Demonstration of microRNA using in situ hybridisation on formalin fixed paraffin wax samples using conventional oligonucleotide probes: a comparison with the use of locked nucleic acid probes.

BACKGROUND: MicroRNAs (miRNAs) regulate the translation of mRNA during gene expression and investigations have highlighted their importance in pathophysiology. qRT-PCR is currently the gold standard method for detecting changes in miRNA expression. However, when used on heterogeneous samples, it cannot identify individual cell types harbouring miRNAs. For this, in situ hybridisation (ISH) can be used. ISH methods using locked nucleic acid (LNA) probes give reliable results in formalin fixed paraffin-embedded (FFPE) samples. In this study their use has been directly compared with conventional oligonucleotide probes (COP) for ISH. METHODS: FFPE samples of colorectal adenocarcinoma, squamous carcinoma of lung and cases of invasive breast carcinoma were used to evaluate COP and LNA methods for the demonstration of miR-126 and miR-205. To demonstrate the utility of the COP method demonstration of miR-21 in 19 Gleason stage 7 prostate biopsy FFPE tissues was also undertaken. The demonstration of miR-21 by ISH in high and low expressing prostate cancer cell lines was also compared with qRT-PCR. RESULTS: Similar results were obtained using the COP and LNA ISH methods for the demonstration of miR-126 and miR-205. miR-21 was successfully demonstrated in the prostate cancer samples by COP ISH and expression levels of the miRNA demonstrated in the cell lines corresponded with qRT-PCR. CONCLUSION: This study has shown that simplification of ISH protocols by the use of COPs provides equivalent results to the use of LNA methods and it can be used to precisely identify cells in which miRNAs are expressed.

Age-dependent hepatic alterations induced by a high-fat high-fructose diet.

OBJECTIVE: The present study aimed to evaluate and clarify how the age at which the intake of a high-fat and high-fructose diet begins can affect animals' livers. METHODS: Thirty-eight male wistar rats aged 6 and 12 weeks were fed a high-fat and high-fructose diet for 13 weeks. Inflammatory cytokines, hepatic glycogen, serum and hepatic triacylglycerol and pAkt protein content in the liver were assessed. Percentage of weight gained, and visceral adiposity were also evaluated. RESULTS: Young animal presented increased hepatic triacylglycerol and decreased glycogen, while adult animals had no significant alterations regarding its contents. IL6 and IL10 to IL6 ratio were also altered in young animals exposed to HFHF, while adult animals fed with HFHF had only increases in TNF-α. Both groups which received HFHF had increased serum triacylglycerol and visceral adiposity. However, only young animals gained more relative weight and had greater final body weight, gains which were related to alterations found in hepatic triacylglycerol and glycogen. CONCLUSION: Age of which consumption begins interferes in how the liver deals with an excess of nutrient and subsequent proinflammatory stimulation, leading to different phenotypes.

Exposure to crack cocaine causes adverse effects on marine mussels Perna perna.

Our study aimed to evaluate crack cocaine effects in different life stages of the marine mussel Perna perna. For this purpose, fertilization rate, embryo-larval development, lysosomal membrane stability and DNA strand breaks were assessed. Effect concentrations in gametes and in larval development were found after 1h (IC50=23.53mg·L-1) and 48h (IC50=16.31mg·L-1), respectively. The highest tested concentration showing no acute toxicity (NOEC) was 10mg·L-1, while the lowest observed effect concentration (LOEC) was 20mg·L-1. NOEC concerning embryo-larval development was 0.625mg·L-1, while the LOEC was 1.25mg·L-1. Cyto-genotoxic effects were evidenced in mussels exposed to crack cocaine concentrations ranging from 5 to 500µg·L-1. Our results report the first data on effects of an illicit drug to marine organisms and should encourage further ecotoxicological studies of these contaminants of emerging concern in coastal ecosystems.

Characterization and biocompatibility of a fibrous glassy scaffold.

Bioactive glasses (BGs) are known for their ability to bond to living bone and cartilage. In general, they are readily available in powder and monolithic forms, which are not ideal for the optimal filling of bone defects with irregular shapes. In this context, the development of BG-based scaffolds containing flexible fibres is a relevant approach to improve the performance of BGs. This study is aimed at characterizing a new, highly porous, fibrous glassy scaffold and evaluating its in vitro and in vivo biocompatibility. The developed scaffolds were characterized in terms of porosity, mineralization and morphological features. Additionally, fibroblast and osteoblast cells were seeded in contact with extracts of the scaffolds to assess cell proliferation and genotoxicity after 24, 72 and 144 h. Finally, scaffolds were placed subcutaneously in rats for 15, 30 and 60 days. The scaffolds presented interconnected porous structures, and the precursor bioglass could mineralize a hydroxyapatite (HCA) layer in simulated body fluid (SBF) after only 12 h. The biomaterial elicited increased fibroblast and osteoblast cell proliferation, and no DNA damage was observed. The in vivo experiment showed degradation of the biomaterial over time, with soft tissue ingrowth into the degraded area and the presence of multinucleated giant cells around the implant. At day 60, the scaffolds were almost completely degraded and an organized granulation tissue filled the area. The results highlight the potential of this fibrous, glassy material for bone regeneration, due to its bioactive properties, non-cytotoxicity and biocompatibility. Future investigations should focus on translating these findings to orthotopic applications. Copyright © 2015 John Wiley & Sons, Ltd.

Grape Juice Concentrate Protects Rat Liver Against Cadmium Intoxication: Histopathology, Cytochrome C and Metalloproteinases Expression.

The aim of this study was to investigate if grape juice concentrate is able to protect rat liver against cadmium toxicity. For this purpose, histopathological analysis, cytochrome C expression and immunoexpresssion of metalloproteinases (MMP) 2 and 9 were investigated. A total of 15 Wistar rats weighing 250 g on the average, and 8 weeks age were distributed into 3 groups (n=5), as follows: Control group (non-treated group, CTRL); Cadmium group (Cd) and grape juice concentrate group (Cd+GJ). Histopathological analysis revealed that liver from animals treated with grape juice concentrate improved tissue degeneration induced by cadmium intoxication. Animals intoxicated with cadmium and treated with grape juice concentrate showed higher cytochrome C gene expression in liver cells. No significant statistically differences (p>0.05) were found to MMP 2 and 9 immunoexpression between groups. Taken together, our results demonstrate that grape juice concentrate is able to prevent tissue degeneration in rat liver as a result of increasing apoptosis.

Gum acacia mitigates genetic damage in adenine-induced chronic renal failure in rats.

BACKGROUND: Subjects with chronic renal failure (CRF) exhibit oxidative genome damage, which may predispose to carcinogenesis, and Gum acacia (GumA) ameliorates this condition in humans and animals. We evaluated here renal DNA damage and urinary excretion of four nucleic acid oxidation adducts namely 8-oxoguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxoguanosine (8-oxoGuo) and 8-hydroxy-2-deoxyguanisone (8-OHdg) in rats with adenine (ADE)-induced CRF with and without GumA treatment. MATERIALS AND METHODS: Twenty-four rats were divided into four equal groups and treated for 4 weeks. The first group was given normal food and water (control). The second group was given normal food and GumA (15% w/v) in drinking water. The third group was fed powder diet containing adenine (ADE) (0·75% w/w in feed). The fourth group was fed like in the third group, plus GumA in drinking water (15%, w/v). RESULTS: ADE feeding induced CRF (as measured by several physiological, biochemical and histological indices) and also caused a significant genetic damage and significant decreases in urinary 8-oxo Gua and 8-oxoGuo, but not in the other nucleic acids. However, concomitant GumA treatment reduced the level of genetic damage in kidney cells as detected by Comet assay and significantly reversed the effect of adenine on urinary 8-oxoGuo. CONCLUSIONS: Treatment with GumA is able to mitigate genetic damage in renal tissues of rats with ADE-induced CRF.

Is lack of sleep capable of inducing DNA damage in aged skin?

Skin naturally changes with age, becoming more fragile. Various stimuli can alter skin integrity. The aim of this study was to evaluate whether sleep deprivation affects the integrity of DNA in skin and exacerbates the effects of aging. Fifteen-month old female Hairless mice underwent 72 h of paradoxical sleep deprivation or 15 days of chronic sleep restriction. Punch biopsies of the skin were taken to evaluate DNA damage by single cell gel (comet) assay. Neither paradoxical sleep deprivation nor sleep restriction increased genetic damage, measured by tail movement and tail intensity values. Taken together, the findings are consistent with the notion that aging overrides the effect of sleep loss on the genetic damage in elderly mice.

Potencial terapêutico e uso de plantas medicinais em uma área de Caatinga no estado do Ceará, nordeste do Brasil / Therapeutic potential and use of medicinal plants in an area of the Caatinga in the state of Ceará, northeastern Brazil

O presente trabalho visou o levantamento etnobotânico das plantas medicinais, verificando a versatilidade das espécies utilizadas e o consenso de uso e/ou conhecimento entre os informantes, do Distrito de Aratama, no Município de Assaré, Ceará. As informações etnobotânicas foram obtidas através de entrevistas estruturadas e semi-estruturadas com os moradores locais. Foram citadas 116 espécies com fins medicinais pertencentes a 103 gêneros e 58 famílias com destaque para Fabaceae (10ssp.), Asteraceae (7spp.) e Lamiaceae (6spp.). Entre as espécies levantadas, Mentha spicata L., Rosmarinus officinalis L., Allium sativum L., Bauhinia cheilantha (Bong.), Ximenea americana L., se destacaram como as mais versáteis dentro da comunidade. As indicações terapêuticas citadas foram agrupadas em 16 categorias de sistemas corporais, dos quais as Desordens mentais e comportamentais, as Afecções ou dores não definidas, os Transtornos do sistema respiratório, as Doenças de pele e do tecido celular subcutâneo, e os Transtornos do sistema sensorial (ouvidos), mostram maior concordância entre os informantes na utilização de espécies para tratar um sistema corporal especifico. Os resultados mostraram elevada riqueza da flora medicinal presente na caatinga. Neste sentido, torna-se necessária a intensificação de estudos que avaliem e consolidem as propriedades químicas e farmacológicas destas espécies.

This study is about an ethnobotanical survey of medicinal plants, checking the versatility of the species used and the consensus of use and/or knowledge among informants from the Aratama District, in the municipality of Assaré, state of Ceará, Brazil. The ethnobotanical information was obtained through structured interviews and semi-structured interviews with local residents. Approximately, 116 species were mentioned for medicinal purposes, belonging to 103 genera and 58 families, especially Fabaceae (10ssp.), Asteraceae (7spp.) and Lamiaceae (6spp.). Among the surveyed species, Mentha spicata L., Rosmarinus officinalis L., Allium sativum L. and Bauhinia cheilantha (Bong.) Ximenea americana L. stood out as the most versatile in the community. The therapeutic indications mentioned were grouped into 16 categories of body systems; the mental and behavioral disorders, disorders or pain not defined, disorder of the respiratory system, skin diseases and subcutaneous tissue disorder, and disorder of the sensory system (ears) showed greater agreement among informants in the use of species to treat a specific body system. The results showed a high species richness of the medicinal flora present in the Brazilian Caatinga. Thus, it is necessary to intensify and consolidate studies assessing the chemical and pharmacological properties of these species.

Insulin resistance is associated with DNA damage in peripheral blood cells in non-diabetic patients with genotype 1 chronic hepatitis C.

BACKGROUND: In chronic liver diseases of different etiologies, including viral hepatitis, genotoxic effects of oxidative stress have been shown, both in clinical and in experimental conditions, suggesting that this mechanism may contribute to the evolution of the disease. AIM: To evaluate DNA damage in the peripheral blood of untreated non-diabetic patients with chronic hepatitis C and control subjects, and its correlation with demographic, anthropometric, biochemical, and histological parameters in the patient sample. PATIENTS AND METHODS: This study comprised 100 subjects of both genders, 60 of whom were treatment-naïve patients with positive serology for genotype 1 hepatitis C. The remaining 40 were blood donors with negative serology for hepatitis who were used as control subjects, and matched by gender, age, weight, and BMI. DNA damage was determined using the comet assay in the total peripheral blood. RESULTS: The DNA damage evaluated by the comet assay revealed higher values in the group of patients with hepatitis compared with that in the control group. The relationships of the comet assay with the studied variables were assessed using multivariate analysis; significant correlations were only identified with insulin (r = 0.343, p = 0.008) and Homeostasis Model Assessment Insulin Resistance (HOMA-IR) (r = 0.331, p = 0.011). CONCLUSION: Patients with genotype 1 chronic hepatitis C have higher rates of DNA damage, as determined by comet assay and this alteration is correlated with the HOMA index of insulin resistance.

Cytogenetic biomonitoring in oral mucosa cells following dental X-ray.

OBJECTIVES: In the past decades, X-rays have been used widely for diagnosis in dentistry. However, it is well known that ionizing radiation causes damage (including single- and double-strand breaks) to deoxyribonucleic acid (DNA) and DNA-protein crosslinks, and induces cellular death. Therefore, outlining the cytogenetic effects induced by X-ray is necessary to identify the degree of cancer risk and minimize potential risks to patients and clinicians. To date, a variety of assays have been proposed in cytogenetic biomonitoring studies, including those that assess metaphase chromosomal aberrations and sister chromatid exchanges, and micronucleus and single-cell gel (comet) assay. METHODS: Cytogenetic biomonitoring studies focusing on oral mucosa cells in individuals exposed to dental X-ray were reviewed. RESULTS: Dental X-ray can induce DNA damage and cytotoxicity in oral mucosa cells. CONCLUSION: These results will contribute to a better understanding of X-ray-induced effects upon the cellular system in individuals continually exposed to known genotoxic/cytotoxic agents.

Expression of apoptosis regulatory proteins p53, bcl-2 and bax in recurrent aphthous ulceration.

BACKGROUND: Recurrent aphthous ulceration (RAU) is considered to be an acute inflammatory disease of unknown pathogenesis. Apoptosis may represent an important event in the control of inflammation. OBJECTIVES: The aim of this study was to investigate apoptosis process in RAU using immunohistochemistry. METHODS: We studied the expression and location of p53, bcl-2 and bax in ulcerated lesions clinically diagnosed as RAU (n = 12) and compared it with that of oral clinically normal mucosa (n = 6) and of other inflammatory chronic disease such as oral fibrous inflammatory hyperplasia (FIH; n = 18). RESULTS: Significant statistically differences (n < 0.05) in p53 expression were noticed in RAU when compared with normal mucosa. No significant statistically differences (P > 0.05) were noticed between FIH and RAU. Bcl-2 and bax did not show remarkable differences between groups. CONCLUSIONS: Taken together, the data suggest that RAU induces p53 immunoexpression. Therefore, the protein might be related to the aetiopathogenesis of the ulcerated oral lesions.

The role of the TP53 gene during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide.

The medium-term tongue carcinogenesis assay is a useful model for studying oral squamous cell carcinomas phase by phase. The aim of the present study was to investigate the expression of p53 by immunohistochemistry and examine the DNA sequence of exons 5, 6, 7, and 8 of Tp53 for mutations during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO). A total of 30 male Wistar rats were treated with 4-nitroquinoline 1-oxide in their drinking water for 4, 12, and 20 weeks at a dose of 50 ppm. Ten animals were used as negative controls. No histopathological changes in the tongue epithelia were observed in the control group or in the treatment group after 4 weeks of 4NQO. Following 12 weeks of treatment, hyperplasia as well as epithelial dysplasia was found in both mild and moderate forms. At 20 weeks, moderate and/or severe oral dysplasia and squamous cell carcinoma of the tongue were found, and the majority of animals had squamous cell carcinoma. The levels of p53 protein were increased (p < 0.05) in pre-neoplastic lesions and in squamous cell carcinomas in some of the tumor cells in squamous cell carcinomas. No mutations were found in any of the exons that were evaluated after the 4-, 12-, or 20-week treatments. Taken together, our results suggest that p53 expression may be an important event in the malignant conversion, whereas Tp53 mutations are not involved in the multi-step tongue carcinogenesis of Wistar rats induced by 4NQO.

Comparison of oral versus sublingual piroxicam during postoperative pain management after lower third molar extraction.

In this study, 53 patients received piroxicam, administered orally or sublingually, after undergoing removal of symmetrically positioned lower third molars, during two separate appointments. This study used a randomized, blind, cross-over protocol. Objective and subjective parameters were recorded for comparison of postoperative results for 7 days after surgery. Patients treated with oral or sublingual piroxicam reported low postoperative pain scores. The patients who received piroxicam orally took a similar average amount of analgesic rescue medication compared with patients who received piroxicam sublingually (p>0.05). Patients exhibited similar values for mouth opening measured just before surgery and immediately following suture removal 7 days later (p>0.05), and showed no significant differences between routes of piroxicam administration for swelling control during the second or seventh postoperative days (p>0.05). In summary, pain, trismus and swelling after lower third molar extraction, independent of surgical difficulty, could be controlled by piroxicam 20mg administered orally or sublingually and no significant differences were observed between the route of delivery used in this study.

Sleep loss and acute drug abuse can induce DNA damage in multiple organs of mice.

The purpose of the present study was to characterize the genetic damage induced by paradoxical sleep deprivation (PSD) in combination with cocaine or ecstasy (3,4-methylenedioxymethamphetamine; MDMA) in multiple organs of male mice using the single cell gel (comet) assay. C57BL/6J mice were submitted to PSD by the platform technique for 72 hours, followed by drug administration and evaluation of DNA damage in peripheral blood, liver and brain tissues. Cocaine was able to induce genetic damage in the blood, brain and liver cells of sleep-deprived mice at the majority of the doses evaluated. Ecstasy also induced increased DNA migration in peripheral blood cells for all concentrations tested. Analysis of damaged cells by the tail moment data suggests that ecstasy is a genotoxic chemical at the highest concentrations tested, inducing damage in liver or brain cells after sleep deprivation in mice. Taken together, our results suggest that cocaine and ecstasy/MDMA act as potent genotoxins in multiple organs of mice when associated with sleep loss.

Putative new heat-stable cytotoxic and enterotoxic factors in culture supernatant of Escherichia coli isolated from drinking water

Enteric infections caused by the ingestion of contaminated water, especially by Escherichia coli, are important to define the virulence properties of these bacteria. Due to frequent infantile diarrhea in the city of Ouro Preto, Minas Gerais state, Brazil, the phenotypic and genotypic diarrheagenic properties of E. coli isolated from drinking water were studied. The culture supernatants of 39 (40 percent) among a total of 97 E. coli isolates from drinking water were positive by suckling mouse assay and induced cytotoxic effects on Vero cells. The enterotoxic and cytotoxic activities were present in the fraction with less than 10 kDa and were not lost when heated up to 60¨¬C and 100¨¬C for 30 minutes. PCR assays showed that among these 39 Vero cytotoxigenic E. coli, four (10.2 percent) were positive for ST II (estB) and two (5 percent) positive for ¥áHly (hlyA). Gene amplification of SLT (stx 1, stx 2), ST I (estA), LT (eltI, eltII), EAST1 (astA), EHly (enhly) and plasmid-encoded enterotoxin (pet) were not observed. This heat-stable cytotoxic enterotoxin of E. coli is probably a new putative diarrheagenic virulence factor, as a toxin presenting these characteristics has not yet been described.

Mutagenicity and cytotoxicity assessment in patients undergoing orthodontic radiographs.

OBJECTIVES: The aim of the present study was to evaluate DNA damage (micronucleus) and cellular death (pyknosis, karyolysis and karyorrhexis) in exfoliated buccal mucosa cells from individuals following radiography. METHODS: Lateral and frontal cephalometric X-ray and panoramic dental X-rays were taken of a total of 18 healthy patients (6 male and 12 female) referred for orthodontic therapy. Exfoliated oral mucosa cells were collected immediately before X-ray exposure and after 10 days. RESULTS: The results revealed no statistically significant difference (P > 0.05) in the frequency micronucleated oral mucosa cells after X-ray exposure. However, X-ray was able to increase other nuclear alterations closely related to cytotoxicity, such as karyorrhexis, pyknosis and karyolysis. CONCLUSIONS: Data indicated that exposure to certain radiography may not be a factor in inducing chromosomal damage, but it does promote cytotoxicity.

Ex vivo assessment of genotoxicity and cytotoxicity in murine fibroblasts exposed to white MTA or white Portland cement with 15% bismuth oxide.

AIMS: To evaluate whether white mineral trioxide aggregate (MTA) or white Portland cement with 15% bismuth oxide were able to induce genetic damage and cellular death ex vivo. METHODOLOGY: Aliquots of 1 × 10(4) murine fibroblasts were incubated at 37 °C for 3 h with MTA (white) or white Portland cement with 15% bismuth oxide, at final concentrations ranging from 10 to 1000 µg mL(-1) individually. Data of three independent repeats from the comet assay and the trypan blue exclusion test were assessed by the one-way anova followed by Tukey's test. RESULTS: Mineral trioxide aggregate or Portland cement containing bismuth oxide did not produce genotoxic effects with respect to the single-cell gel (comet) assay data for all concentrations evaluated. Furthermore, no cytotoxicity was observed for MTA or Portland cement. CONCLUSION: White MTA or white Portland cement containing 15% bismuth oxide were not genotoxic and cytotoxic.