RESUMO
Accumulating evidence indicates that chronic circadian rhythm disruption is associated with the development of neurodegenerative diseases induced by exposure to neurotoxic chemicals. Herein, we examined the relationship between cellular circadian rhythm disruption and cytotoxicity in neural cells. Moreover, we evaluated the potential application of an in vitro cellular circadian rhythm assay in determining circadian rhythm disruption as a sensitive and early marker of neurotoxicant-induced adverse effects. To explore these objectives, we established an in vitro cellular circadian rhythm assay using human glioblastoma (U87 MG) cells stably transfected with a circadian reporter vector (PER2-dLuc) and determined the lowest-observed-adverse-effect levels (LOAELs) of several common neurotoxicants. Additionally, we determined the LOAEL of each compound on multiple cytotoxicity endpoints (nuclear size [NC], mitochondrial membrane potential [MMP], calcium ions, or lipid peroxidation) using a multiparametric high-content screening (HCS) assay using transfected U87 MG cells treated with the same neurotoxicants for 24 and 72 h. Based on our findings, the LOAEL for cellular circadian rhythm disruption for most chemicals was slightly higher than that for most cytotoxicity indicators detected using HCS, and the LOAEL for MMP in the first 24 h was the closest to that for cellular circadian rhythm disruption. Dietary antioxidants (methylselenocysteine and N-acetyl-l-cysteine) prevented or restored neurotoxicant-induced cellular circadian rhythm disruption. Our results suggest that cellular circadian rhythm disruption is as sensitive as cytotoxicity indicators and occurs early as much as cytotoxic events during disease development. Moreover, the in vitro cellular circadian rhythm assay warrants further evaluation as an early screening tool for neurotoxicants.
Assuntos
Ritmo Circadiano , Neurônios , HumanosRESUMO
BACKGROUND: Citric acid (CA) and sodium hypochlorite (NaOCl) have been used to disinfect animals to protect them against avian influenza and foot-and-mouth disease. OBJECTIVES: We performed a good laboratory practice (GLP)-compliant animal toxicity study to assess the acute toxic effects of CA and NaOCl aerosol exposure in Sprague-Dawley rats. METHODS: Groups of five rats per sex were exposed for 4 h to four concentrations of the two chemicals, i.e., 0.00, 0.22, 0.67, and 2.00 mg/L, using a nose-only exposure. After a single exposure to the chemicals, clinical signs, body weight, and mortality was observed during the observation period. On day 15, an autopsy, and then gross findings, and histopathological analysis were performed. RESULTS: After exposure to CA and NaOCl, body weight loss was observed but recovered. Two males died in the CA 2.00 mg/L group and, two males and one female died in the 2.00 mg/L NaOCl group. In the gross findings and histopathological analysis, discoloration of the lungs was observed in the CA exposed group and inflammatory lesions with discoloration of the lungs were observed in the NaOCl exposed group. These results suggest that the lethal concentration 50 (LC50) of CA is 1.73390 mg/L for males and > 1.70 mg/L for females. For NaOCl, the LC50 was 2.22222 mg/L for males and 2.39456 mg/L for females. CONCLUSIONS: The Globally Harmonized System is category 4 for both CA and NaOCl. In this study, the LC50 results were obtained through a GLP-based acute inhalation toxicity assessment. These results provide useful data to reset safety standards for CA and NaOCl use.
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Pulmão , Hipoclorito de Sódio , Masculino , Ratos , Feminino , Animais , Ratos Sprague-Dawley , Hipoclorito de Sódio/toxicidadeRESUMO
On September 27, 2012, an explosion from hydrofluoric acid occurred in Gumi city of Gyeongbuk province, Republic of Korea, exposing livestock animals nearby to Hydrofluoric acid (HF). This study aimed at evaluating the HF exposure among cattle raised near the accident site by determining the fluoride ion (F-1) levels and other biochemical parameters in the animals' urine and serum. The study groups included 90 cattle raised on farms near the accident site and, as controls, 21 cattle raised on a farm more than 100 km away from the accident site. Urine and blood serum samples were taken from 10% to 20% of the cattle on each farm that were present 17 days after the accident. The F-1 concentrations in the samples were analysed by the fluoride-ion-selective electrode method or a biochemistry analyser. The mean F-1 levels in the cattle serum samples (expressed as mg/L) were 0.23 (100 m), 0.15 (500 m), 0.23 (800 m), 0.11 (900 m), 0.07 (1.2 km), 0.16 (1.5 km), and 0.10 in the control group. The mean F-1 levels in the cattle urine samples (expressed as F-1 mg/g creatinine) were 27.8 (100 m), 24.4 (500 m), 11.1 (800 m), 16.3 (900 m), 3.02 (1.2 km), 9.16 (1.5 km), and 3.58 in the control group. The mean ± SD concentrations of calcium ions in serum (expressed as mg/dL) were 9.72 ± 0.41 (100 m), 9.54 ± 0.57 (500 m), 8.31 ± 0.44 (800 m), 9.06 ± 0.40 (900 m), 8.36 ± 0.89 (1.2 km), 9.13 ± 0.98 (1.5 km), and 10.48 ± 1.43 in the control group. The serum and urine F-1 levels in cattle exposed to HF decreased with the distance from the accident site, suggesting that the relative F-1 levels in urine after normalization through concentration of urinary creatinine could be a more reliable biomarker for HF exposure in cattle than the urine F-1 level alone.
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Stem cell-based products (SCPs) are an emerging field of veterinary medicine that focuses on the regeneration, repair, or replacement of damaged tissues or organs. However, there are some issues in applying the traditional regulatory guideline for the approval of SCPs as veterinary medicinal products. This article describes the positions of Korea, US, and EU regarding SCPs, and compares the regulatory guidelines of each country for their safety evaluation. Although there are some differences in the regulatory guidelines, similar considerations in identifying the quality of SCPs and their safety has adopted. Overall, these guidelines need to be harmonized among countries.
Assuntos
Legislação Veterinária , Transplante de Células-Tronco/legislação & jurisprudência , Células-Tronco , Medicina Veterinária/métodos , Animais , União Europeia , República da Coreia , Estados UnidosRESUMO
BACKGROUND: Although previous in vivo studies explored urinary microRNA (miRNA), there is no agreement on nephrotoxicity-specific miRNA biomarkers. OBJECTIVES: In this study, we assessed whether urinary miRNAs could be employed as biomarkers for nephrotoxicity. METHODS: For this, literature-based candidate miRNAs were identified by reviewing the previous studies. Female Sprague-Dawley rats received subcutaneous injections of a single dose or repeated doses (3 consecutive days) of gentamicin (GEN; 137 or 412 mg/kg). The expression of miRNAs was analyzed by real-time reverse transcription-polymerase chain reaction in 16 h pooled urine from GEN-treated rats. RESULTS: GEN-induced acute kidney injury was confirmed by the presence of tubular necrosis. We identified let-7g-5p, miR-21-3p, 26b-3p, 192-5p, and 378a-3p significantly upregulated in the urine of GEN-treated rats with the appearance of the necrosis in proximal tubules. Specifically, miR-26-3p, 192-5p, and 378a-3p with highly expressed levels in urine of rats with GEN-induced acute tubular injury were considered to have sensitivities comparable to clinical biomarkers, such as blood urea nitrogen, serum creatinine, and urinary kidney injury molecule protein. CONCLUSIONS: These results indicated the potential involvement of urinary miRNAs in chemical-induced nephrotoxicity, suggesting that certain miRNAs could serve as biomarkers for acute nephrotoxicity.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Antibacterianos/toxicidade , Modelos Animais de Doenças , Gentamicinas/toxicidade , MicroRNAs/urina , Animais , Biomarcadores/urina , Feminino , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-DawleyRESUMO
Circadian gene expression is defined by the gene-specific phase and amplitude of daily oscillations in mRNA and protein levels. D site-binding protein mRNA (Dbp mRNA) shows high-amplitude oscillation; however, the underlying mechanism remains elusive. Here, we demonstrate that heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a key regulator that activates Dbp transcription via the poly(C) motif within its proximal promoter. Biochemical analyses identified hnRNP K as a specific protein that directly associates with the poly(C) motif in vitro Interestingly, we further confirmed the rhythmic binding of endogenous hnRNP K within the Dbp promoter through chromatin immunoprecipitation as well as the cycling expression of hnRNP K. Finally, knockdown of hnRNP K decreased mRNA oscillation in both Dbp and Dbp-dependent clock genes. Taken together, our results show rhythmic protein expression of hnRNP K and provide new insights into its function as a transcriptional amplifier of Dbp.
Assuntos
Ritmo Circadiano/genética , Proteínas de Ligação a DNA/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Células 3T3 , Animais , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Poli C/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genéticaRESUMO
The D site-binding protein (Dbp) supports the rhythmic transcription of downstream genes, in part by displaying high-amplitude cycling of its own transcripts compared to other circadian-clock genes. However, the underlying mechanism remains elusive. Here, we demonstrated that the poly(C) motif within the Dbp proximal promoter, in addition to an E-box element, provoked transcriptional activation. Furthermore, we generated a cell line with poly(C) deleted to demonstrate the endogenous effect of the poly(C) motif within the Dbp promoter. We investigated whether RNA polymerase 2 (Pol2) recruitment on the Dbp promoter was decreased in the cell line with poly(C) deleted. Next, assay for transposase-accessible chromatin (ATAC)-quantitative PCR (qPCR) showed that the poly(C) motif induced greater chromatin accessibility within the region of the Dbp promoter. Finally, we determined that the oscillation amplitude of endogenous Dbp mRNA of the cell line with poly(C) deleted was decreased, which affected the oscillation of other clock genes that are controlled by Dbp Taken together, our results provide new insights into the function of the poly(C) motif as a novel cis-acting element of Dbp, along with its significance in the regulation of circadian rhythms.
Assuntos
Relógios Circadianos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , RNA Polimerase II/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Motivos de Aminoácidos , Animais , Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Camundongos , Células NIH 3T3 , Regiões Promotoras Genéticas , Ligação Proteica , Deleção de Sequência , Fatores de Transcrição/metabolismoRESUMO
Most living creatures have a circadian rhythm that is generated by a precisely regulated transcriptional-translational feedback loop of clock genes. Brain and muscle ARNT-like 1 (BMAL1) is one of the core clock genes and transcription factors that represents a positive arm of this autoregulatory circadian clock system. Despite the indispensable role of BMAL1 in the circadian rhythm, the molecular mechanisms underlying translational control of BMAL1 are largely unknown. Here, using murine NIH-3T3 cells, gene constructs, and a variety of biochemical approaches, including RNAi- and luciferase reporter gene-based assays, along with immunoblotting, in vitro transcription, quantitative real-time PCR, and real-time bioluminescence experiments, we show that translation of Bmal1 is negatively regulated by an RNA-binding protein, heterogeneous nuclear ribonucleoprotein Q (hnRNP Q). Interestingly, we found that hnRNP Q rhythmically binds to a specific region of the Bmal1 mRNA 5' UTR and controls its time-dependent expression. Moreover, we demonstrate that knockdown of hnRNP Q modulates BMAL1 protein oscillation amplitude without affecting mRNA rhythmic patterns. Furthermore, hnRNP Q depletion increases the mRNA oscillation amplitudes of BMAL1-regulated target genes. Together, our results suggest that hnRNP Q plays a pivotal role in both Bmal1 translation and BMAL1-regulated gene expression.
Assuntos
Regiões 5' não Traduzidas , Fatores de Transcrição ARNTL/biossíntese , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Fatores de Transcrição ARNTL/genética , Animais , Ribonucleoproteínas Nucleares Heterogêneas/genética , Camundongos , Células NIH 3T3 , Transporte Proteico/genética , RNA Mensageiro/genéticaRESUMO
Acetylcholinesterase (AChE) activity level can be used as a diagnostic marker for anticholinesterase pesticide poisoning. In this study, we aimed to establish a baseline level of normal brain AChE activity in wild birds. AChE activity was measured in the brains of 87dead wild birds (26 species). The level of AChE activity ranged from 6.40 to 15.9 µmol/min/g of brain tissue in normal wild birds. However, the brain tissue AChE activity level in wild birds exposed to organophosphate (OP) pesticide was 48.0%-96.3% of that in the normal birds. These results may serve as reference values to facilitate routine diagnosis and monitoring of OP-poisoned wild birds.
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Acetilcolinesterase/metabolismo , Doenças das Aves/induzido quimicamente , Aves/metabolismo , Encéfalo/enzimologia , Intoxicação por Organofosfatos/veterinária , Animais , Animais Selvagens , Doenças das Aves/diagnóstico , Doenças das Aves/enzimologia , Intoxicação por Organofosfatos/diagnóstico , Intoxicação por Organofosfatos/enzimologia , Valores de Referência , República da CoreiaRESUMO
With the increased use of cell therapy in the veterinary sector, there is a growing demand for the development of cell-based medicinal products and the determination of their safety. Currently, the Korean Animal and Plant Quarantine Agency has established a guideline for evaluating the safety of cell-based medicinal products for animal use. The guideline includes items related to definition, classification, management, manufacturing procedure and quality control (standard and test method), stability testing, toxicity testing, pharmacological testing, and performance of clinical trials. In addition, testing protocols related to safety assessment of animal cell-based products such as chromosome karyotyping, tumorigenicity testing, confirmatory testing of biodistribution and kinetics, and target animal safety testing are described in detail. Moreover, because cell-based medicinal products are novel therapies, deviations from traditional designs may be justified in order to obtain relevant safety information on the treatment. Additionally, this guideline can be amended on the basis of new scientific findings.
Assuntos
Produtos Biológicos/normas , Testes de Toxicidade/veterinária , Animais , Produtos Biológicos/efeitos adversos , Produtos Biológicos/uso terapêutico , Ensaios Clínicos Veterinários como Assunto , Testes de Toxicidade/métodos , Testes de Toxicidade/normasRESUMO
In vitro prediction of hepatotoxicity can enhance the performance of non-clinical animal testing for identifying chemical hazards. In this study, we assessed high-content analysis (HCA) using multi-parameter cell-based assays as an in vitro hepatotoxicity testing model using various hepatotoxicants and human hepatocytes such as HepG2 cells and human primary hepatocytes (hPHs). Both hepatocyte types were exposed separately to multiple doses of ten hepatotoxicants associated with liver injury whose mechanisms of action have been described. HCA data were obtained using fluorescence probes for nuclear size (Hoechst), mitochondrial membrane potential (TMRM), cytosolic free calcium (Fluo-4AM), and lipid peroxidation (BODIPY). Cellular alterations were observed in response to all hepatotoxicants tested. The most sensitive parameter was TMRM, with high sensitivity at a low dose, next was BODIPY, followed by Fluo-4AM. HCA data from HepG2 cells and hPHs were generally concordant, although some inconsistencies were noted. Both hepatocyte types showed mild or severe mitochondrial impairment and lipid peroxidation in response to several hepatotoxicants. The results demonstrate that the application of HCA to in vitro hepatotoxicity testing enables more efficient hazard identification, and further, they suggest that certain parameters could serve as sensitive endpoints for predicting the hepatotoxic potential of chemical compounds.
Assuntos
Cálcio/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Hepatócitos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Células Hep G2 , Humanos , Testes de ToxicidadeRESUMO
Biofluid-based biomarkers provide an efficient tool for hazard identification of chemicals. Here, we explored the potential of microRNAs (miRNAs) as biomarkers for hepatotoxicity of chemicals by linking in vitro to in vivo animal models. A search of the literature identified candidate circulating miRNA biomarkers of chemical-induced hepatotoxicity. The expression of candidate miRNAs (miR-122, miR-151a, miR-192, miR-193a, miR-194, miR-21, miR-29c), was determined by real-time reverse transcription-polymerase chain reaction in in vivo acute liver injury induced by acetaminophen, and then were further compared with those of in vitro cell assays. Candidate miRNAs, except miR-29c, were significantly or biologically upregulated by acetaminophen, at a dose that caused acute liver injury as confirmed by hepatocellular necrosis. Except miR-122 and miR-193a, other miRNAs elevated in in vivo models were confirmed by in vitro models using HepG2 cells, whereas they failed by in vitro models using human primary hepatocytes. These findings indicate that certain miRNAs may still have the potential of toxicological biomarkers in linking in vitro to in vivo hepatotoxicity.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/sangue , Expressão Gênica/efeitos dos fármacos , Substâncias Perigosas/toxicidade , Hepatócitos/efeitos dos fármacos , MicroRNAs/sangue , Animais , Biomarcadores/sangue , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Masculino , MicroRNAs/genética , Ratos Sprague-Dawley , Regulação para CimaRESUMO
INTRODUCTION: Veterinarians use flumequine (FLU) widely but its toxicological effects are still unclear. MATERIAL AND METHODS: FLU doses of 53, 200, or 750 mg/kg were administered orally for six weeks to pubertal male rats for evaluation of their toxicity. RESULTS: Weight gain was poorer after seven days of exposure to FLU 750, but relative weights of the brain, adrenal and thyroid glands, and testes were notably higher. Haematological and lipid profile parameters, cardiac markers, and inorganic phosphate significantly increased in the FLU 750 group. Blood glucose, oestradiol and serum concentrations of immunoglobulins G (IgG) and E (IgE) significantly decreased after treatment. The levels of interleukins 10 (IL-10) and 6 (IL-6) fell significantly in the FLU 200 and FLU 750 groups. Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) and cyclooxygenase-2 (Cox-2) expression amplified after treatment. Serum levels of free triiodothyronine (fT3) and free thyroxine (fT4) reduced in the FLU 200 and FLU 750 groups without changes in total T3 or T4 level. All doses of FLU significantly depressed concentrations of thyroid-stimulating hormone (TSH) and testosterone. Histopathology of thyroid glands from rats treated with FLU 750 showed degeneration and depletion of thyroid follicular epithelial cells. Expression of 8-hydroxydeoxyguanosine (8-OHdG) was increased in a dose-dependent manner in the brain, but decreased in the testes. Expression of CYP1A1 increased in the adrenal and pituitary glands. CONCLUSION: The results of this study suggest that the toxicity of FLU in rats is an effect of its disruptive influence on the pituitary-thyroid hormonal system and on the dysfunction of the immune system.
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BACKGROUND: Pluripotent stem cells (PSCs) such as embryonic stem cells and induced pluripotent stem cells are promising target cells for cell regenerative medicine together with recently advanced technology of in-vitro differentiation. However, residual undifferentiated stem cells (USCs) during in-vitro differentiation are considered a potential risk for development of cancer cells and nonspecific lineage cell types. In this study we observed that USCs still exist during hepatic differentiation, consequently resulting in poor quality of the hepatic population and forming teratoma in vivo. Therefore, we hypothesized that effectively removing USCs from in-vitro differentiation could improve the quality of the hepatic population and guarantee safety from risk of teratoma formation. METHODS: Human PSCs were differentiated to hepatocytes via four steps. YM155, a known BIRC5 inhibitor, was applied for removing the residual USCs on the hepatic differentiation. After YM155 treatment, hepatocyte development was evaluated by measuring gene expression, immunostaining and hepatic functions at each stage of differentiation, and forming teratomas were confirmed by cell transplantation with or without YM155. RESULTS: The selected concentrations of YM155 removed USCs (NANOG+ and OCT4+) in a dose-dependent manner. As a result, expression of endodermal markers (SOX17, FOXA2 and CXCR4) at stage II of differentiation and hepatic markers (ALB, AFP and HNF4A) at stage III was up-regulated by YM155 treatment as well as the hepatic population (ALB+), and functions (ALB/urea secretion and CYP450 enzyme activity) were enhanced at the final stage of differentiation (stage IV). Furthermore, we demonstrated that NANOG and OCT4 expression remaining until stage III (day 15 of differentiation) completely disappeared when treated with YM155 and teratoma formation was effectively prevented by YM155 pretreatment in the in-vitro study. CONCLUSIONS: We suggest that the removal of USCs using YM155 could improve the quantity and quality of induced hepatocytes and eliminate the potential risk of teratoma formation.
Assuntos
Diferenciação Celular , Imidazóis/farmacologia , Fígado/citologia , Naftoquinonas/farmacologia , Células-Tronco Pluripotentes/citologia , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes/efeitos dos fármacos , Teratoma/patologiaRESUMO
Genomic analysis in the local lymph node assays (LLNAs) is useful for assessing skin sensitization of chemicals and providing insights into mechanisms of sensitization. In this study, we collected 1406 genes from previous microarray findings, validated changes in their expression by RT-PCR analysis in local lymph nodes draining skin exposed to different sensitizers, and interpreted their biological function through pathway-based genomic analysis, in which 468 genes were identified as being in the KEGG pathway database. The top-ranked functions (P < 0.01) identified as being affected by the sensitizers were associated with aspects of cell growth, such as DNA replication, cell cycle regulation and pyrimidine metabolism. All the sensitizers tested (DNCB, OXA and TDI) induced significant up-regulation of Psme4, which is associated with DNA replication; Tfdp1, which is related to cell cycle regulation; and Dut, which is involved in pyrimidine metabolism. Specific changes were also shown in functional categories related to the immune response, including cytokines and their receptors. Genes identified in these functional categories, such as Ccl21c, Cxcl9, Cxcl10, Ifng and Il12rb1, were found to have functional relevance. These findings may enhance our understanding and assessment of chemical sensitizers, and enable us to distinguish sensitizers from irritants and to classify chemicals as contact sensitizers.
Assuntos
Alérgenos/toxicidade , Expressão Gênica/efeitos dos fármacos , Ensaio Local de Linfonodo , Linfonodos/efeitos dos fármacos , Administração Tópica , Animais , Óleo de Cróton/toxicidade , Dinitroclorobenzeno/toxicidade , Genômica , Linfonodos/metabolismo , Linfonodos/patologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Oxazolona/toxicidade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tolueno 2,4-Di-Isocianato/toxicidadeRESUMO
Forty-one Enterococcus faecalis (E. faecalis) isolates from feces of pigs and chickens in Korea were screened for the presence of virulence factors. Gelatinase activity (85.4%, 35/41) was the more commonly observed phenotype of virulence in E. faecalis, compared with hemolytic activity (12.2%, 5/41). Thirty-one of 35 (88.6%) gelatinase-positive E. faecalis isolates harbored the gelE and fsrABC genes. A gene encoding for the enterococcal surface protein (Esp) was detected in 24.4% (10/41) of the isolates. All betahemolysin- producing isolates harbored the esp gene.
Assuntos
Proteínas de Bactérias/genética , Enterococcus faecalis/isolamento & purificação , Fezes/microbiologia , Infecções por Bactérias Gram-Positivas/veterinária , Doenças das Aves Domésticas/microbiologia , Doenças dos Suínos/microbiologia , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/metabolismo , Galinhas , Enterococcus faecalis/classificação , Enterococcus faecalis/enzimologia , Enterococcus faecalis/genética , Gelatinases/genética , Gelatinases/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , República da Coreia , Suínos , Fatores de Virulência/metabolismoRESUMO
Limb bud (LB) and central nerve system (CNS) cells were prepared from 12.5 day old pregnant female Crj:CD (SD) rats and treated with olaquindox and vitamin A. Cytotoxicity and inhibition on differentiation were measured in each cell. Three doses of olaquindox (4, 21 and 100 mgkg) , and 0.2 and 75 mg/kg of vitamin A were administered to pregnant rat for 11 days from 6(th) to 16(th) of pregnancy. IC50 values of olaquindox for proliferation and differentiation in CNS cell were 22.74 and 28.32 µg/ml and 79.34 and 23.29 µg/ml in LB cell and those values of vitamin A were 8.13 and 5.94 µg/ml in CNS cell and 0.81 and 0.05 µg/ml in LB cell, respectively. Mean body weights of pregnant rats were decreased at high dose of olaquindox (110 mg/kg) but relative ovary weight, number of corpus lutea, and number of implantation were not changed. Resorption and dead fetus were increased at high dose of olaquindox, and relative ovary weight, the number of corpus lutea and implantation, and sex ratio of male to female were not significantly changed in all dose of olaquindox. Mean fetal and placenta weights were significantly (p < 0.01) decreased in rats of high group. Seven fetuses out of 103 showed external anomaly like bent tail, and 10 out of 114 fetuses showed visceral anomalies at high group. The ossification of sternebrae and metacarpals were significantly (p < 0.01) increased by low and middle dose of olaquindox but it was significantly (p < 0.01) prohibited by high dose of olaquindox. In rats treated with vitamin A, the resorption and dead fetus were increased by high dose. Mean fetal weights were significantly (p < 0.01) increased by low dose but significantly (p < 0.01) decreased by high dose. Thirty four fetuses out of 52 showed external anomaly; bent tail (1) , cranioarchschisis (14) , exencephaly (14) , dome shaped head (22) , anophthalmia (15) , brcahynathia (10) and others (19) . Forty five fetuses out of 52 showed soft tissue anomaly; cleft palate (42/52) and anophthalmia (22/52) by high dose of vitamin A. Sixty one fetuses out of 61 (85.2%) showed skull anomaly; defect of frontal, partial and occipital bone (21/61) , defect of palatine bone (52/61) and others (50/61) . In summary, we support that vitamin A is strong teratogen based on our micromass and in vivo data, and olaquindox has a weak teratogenic potential in LB cell but not in CNS cell. We provide the in vivo evidence that a high dose of olaquindox could have weak embryotoxic potential in rats.
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Clove (Eugenia caryophyllata) is a well-known medicinal plant used for diarrhea, digestive disorders, or in antiseptics in Korea. Eugenol is the main active ingredient of clove and has been chosen as a marker compound for the chemical evaluation or QC of clove. This paper reports the development and validation of an HPLC-diode array detection (DAD) method for the determination of eugenol in clove. HPLC separation was accomplished on an XTerra RP18 column (250 x 4.6 mm id, 5 microm) with an isocratic mobile phase of 60% methanol and DAD at 280 nm. Calibration graphs were linear with very good correlation coefficients (r2 > 0.9999) from 12.5 to 1000 ng/mL. The LOD was 0.81 and the LOQ was 2.47 ng/mL. The method showed good intraday precision (%RSD 0.08-0.27%) and interday precision (%RSD 0.32-1.19%). The method was applied to the analysis of eugenol from clove cultivated in various countries (Indonesia, Singapore, and China). Quantitative analysis of the 15 clove samples showed that the content of eugenol varied significantly, ranging from 163 to 1049 ppb. The method of determination of eugenol by HPLC is accurate to evaluate the quality and safety assurance of clove, based on the results of this study.
Assuntos
Eugenol/análise , Syzygium/química , Calibragem , Cromatografia Líquida de Alta Pressão , Indicadores e Reagentes , Coreia (Geográfico) , Extratos Vegetais/análise , Reprodutibilidade dos Testes , SolventesRESUMO
A total of 147 Enterococcus faecium and 165 Enterococcus faecalis isolates from fecal samples of chickens and pigs at slaughterhouses in Korea were tested for their resistance to 8 growth-promoting antimicrobials commonly used in animals and quinupristin and dalfopristin. Resistance to most antimicrobials was very common among both E. faecalis and E. faecium. In particular, E. faecalis showed almost no susceptibility to all the antimicrobials tested except penicillin and flavomycin, to which 1.4% and less than 24% showed resistance, respectively. Although the prevalence of resistance was lower than in E. faecalis, E. faecium showed relatively uniform resistance to all the agents tested. Among the antimicrobials tested, virginiamycin and penicillin were the most effective against E. faecium isolates: less than 31% and 41% showed resistance to those 2 antimicrobials, respectively. Penicillin was the only agent that showed relatively strong activity against both E. faecalis and E. faecium. Resistance observed in E. faecalis and E. faecium against most antimicrobials used for growth promotion was more prevalent in Korea than in European countries. The current study is the first report of resistance against feed additive antimicrobials in enterococcal isolates from livestock in Korea.
Assuntos
Galinhas/microbiologia , Enterococcus/isolamento & purificação , Infecções por Bactérias Gram-Positivas/veterinária , Suínos/microbiologia , Animais , Antibacterianos/farmacologia , Enterococcus/crescimento & desenvolvimento , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/isolamento & purificação , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/epidemiologia , Imunidade Inata , Coreia (Geográfico)/epidemiologia , Testes de Sensibilidade Microbiana/veterinária , Doenças das Aves Domésticas/tratamento farmacológico , Doenças das Aves Domésticas/epidemiologia , Especificidade da Espécie , Doenças dos Suínos/tratamento farmacológico , Doenças dos Suínos/epidemiologiaRESUMO
It is often difficult to discriminate between chemically induced skin irritation and sensitization due to their similar clinical, pathological, and immunological responses. More information than that currently available from local lymph node assays (LLNAs), such as data from gene expression and pathway analysis, can provide more insightful data than the assay itself for distinguishing skin sensitization from skin irritation. This study investigated the gene expression profiles and pathways in ear skins of mice topically exposed daily for three consecutive days to the known strong contact sensitizer 1-chloro-2,4-dinitrobenzene, the skin contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone, the skin or respiratory sensitizer toluene 2,4-diisocyanate, or to the non-sensitizing irritant croton oil. All the sensitizers induced histological changes in ear tissues similar to those induced by the croton oil. In gene expression microarrays, sensitizers up-regulated 193 genes and down-regulated 61 genes in ear skin following chemical exposure. 13 genes whose expression was affected by more than two-fold by all three of the sensitizers, but not by the irritant, were selected by microarray analysis. Microarray and real-time RT-PCR analyses revealed that, of these genes, the allergic inflammation-related genes Oasl2 and Zbp1 were up-regulated in skin inflammation by the sensitizers. In gene expression pathway analysis of all the sensitizers and the croton oil, the top functions of the 48 genes were related to cytokine and cytokine receptors interactions, and only two genes (Cxcl9 and Cxcl10) were specific to skin sensitizer-induced skin inflammation. Thus, although contact sensitizer-induced skin inflammation is similar to irritant-induced responses in terms of histological changes and gene expression profiles, the regulation of allergic inflammation-related gene transcripts, such as those of Oasl2 and Zbp1 or Cxcl9 and Cxcl10, could help to discriminate skin sensitization from chemically induced skin inflammation.