MOPIPP, does not activate these pathways. Pharmacologic inhibition of JNK activity promotes survival, even when cells are extensively vacuolated, but suppression of c-Jun transcriptional activity offers no protection. MOMIPP readily penetrates the blood-brain barrier and is moderately effective in suppressing progression of intracerebral glioblastoma xenografts. CONCLUSIONS: The results suggest that interference with glucose uptake and induction of JNK-mediated phosphorylation of pro-survival members of the Bcl-2 family represent key events in the methuosis death process. In addition to providing new insights into the underlying molecular mechanism of methuosis, the results indicate that compounds of the cytotoxic IPP class may have potential for further development as therapeutic agents for brain tumors.
Assuntos
Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Indóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Piridinas/farmacologia , Adulto , Animais , Antineoplásicos/uso terapêutico , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Indóis/uso terapêutico , Camundongos , Piridinas/uso terapêutico , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: 3-(6-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (6-MOMIPP) is a novel indole-based chalcone that disrupts microtubules. The present study aims to define the mechanism through which 6-MOMIPP induces cell death and to evaluate the efficacy of the compound in penetrating the blood-brain barrier and inhibiting growth of glioblastoma xenografts. METHODS: The effects of 6-MOMIPP were evaluated in cultured U251 glioblastoma cells, using viability, flow cytometry, and tubulin polymerization assays. Scintillation proximity and tubulin crosslinking methods were used to identify the binding site of 6-MOMIPP on tubulin, and western blots were performed to define the signaling pathways that contribute to cell death. LC/MS assays were used to study the pharmacokinetic behavior of 6-MOMIPP in mice. Subcutaneous and intracerebral xenograft models were utilized to assess the effects of 6-MOMIPP on growth of U251 glioblastoma in vivo. RESULTS: The findings indicate that 6-MOMIPP targets the colchicine site on ß-tubulin. At concentrations ≥ 250 nm, 6-MOMIPP induces mitotic arrest, caspase activation and loss of cell viability. Cells are protected by caspase inhibitors, pointing to an apoptotic mechanism of cell death. Loss of cell viability is preceded by activation of Cdk1(Cdc2) and phosphorylation of Bcl-2 and Bcl-xL. Inhibition of both events with a Cdk1 inhibitor prevents cell death. 6-MOMIPP has broad activity against the viability of multiple glioblastoma, melanoma and lung carcinoma cell lines. Viability of normal cells, including differentiated neurons, is not significantly affected at a drug concentration (1 µM) that reduces viability in most cancer lines. Pharmacokinetic studies in mice show that concentrations of 6-MOMIPP in the brain mirror those in the plasma, indicating that 6-MOMIPP readily penetrates the blood-brain barrier. Studies with mice bearing human U251 glioblastoma xenografts demonstrate that 6-MOMIPP is effective in suppressing growth of subcutaneous and intracerebral tumors without causing general toxicity. CONCLUSIONS: The results indicate that 6-MOMIPP is a novel microtubule disruptor that targets the colchicine binding site on ß-tubulin to induce mitotic arrest and cell death. The ability of 6-MOMIPP to penetrate the blood-brain barrier and inhibit growth of glioblastoma xenografts suggests that it warrants further preclinical evaluation as potential small-molecule therapeutic that may have advantages in treating primary and metastatic brain tumors.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Indóis/farmacologia , Mitose , Piridinas/farmacologia , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Ciclo Celular/efeitos dos fármacos , Feminino , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Indóis/farmacocinética , Camundongos , Camundongos Nus , Piridinas/farmacocinética , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The rise in organisms resistant to existing drugs has added urgency to the search for new antimicrobial agents. Aspartate ß-semialdehyde dehydrogenase (ASADH) catalyzes a critical step in an essential microbial pathway that is absent in mammals. Our laboratory is using fragment library screening to identify efficient and selective ASADH inhibitors. These preliminary agents are then tested to identify compounds with desired antimicrobial properties for further refinement. Toward this end, we have established a microplate-based, dual-assay approach using a single reagent to evaluate antibiotic activity and mammalian cell toxicity during early stage development. The bacterial assay uses nonpathogenic bacteria to allow efficacy testing without a dedicated microbial laboratory. Toxicity assays are performed with a panel of mammalian cells derived from representative susceptible tissues. These assays can be adapted to target other microbial systems, such as fungi and biofilms, and additional mammalian cell lines can be added as needed. Application of this screening approach to antibiotic standards demonstrates the ability of these assays to identify bacterial selectivity and potential toxicity issues. Tests with selected agents from the ASADH inhibitor fragment library show some compounds with antibiotic activity, but as expected, most of these early agents display higher than desired mammalian cell toxicity.
Assuntos
Antibacterianos/farmacologia , Aspartato-Semialdeído Desidrogenase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Antibacterianos/toxicidade , Linhagem Celular , Inibidores Enzimáticos/toxicidade , Humanos , Concentração Inibidora 50 , Reprodutibilidade dos TestesRESUMO
A 14-step biomimetic synthetic route to glyceollin I (1.5% overall yield) was developed and deployed to produce the natural enantiomeric form in soy, its unnatural stereoisomer, and a racemic mixture. Enantiomeric excess was assessed by asymmetric NMR shift reagents and chiral HPLC. Antiproliferative effects were measured in human breast, ovarian, and prostate cancer cell lines, with all three chiral forms exhibiting growth inhibition (GI) in the low to mid µM range for all cells. The natural enantiomer, and in some cases the racemate, gave significantly greater GI than the unnatural stereoisomer for estrogen receptor positive (ER(+)) versus ER(-) breast/ovarian cell lines as well as for androgen receptor positive (AR(+)) versus AR(-) prostate cancer cells. Surprisingly, differences between ER(+) and ER(-) cell lines were not altered by media estrogen conditions. These results suggest the antiproliferative mechanism of glyceollin I stereoisomers may be more complicated than strictly ER interactions.
Assuntos
Antineoplásicos/farmacologia , Biomimética , Pterocarpanos/química , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Cromatografia Líquida de Alta Pressão/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Humanos , Espectroscopia de Ressonância Magnética/métodos , Masculino , Modelos Químicos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Receptores de Estrogênio/metabolismo , EstereoisomerismoRESUMO
A practical synthesis of 2,3-diarylated 2H-benzo[e][1,2]thiazine 1,1-dioxides and their 3,4-dihydro derivatives was developed. ortho-Methyl lithiation of N-aryl-o-toluenesulfonamide followed by reaction with aryl aldehydes gave carbinol sulfonamides, which were either converted directly, or first oxidized to their ketones and converted, to 2,3-diarylated six-membered benzosultams via a TMSCl-NaI-MeCN mediated cyclization. A library of benzosultams was synthesized and evaluated for inhibitory activity against MCF-7 cells. Compound 3 in the 3,4-dihydro (saturated) series and compound 8 in the unsaturated series exhibited the highest potencies with growth inhibition (GI50) values of 0.8 and 18.0 µM, respectively. Molecular modeling studies suggest that these compounds can associate with the colchicine binding site on microtubules. However, experimental assessments of that and other mechanistic possibilities are still ongoing.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Tiazinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Relação Estrutura-Atividade , Tiazinas/síntese química , Tiazinas/químicaRESUMO
A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the simultaneous determination of each of esmolol's enantiomers at the 25-1000 ng/ml concentrations observed in human plasma upon intravenous administration of this rapidly metabolized beta-adrenergic receptor blocking agent. Alternatively, a high performance liquid chromatography (HPLC) UV detection method has been developed for the simultaneous determination of each of the enantiomers for esmolol's metabolite which, in turn, achieve 2.5-50 microg/ml concentrations in human plasma. Utilizing chiral columns, these methods do not require a precolumn asymmetric derivatization step. Linearity in all cases was >0.99. Precision and accuracy at all but the lowest concentrations were within +/-6% for the esmolol enantiomers and within +/-2.5% for the esmolol metabolite enantiomers. These values should be suitable for performing thorough pharmacokinetic studies for all of the stereoisomers of this prototypical soft drug and its corresponding metabolite.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 1/sangue , Cromatografia Líquida de Alta Pressão/métodos , Propanolaminas/sangue , Antagonistas de Receptores Adrenérgicos beta 1/química , Antagonistas de Receptores Adrenérgicos beta 1/farmacocinética , Humanos , Modelos Lineares , Propanolaminas/química , Propanolaminas/farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , EstereoisomerismoRESUMO
Peptidylglycine alpha-amidating monooxygenase (PAM) converts inactive terminal-glycine prohormones into their activated alpha-amidated forms. PAM is thought to play a role in the development of antiandrogen drug resistance in prostate cancer (CaP) through PAMactivated autocrine growth. On the basis of the previous finding that many lung cancer cell lines excrete PAM into their culture media, this study investigates PAM levels in media collected from human CaP cell line cultures. Androgen-independent DU145 and PC-3 prostate cancer cell lines exhibited readily detectable levels of PAM activity in extracts and media, whereas the androgen-dependent LNCaP cell line showed little or no activity. Because of the much larger volume of media versus cell extracts, more than 90% of the total PAM activity was located in the media for both the PC-3 and DU145 cell lines, providing a readily accessible source of CaP PAM. A simple, scalable method to obtain PAM from the culture media of androgen-independent human prostate cancer cell lines is described in this article. This approach provides a much easier means of collecting CaP-derived PAM than previously described cell fractionation procedures and should facilitate the investigations of the role and targeting of PAM in hormone-independent CaP.
Assuntos
Meios de Cultura/química , Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos/metabolismo , Neoplasias da Próstata/enzimologia , Animais , Linhagem Celular Tumoral , Dissulfiram/metabolismo , Dopamina/metabolismo , Inibidores Enzimáticos/metabolismo , Humanos , MasculinoRESUMO
A sensitive and rapid high-performance liquid chromatography method has been developed for simultaneous determination of procaine and its metabolite p-aminobenzoic acid (PABA) from human and rat liver tissue extracts. The method has been validated according to ICH guidelines in terms of selectivity, linearity, lower limit of detection, lower limit of quantitation, accuracy, precision and recovery from human and rat liver tissue extracts. Chromatography was carried out on a Discovery C(18) column using 10mM ammonium acetate at pH 4.0 and acetonitrile as mobile phase. Retention times for procaine and PABA were 6.6 and 5.3 min, respectively. Linearity for each calibration curve in both tissue extracts was observed across a range from 10 microM to 750 microM for procaine and PABA. The lower limit of detection for both procaine and PABA was 5 microM and the lower limit of quantitation was 10 microM in both tissue extracts. The intra- and inter-day relative standard deviations (R.S.D.) for both procaine and PABA were <6%. Recoveries of procaine and PABA from human and rat liver tissue extracts were determined by two different methods with a single-step protein precipitation technique being employed in both methods. Recoveries for both procaine and PABA were greater than 80% from both human and rat liver tissue extracts.
Assuntos
Ácido 4-Aminobenzoico/análise , Cromatografia Líquida de Alta Pressão/métodos , Fígado/química , Procaína/análise , Animais , Humanos , Ratos , Sensibilidade e EspecificidadeRESUMO
A sensitive high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for simultaneous determination of procaine and its metabolite p-aminobenzoic acid (PABA). N-Acetylprocainamide (NAPA) was used as an internal standard for procaine and PABA analysis. This assay method has also been validated in terms of linearity, lower limit of detection, lower limit of quantitation, accuracy and precision as per ICH guidelines. Chromatography was carried out on an XTerra MS C(18) column and mass spectrometric analysis was performed using a Quattro Micro mass spectrometer working with electro-spray ionization (ESI) source in the positive ion mode. Enhanced selectivity was achieved using multiple reaction monitoring (MRM) functions, m/z 237-->100, m/z 138-->120, and m/z 278-->205 for procaine, PABA and NAPA, respectively. Retention times for PABA, procaine and NAPA were 4.0, 4.7 and 5.8min, respectively. Linearity for each calibration curve was observed across a range from 100nM to 5000nM for PABA, and from 10nM to 5000nM for procaine. The intra- and inter-day relative standard deviations (RSD) were <5%.
Assuntos
Ácido 4-Aminobenzoico/análise , Cromatografia Líquida de Alta Pressão/métodos , Procaína/análise , Espectrometria de Massas em Tandem/métodos , Ácido 4-Aminobenzoico/química , Estrutura Molecular , Procaína/química , Reprodutibilidade dos TestesRESUMO
A rapid high-performance liquid chromatography method has been developed for simultaneous determination of capecitabine and its metabolites: 5'-deoxy-5-fluorocytidine (5'-DFCR), 5'-deoxy-5-fluorouridine (5'-DFUR) and 5-fluorouracil (5-FU). 5'-DFCR was synthesized by hydrolyzing capecitabine using commercially available carboxyl esterase (CES) and characterized by NMR, mass spectrometry and elemental analysis. Base-line separations between capecitabine, 5'-DFCR, 5'-DFUR and 5-FU were found with symmetrical peak shapes on a Discovery RP-amide C16 column using 10 mM ammonium acetate at pH 4.0 and methanol as the mobile phase. The retention times of capecitabine, 5'-DFCR, 5'-DFUR and 5-FU were 8.9, 5.0, 5.3 and 3.0 min, respectively. Linear calibration curves were obtained for each compound across a range from 1 to 500 microg ml(-1). The intra- and inter-day relative standard deviations (%RSD) were <5%. A single-step protein precipitation method was employed for separation of the analytes from bio-matrices. Greater than 85% recoveries were obtained for capecitabine, 5'-DFCR, 5'-DFUR and 5-FU from bio-fluids including mouse plasma, mouse serum and rabbit bile.
Assuntos
Bile/química , Cromatografia Líquida de Alta Pressão/métodos , Desoxicitidina/análogos & derivados , Fluoruracila/análogos & derivados , Plasma/química , Soro/química , Animais , Calibragem , Capecitabina , Desoxicitidina/análise , Desoxicitidina/sangue , Desoxicitidina/metabolismo , Floxuridina/análise , Floxuridina/sangue , Fluoruracila/análise , Fluoruracila/sangue , Fluoruracila/metabolismo , Camundongos , Coelhos , Reprodutibilidade dos TestesRESUMO
The effects of TCDD on the distribution of biogenic amines and production of superoxide anion (SA) in different brain regions of rats have been studied after subchronic exposure. Groups of females Sprague-Dawley rats were administered daily dose of 46ng TCDD/(kgday) (treated groups), or the vehicle used to dissolve TCDD (control group), for 90 days. The rats were sacrificed at the end of the exposure period and their brains were dissected into different regions including, hippocampus (H), cerebral cortex (Cc), cerebellum (C), and brain stem (Bs). The levels of different biogenic amines and some of their metabolites, including, norepinephrine (NE), dopamine (DA), 3,4-dihydroxy phenyl acetic acid (DOPAC), 4-hydroxy-3-methoxy-phenyl acetic acid (HVA), 5-hydroxy tryptamine (5-HT), and 5-hydroxy indole 3-acetic acid (5-HIAA), were determined in those brain regions, using a high performance liquid chromatography (HPLC) system with an electrochemical detector. SA production was also determined in those regions, using the cytochrome c reduction method. Results of analyses indicate significant increases in the levels of DA, NE and DOPAC in H, NE and HVA in Cc, NE and DA in Bs and NE in C. SA production was significantly increased in H and Cc, but not in Bs or C. The results also indicated strong correlations between DA and DOPAC, and SA and NE in all of the brain regions, and also between SA and 5-HT/HIAA in H and Cc. These results may indicate the contribution of biogenic amines, especially NE and 5-HT/HIAA to SA overproduction in some brain regions and may also indicate the potential of long term neurotoxic effects of those biogenic amines, in response to subchronic exposure to TCDD.
Assuntos
Aminas Biogênicas/metabolismo , Química Encefálica/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Dibenzodioxinas Policloradas/toxicidade , Superóxidos/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Citocromos c/metabolismo , Dopamina/metabolismo , Feminino , Ácido Homovanílico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Norepinefrina/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Serotonina/metabolismoRESUMO
A microplate screening method has been developed to evaluate the effects of test agents on the accumulation of the fluorescent P-glycoprotein (Pgp) substrates Hoechst 33342, rhodamine 123, and rhodamine 6G in multidrug-resistant (MDR) breast cancer cells that overexpress Pgp. All three substrates exhibit substantially higher accumulation in MCF7 non-MDR cells versus NCI/ADR-RES MDR cells, while incubation with 50 microM reserpine significantly reduces or eliminates these differences. Rhodamine 123 shows the lowest substrate accumulation efficiency in non-MDR cells relative to the substrate incubation level. The effects of several chemosensitizing agents and a series of paclitaxel analogs on the accumulation of each fluorescent substrate suggest that there are distinct differences in the substrate interaction profiles exhibited by these different agents. The described methods may be useful in Pgp-related research in the areas of cancer MDR, oral drug absorption, the blood-brain barrier, renal/hepatic transport processes, and drug-drug interactions.