Investigations of the Toxic Effect of Silver Nanoparticles on Mammalian Cell Lines

Silver nanoparticles are widely used for many applications. In this study silver nanoparticles have been tested for their toxic effect on fibroblasts (NIH-3T3), on a human lung adenocarcinoma epithelial cell line (A-549), on PC-12-cells, a rat adrenal pheochromocytoma cell line, and on HEP-G2-cells, a human hepatocellular carcinoma cell line. The viability of the cells cultivated with different concentrations of silver was determined by the MTT assay, a photometric method to determine cell metabolism. Dose-response curves were extrapolated and IC50, total lethal concentration (TLC), and no observable adverse effect concentration (NOAEC) values were calculated for each cell line. As another approach, ECIS (electric-cell-substrate-impedance-sensing) an automated method to monitor cellular behavior in real-time was applied to observe cells cultivated with silver nanoparticles. To identify the type of cell death the membrane integrity was analyzed by measurements of the lactate dehydrogenase releases and by determination of the caspase 3/7 activity. To ensure that the cytotoxic effect of silver nanoparticles is not traced back to the presence of Ag+ ions in the suspension, an Ag+ salt (AgNO3) has been examined at the same concentration of Ag+ present in the silver nanoparticle suspension that is assuming that the Ag particles are completely available as Ag+ ions.

Granulocyte-colony stimulating factor in experimental stroke and its effects on infarct size and functional outcome: a systematic review

Background Granulocyte-colony stimulating factor (G-CSF) shows promise as a treatment for stroke. This systematic review assesses G-CSF in experimental ischaemic stroke. Methods Relevant studies were identified with searches of Medline, Embase and PubMed. Data were extracted on stroke lesion size, neurological outcome and quality, and analysed using Cochrane Review Manager using random effects models; results are expressed as standardised mean difference (SMD) and odds ratio (OR). Results Data were included from 19 publications incorporating 666 animals. G-CSF reduced lesion size significantly in transient (SMD -1.63, p<0.00001) but not permanent (SMD -1.56, p=0.11) focal models of ischaemia. Lesion size was reduced at all doses and with treatment commenced within 4 hours of transient ischaemia. Neurological deficit (SMD -1.37, p=0.0004) and limb placement (SMD -1.88, p=0.003) improved with G-CSF; however, locomotor activity (>4 weeks post ischaemia) was not (SMD 0.76, p=0.35). Death (OR 0.27, p<0.0001) was reduced with G-CSF. Median study quality was 4 (range 0-7/8); Egger’s test suggested significant publication bias (p=0.001). Conclusions G-CSF significantly reduced lesion size in transient but not permanent models of ischaemic stroke. Motor impairment and death were also reduced. Further studies assessing dose-response, administration time, length of ischaemia and long-term functional recovery are needed.

Alcohol consumption and lung cancer risk in never smokers

Objective: The main objective of this study is to analyse the role of alcohol consumption on lung cancer risk in people who have never smoked. Methods: We conducted a systematic review of the scientific literature following the PRISMA statement. We searched Medline, EMBASE and CINAHL using different combinations of MeSH terms and free text. We included cohort studies, pooled cohort studies and case-control studies comprising at least 25 anatomopathologically-confirmed diagnoses of lung cancer cases, a sample size larger than 100 individuals and more than five years of follow-up for cohort studies. We excluded studies that did not specifically report results for never smokers. We developed a quality score to assess the quality of the included papers and we ultimately included 14 investigations with a heterogeneous design and methodology. Results: Results for alcohol consumption and lung cancer risk in never smokers are inconclusive; however, several studies showed a dose-response pattern for total alcohol consumption and for spirits. Heterogeneous results were found for wine and beer. Conclusion: No clear effect is observed for alcohol consumption. Due to the limited evidence, no conclusion can be drawn for beer or wine consumption. There is little research available on the effect of alcohol on lung cancer risk for people who have never smoked, and more studies are urgently needed on this topic.

Avaliação ecotoxicológica de fármacos psicotrópicos e suas possíveis interações com nanomateriais usando embriões de peixe-zebra

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2016.

Toxicity of polychlorinated biphenyls and N-phenyl-1-naphthylamine in the juvenile turtle, Chelydra serpentina

Polychlorinated biphenyls (PCBs) and substituted phenylamine antioxidants (SPAs) are two chemical groups that have been used in multiple Canadian industrial processes. Despite the production ban of PCBs in North America in 1977, they are still ubiquitous in the environment and in wildlife tissues. Previous studies of fish, amphibians, birds, and mammals have shown that PCBs are toxic and act as endocrine disruptors. In contrast, SPAs, specifically N-phenyl-1-naphthylamine (PANA), have received very little attention despite their current use in Canada and their expected environmental releases. The effects of PCB and PANA exposures in reptiles remain unknown thus, juvenile Chelydra serpentina were used in this thesis as a model vertebrate to fill in missing toxicity research gaps due to their importance as an environmental indicator. First, food pellets were spiked at an environmentally relevant concentration of the PCB mixture Aroclor 1254 (A1254) to model hepatic bioaccumulation (0.45 μg/g A1254 for 31 days) and depuration (clean food for 50 days) of PCBs in turtles. No significant differences in PCB concentrations were observed between the control and treated animals, suggesting that juvenile turtles exposed to environmentally relevant concentrations of PCBs can likely detoxify low concentrations of PCBs. Additionally, two dose-response experiments were performed using A1254 or PANA spiked food (0-12.7 μg/g and 0-3,446 μg/g, respectively) to determine hepatic toxicity and bioaccumulation in juvenile C. serpentina. An increase in hepatic cyp1a was observed when exposed to the highest dose of both chemicals: 1) for A1254, induction correlated to the significant increase in hepatic PCB congeners that are known to be metabolized by CYP1A; and 2) for PANA, induction suggested that CYP1A has a potential role in its detoxification. PCBs are known endocrine disruptors, but no significant changes were observed for both thyroid receptors (alpha and beta) or by estrogen and androgen receptors. This lack of response, also noted in the PANA exposure, suggests that C. serpentina is less sensitive to endocrine disruption than other vertebrates. Furthermore, the expression of genes involved in cellular stress was not altered in PCB and PANA exposed animals, supporting the resilience of turtles to oxidative stress. This is the first study to demonstrate the toxicity of PCBs and PANA in C. serpentina, demonstrating the turtle’s high tolerance to contamination.

Edoxaban versus enoxaparin–warfarin in patients undergoing cardioversion of atrial fibrillation (ENSURE-AF): a randomised, open-label, phase 3b trial

Background Edoxaban, an oral factor Xa inhibitor, is non-inferior for prevention of stroke and systemic embolism in patients with atrial fibrillation and is associated with less bleeding than well controlled warfarin therapy. Few safety data about edoxaban in patients undergoing electrical cardioversion are available. Methods We did a multicentre, prospective, randomised, open-label, blinded-endpoint evaluation trial in 19 countries with 239 sites comparing edoxaban 60 mg per day with enoxaparin–warfarin in patients undergoing electrical cardioversion of non-valvular atrial fibrillation. The dose of edoxaban was reduced to 30 mg per day if one or more factors (creatinine clearance 15–50 mL/min, low bodyweight [≤60 kg], or concomitant use of P-glycoprotein inhibitors) were present. Block randomisation (block size four)—stratified by cardioversion approach (transoesophageal echocardiography [TEE] or not), anticoagulant experience, selected edoxaban dose, and region—was done through a voice-web system. The primary efficacy endpoint was a composite of stroke, systemic embolic event, myocardial infarction, and cardiovascular mortality, analysed by intention to treat. The primary safety endpoint was major and clinically relevant non-major (CRNM) bleeding in patients who received at least one dose of study drug. Follow-up was 28 days on study drug after cardioversion plus 30 days to assess safety. This trial is registered with ClinicalTrials.gov, number NCT02072434. Findings Between March 25, 2014, and Oct 28, 2015, 2199 patients were enrolled and randomly assigned to receive edoxaban (n=1095) or enoxaparin–warfarin (n=1104). The mean age was 64 years (SD 10·54) and mean CHA2DS2-VASc score was 2·6 (SD 1·4). Mean time in therapeutic range on warfarin was 70·8% (SD 27·4). The primary efficacy endpoint occurred in five (<1%) patients in the edoxaban group versus 11 (1%) in the enoxaparin–warfarin group (odds ratio [OR] 0·46, 95% CI 0·12–1·43). The primary safety endpoint occurred in 16 (1%) of 1067 patients given edoxaban versus 11 (1%) of 1082 patients given enoxaparin–warfarin (OR 1·48, 95% CI 0·64–3·55). The results were independent of the TEE-guided strategy and anticoagulation status. Interpretation ENSURE-AF is the largest prospective randomised clinical trial of anticoagulation for cardioversion of patients with non-valvular atrial fibrillation. Rates of major and CRNM bleeding and thromboembolism were low in the two treatment groups. Funding Daiichi Sankyo provided financial support for the study. © 2016 Elsevier Ltd

Dolor osteo-muscular y factores asociados en el personal de enfermería de un centro hospitalario de alta complejidad en Colombia

Antecedentes: Los trastornos musculo-esqueléticos son una de las primeras causas de ausentismo laboral y afectan con mayor frecuencia columna y miembros. El personal de enfermería está expuesto a riesgo biomecánico superior dado por la manipulación manual de pacientes. Estimar la magnitud de asociación entre dolor osteo-muscular y carga biomecánica por movilización de pacientes en personal de enfermería y, los factores que modifican tal efecto, es de gran importancia en el ámbito laboral en busca de estrategias de prevención de enfermedades de origen laboral. Objetivo: determinar los factores predictores de dolor osteo-muscular en trabajadores de enfermería en un centro hospitalario de alta complejidad. Materiales y métodos: Estudio analítico transversal, en el que participaron 141 personas, quienes otorgaron su consentimiento informado. Los criterios de inclusión fueron: edad≥ 18 años, estudiantes, practicantes, auxiliares y jefes de enfermería asistenciales de salas de cirugía y hospitalización, antigüedad mínima de 2 meses en el cargo. Se utilizaron como criterios de exclusión: personal de enfermería en cargos administrativos y de las áreas de urgencias y cuidados intensivos, trabajadores con diagnóstico establecido artrosis, osteoartritis o artritis reumatoide, mujeres gestantes. Para lograr los objetivos aplicó el Cuestionario Nórdico, el cuestionario internacional de actividad física IPAQ y la metodología MAPO. Resultados: La frecuencia global de dolor osteo-muscular en el último año fue de 55%, implicando más frecuentemente columna lumbar, columna dorsal y miembro superior. Se evaluaron los servicios de onco-hematología, hospitalización y salas de cirugía y las clasificaciones de MAPO fueron medio a alto, no hubo áreas con riesgo irrelevante. Mediante análisis de regresión logística multivariada, se pudo determinar que los principales predictores de dolor osteo-muscular son trabajar en un área con MAPO nivel alto (OR=2,236, con respecto a MAPO medio) y en el turno de la mañana (OR=2,355, con respecto a turno en la tarde/noche).

New cholinesterase inhibitors for Alzheimer's disease: Structure Activity Studies (SARs) and molecular docking of isoquinolone and azepanone derivatives

A library of isoquinolinone and azepanone derivatives were screened for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity. The strategy adopted included (a) in vitro biological assays, against eel AChE (EeAChE) and equine serum BuChE (EqBuChE) in order to determine the compounds IC50 and their dose-response activity, consolidated by (b) molecular docking studies to evaluate the docking poses and interatomic interactions in the case of the hit compounds, validated by STD-NMR studies. Compound (1f) was identified as one of these hits with an IC50 of 89.5 mu M for EeAChE and 153.8 mu M for EqBuChE, (2a) was identified as a second hit with an IC50 of 108.4 mu M (EeAChE) and 277.8 mu M (EqBuChE). In order to gain insights into the binding mode and principle active site interactions of these molecules, (R)-(1f) along with 3 other analogues (also as the R-enantiomer) were docked into both RhAChE and hBuChE models. Galantamine was used as the benchmark. The docking study was validated by performing an STD-NMR study of (1f) with EeAChE using galantamine as the benchmark.

Long-term citalopram administration reduces responsiveness of HPA axis in patients with major depression: relationship with S-citalopram concentrations in plasma and cerebrospinal fluid (CSF) and clinical response.

RATIONALE: A dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is a well-documented neurobiological finding in major depression. Moreover, clinically effective therapy with antidepressant drugs may normalize the HPA axis activity. OBJECTIVE: The aim of this study was to test whether citalopram (R/S-CIT) affects the function of the HPA axis in patients with major depression (DSM IV). METHODS: Twenty depressed patients (11 women and 9 men) were challenged with a combined dexamethasone (DEX) suppression and corticotropin-releasing hormone (CRH) stimulation test (DEX/CRH test) following a placebo week and after 2, 4, and 16 weeks of 40 mg/day R/S-CIT treatment. RESULTS: The results show a time-dependent reduction of adrenocorticotrophic hormone (ACTH) and cortisol response during the DEX/CRH test both in treatment responders and nonresponders within 16 weeks. There was a significant relationship between post-DEX baseline cortisol levels (measured before administration of CRH) and severity of depression at pretreatment baseline. Multiple linear regression analyses were performed to identify the impact of psychopathology and hormonal stress responsiveness and R/S-CIT concentrations in plasma and cerebrospinal fluid (CSF). The magnitude of decrease in cortisol responsivity from pretreatment baseline to week 4 on drug [delta-area under the curve (AUC) cortisol] was a significant predictor (p<0.0001) of the degree of symptom improvement following 16 weeks on drug (i.e., decrease in HAM-D21 total score). The model demonstrated that the interaction of CSF S-CIT concentrations and clinical improvement was the most powerful predictor of AUC cortisol responsiveness. CONCLUSION: The present study shows that decreased AUC cortisol was highly associated with S-CIT concentrations in plasma and CSF. Therefore, our data suggest that the CSF or plasma S-CIT concentrations rather than the R/S-CIT dose should be considered as an indicator of the selective serotonergic reuptake inhibitors (SSRIs) effect on HPA axis responsiveness as measured by AUC cortisol response.

Allopurinol hypersensitivity is primarily mediated by dose-dependent oxypurinol-specific T cell response

BACKGROUND Allopurinol is a main cause of severe cutaneous adverse reactions (SCAR). How allopurinol induces hypersensitivity remains unknown. Pre-disposing factors are the presence of the HLA-B*58:01 allele, renal failure and possibly the dose taken. OBJECTIVE Using an in vitro model, we sought to decipher the relationship among allopurinol metabolism, HLA-B*58:01 phenotype and drug concentrations in stimulating drug-specific T cells. METHODS Lymphocyte transformation test (LTT) results of patients who had developed allopurinol hypersensitivity were analysed. We generated allopurinol or oxypurinol-specific T cell lines (ALP/OXP-TCLs) from allopurinol naïve HLA-B*58:01(+) and HLA-B*58:01(-) individuals using various drug concentrations. Their reactivity patterns were analysed by flow cytometry and (51) Cr release assay. RESULTS Allopurinol allergic patients are primarily sensitized to oxypurinol in a dose-dependent manner. TCL induction data show that both the presence of HLA-B*58:01 allele and high concentration of drug are important for the generation of drug-specific T cells. The predominance of oxypurinol-specific lymphocyte response in allopurinol allergic patients can be explained by the rapid conversion of allopurinol to oxypurinol in vivo rather than to its intrinsic immunogenicity. OXP-TCLs do not recognize allopurinol and vice versa. Finally, functional avidity of ALP/OXP-TCL is dependent on both the induction dose and HLA-B*58:01 status. CONCLUSIONS AND CLINICAL RELEVANCE This study establishes the important synergistic role of drug concentration and HLA-B*58:01 allele in the allopurinol or oxypurinol-specific T cell responses. Despite the prevailing dogma that Type B adverse drug reactions are dose independent, allopurinol hypersensitivity is primarily driven by oxypurinol-specific T cell response in a dose-dependent manner, particular in the presence of HLA-B*58:01 allele.

Androgen deprivation results in time-dependent hypoxia in LNCaP prostate tumours; informed scheduling of the bioreductive drug AQ4N improves treatment response

Androgen withdrawal induces hypoxia in androgen-sensitive tissue; this is important as in the tumour microenvironment hypoxia is known to drive malignant progression. This study examined the time-dependent effect of androgen deprivation therapy (ADT) on tumour oxygenation and investigated the role of ADT-induced hypoxia on malignant progression in prostate tumours. LNCaP xenografted tumours were treated with anti-androgens and tumour oxygenation measured. Dorsal skin fold chambers (DSF) were used to image tumour vasculature in vivo. Quantitative PCR (QPCR) identified differential gene expression following treatment with bicalutamide. Bicalutamide and vehicle-only treated tumours were re-established in vitro and invasion and sensitivity to docetaxel were measured. Tumour growth delay was calculated following treatment with bicalutamide combined with the bioreductive drug AQ4N. Tumour oxygenation measurements showed a precipitate decrease following initiation of ADT. A clinically relevant dose of bicalutamide (2mg/kg/day) decreased tumour oxygenation by 45% within 24h, reaching a nadir of 0.09% oxygen (0.67±0.06 mmHg) by day 7; this persisted until day 14 when it increased up to day 28. Using DSF chambers, LNCaP tumours treated with bicalutamide showed loss of small vessels at days 7 and 14 with revascularization occurring by day 21. QPCR showed changes in gene expression consistent with the vascular changes and malignant progression. Cells from bicalutamide-treated tumours were more malignant than vehicle-treated controls. Combining bicalutamide with AQ4N (50mg/kg; single dose) caused greater tumour growth delay than bicalutamide alone. This study shows that bicalutamide-induced hypoxia selects for cells that show malignant progression; targeting hypoxic cells may provide greater clinical benefit.

Toxicidade do cloridrato de doxorrubicina na dose de 120mg/m², em cadelas com tumor venéreo transmissível

Pós-graduação em Cirurgia Veterinária - FCAV

ABCB1 haplotype is associated with major molecular response in chronic myeloid leukemia patients treated with standard-dose of imatinib

Background: Imatinib mesylate (IM) is a selective tyrosine kinase inhibitor used for treating chronic myeloid leukemia (CML). IM has high efficacy, however some individuals develop a resistance due to impaired bio-availability. Polymorphisms in genes encoding membrane transporters such as ABCB1 have been associated with differences in protein expression and function that influence the response to several drugs. Aim: To investigate the relationship of ABCB1 polymorphisms with markers of response to IM in patients with CML Methods: One hundred eighteen CML patients initially treated with a standard dose of IM (400 mg/day) for 18 months were selected at two health centers in Sao Paulo City, Brazil. The response criteria were based on the European LeukemiaNet recommendations. ABCB1 polymorphisms c.1236C>T (rs1128503), c.3435C>T (rs1045642) and c.2677G>T/A (rs2032582) were evaluated by PCR-RFLP. Results: ABCB1 polymorphisms were not related with a risk for CML in this sample population (p<0.05). In the CML group, frequencies of ABCB1 SNPs were similar between responder and non-responder patients (p>0.05). In the responder group, the frequency of ABCB11236CT/2677GT/3435CT haplotype was higher in patients with major molecular response (MMR) (51.7%) than in patients without MMR (8.3%, p = 0.010). Furthermore, carriers of this haplotype had increased the probability of reaching the MMR compared with the non-carriers (OR: 11.8; 95% CI: 1.43-97.3, p = 0.022). Conclusions: The ABCB1 1236CT/2677GT/3435CT haplotype is positively associated with the major molecular response to IM in CML patients. (C) 2011 Elsevier Inc. All rights reserved.

Drug antigenicity, immunogenicity, and costimulatory signaling: evidence for formation of a functional antigen through immune cell metabolism

Recognition of drugs by immune cells is usually explained by the hapten model, which states that endogenous metabolites bind irreversibly to protein to stimulate immune cells. Synthetic metabolites interact directly with protein-generating antigenic determinants for T cells; however, experimental evidence relating intracellular metabolism in immune cells and the generation of physiologically relevant Ags to functional immune responses is lacking. The aim of this study was to develop an integrated approach using animal and human experimental systems to characterize sulfamethoxazole (SMX) metabolism-derived antigenic protein adduct formation in immune cells and define the relationship among adduct formation, cell death, costimulatory signaling, and stimulation of a T cell response. Formation of SMX-derived adducts in APCs was dose and time dependent, detectable at nontoxic concentrations, and dependent on drug-metabolizing enzyme activity. Adduct formation above a threshold induced necrotic cell death, dendritic cell costimulatory molecule expression, and cytokine secretion. APCs cultured with SMX for 16 h, the time needed for drug metabolism, stimulated T cells from sensitized mice and lymphocytes and T cell clones from allergic patients. Enzyme inhibition decreased SMX-derived protein adduct formation and the T cell response. Dendritic cells cultured with SMX and adoptively transferred to recipient mice initiated an immune response; however, T cells were stimulated with adducts derived from SMX metabolism in APCs, not the parent drug. This study shows that APCs metabolize SMX; subsequent protein binding generates a functional T cell Ag. Adduct formation above a threshold stimulates cell death, which provides a maturation signal for dendritic cells.

Active intravenous drug use during chronic hepatitis C therapy does not reduce sustained virological response rates in adherent patients

Reluctance has been expressed about treating chronic hepatitis C in active intravenous (IV) drug users (IDUs), and this is found in both international guidelines and routine clinical practice. However, the medical literature provides no evidence for an unequivocal treatment deferral of this risk group. We retrospectively analyzed the direct effect of IV drug use on treatment outcome in 500 chronic hepatitis C patients enrolled in the Swiss Hepatitis C Cohort Study. Patients were eligible for the study if they had their serum hepatitis C virus (HCV) RNA tested 6 months after the end of treatment and at least one visit during the antiviral therapy, documenting the drug use status. Five hundred patients fulfilled the inclusion criteria (199 were IDU and 301 controls). A minimum exposure to 80% of the scheduled cumulative dose of antivirals was reached in 66.0% of IDU and 60.5% of controls (P = NS). The overall sustained virological response (SVR) rate was 63.6%. Active IDU reached a SVR of 69.3%, statistically not significantly different from controls (59.8%). A multivariate analysis for treatment success showed no significant negative influence of active IV drug use. In conclusion, our study shows no relevant direct influence of IV drugs on the efficacy of anti-HCV therapy among adherent patients.