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.

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.

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.

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.

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.

Chronic renal failure induces genetic instability in multiple organs of Wistar rats.

BACKGROUND: Taking into consideration the strong evidence for a relationship between DNA damage and carcinogenesis, the aim of this study was to investigate whether blood, liver, heart, kidney and brain are particularly sensitive organs for DNA damaging during chronic renal disease by the single-cell gel (comet) assay to predict genetic instability induced by this pathological condition. METHODS: A total of 18 male Wistar rats were divided into two groups: negative control (n = 8) and experimental (n = 10), in which was submitted to the 5/6 renal mass ablation by ligation of two or three branches of the left renal artery and total right nephrectomy during 8 weeks. RESULTS: The results showed that chronic renal disease was able to induce genetic damage in blood, heart, liver and kidney cells as depicted by the mean tail moment. No genetic damage was induced in brain cells, i.e. no significant statistically differences (P > 0.05) were noticed when compared to negative control. CONCLUSION: In conclusion, our results suggest that chronic renal failure could contribute to the damage of DNA at all organs evaluated, except to the brain cells. As DNA damage is an important step in events leading to carcinogenesis, this study represents a relevant contribution to the correct evaluation of the potential health risks associated with kidney disease.

Distinct effects of acute and chronic sleep loss on DNA damage in rats.

The aim of this investigation was to evaluate genetic damage induced in male rats by experimental sleep loss for short-term (24 and 96 h) and long-term (21 days) intervals, as well as their respective recovery periods in peripheral blood, brain, liver and heart tissue by the single cell gel (comet) assay. Rats were paradoxically deprived of sleep (PSD) by the platform technique for 24 or 96 h, or chronically sleep-restricted (SR) for 21 days. We also sought to verify the time course of their recovery after 24 h of rebound sleep. The results showed DNA damage in blood cells of rats submitted to PSD for 96 h. Brain tissue showed extensive genotoxic damage in PSD rats (both 24 and 96 h), though the effect was more pronounced in the 96 h group. Rats allowed to recover from the PSD-96 h and SR-21 days treatments showed DNA damage as compared to negative controls. Liver and heart did not display any genotoxicity activity. Corticosterone concentrations were increased after PSD (24 and 96 h) relative to control rats, whereas these levels were unaffected in the SR group. Collectively, these findings reveal that sleep loss was able to induce genetic damage in blood and brain cells, especially following acute exposure. Since DNA damage is an important step in events leading to genomic instability, this study represents a relevant contribution to the understanding of the potential health risks associated with sleep deprivation.

Radiopacifiers do not induce genetic damage in murine fibroblasts: an in vitro study.

AIM: To evaluate whether several radiopacifiers are able to induce genetic damage in a laboratory cell culture study. METHODOLOGY: Murine fibroblasts were exposed to barium sulphate, bismuth oxide or zirconium oxide, at final concentrations ranging from 10 to 1000 microg mL(-1) for 1 h at 37 degrees C. The negative control group was treated with a vehicle control [phosphate buffered solution (PBS)] for 1 h at 37 degrees C and the positive control group was treated with hydrogen peroxide (at 10 microM) for 5 min on ice. Genotoxicity data were assessed by the single-cell gel (comet) assay. RESULTS: All the tested compounds did not induce DNA breakage as depicted by the mean tail moment in all the concentrations analysed. CONCLUSION: Exposure to the tested radiopacifiers may not be a factor that increases the level of DNA lesions in mammalian cells as detected by a single-cell gel (comet) assay.

DNA damage in lymphocytes and buccal mucosa cells of children with malignant tumours undergoing chemotherapy.

The aim of the present study was to evaluate DNA damage (micronucleus) in cytokinesis-blocked lymphocytes and exfoliated buccal mucosa cells from children with malignant tumours and under chemotherapy. Micronucleated cells (MNCs) were assessed from children before and during chemotherapy. A total of 21 healthy children (controls), matched for gender and age, were used as control. The results pointed out higher frequencies of micronucleated lymphocytes in children with malignant tumour before any therapy when compared to healthy probands. Furthermore an increase of micronucleated lymphocytes during chemotherapy was detected when compared to the data obtained before chemotherapy. No statistically significant increases of MNCs were noticed in buccal mucosa cells at any of the timepoints evaluated. Taken together, these data indicate that the presence of malignant tumours may increase the frequency of DNA damage in circulating lymphocytes, these cells being more sensitive for detecting chromosome aberrations caused by anti-cancer drugs.

Antigenotoxicity and antimutagenicity of lycopene in HepG2 cell line evaluated by the comet assay and micronucleus test.

Epidemiological studies have provided evidence that high consumption of tomatoes effectively reduces the risk of reactive oxygen species (ROS)-mediated diseases such as cancer. Tomatoes are rich sources of lycopene, a potent singlet oxygen-quenching carotenoid. In addition to its antioxidant properties, lycopene shows an array of biological effects including antimutagenic and anticarcinogenic activities. In the present study, the chemopreventive action of lycopene was examined on DNA damage and clastogenic or aneugenic effects of H2O2 and n-nitrosodiethylamine (DEN) in the metabolically competent human hepatoma cell line (HepG2 cells). Lycopene at concentrations of 10, 25, and 50 microM, was tested under three protocols: before, simultaneously, and after treatment with the mutagen, using the comet and micronucleus assays. Lycopene significantly reduced the genotoxicity and mutagenicity of H2O2 in all of the conditions tested. For DEN, significant reductions of primary DNA damage (comet assay) were detected when the carotenoid (all of the doses) was added in the cell culture medium before or simultaneously with the mutagen. In the micronucleus test, the protective effect of lycopene was observed only when added prior to DEN treatment. In conclusion, our results suggest that lycopene is a suitable agent for preventing chemically-induced DNA and chromosome damage.

Low-level laser therapy improves bone repair in rats treated with anti-inflammatory drugs.

Nowadays, selective cyclooxygenase-2 non-steroidal anti-inflammatory drugs have been largely used in surgical practice for reducing oedema and pain. However, the association between these drugs and laser therapy is not known up to now. Herein, the aim of this study was to evaluate the action of anti-COX-2 selective drug (celecoxib) on bone repair associated with laser therapy. A total of 64 rats underwent surgical bone defects in their tibias, being randomly distributed into four groups: Group 1) negative control; Group 2) animals treated with celecoxib; Group 3) animals treated with low-level power laser and Group 4) animals treated with celecoxib and low-level power laser. The animals were killed after 48 h, 7, 14 and 21 days. The tibias were removed for morphological, morphometric and immunohistochemistry analysis for COX-2. Statistical significant differences (P < 0.05) were observed in the quality of bone repair and quantity of formed bone between groups at 14 days after surgery for Groups 3 and 4. COX-2 immunoreactivity was more intense in bone cells for intermediate periods evaluated in the laser-exposed groups. Taken together, such results suggest that low-level laser therapy is able to improve bone repair in the tibia of rats as a result of an up-regulation for cyclooxygenase-2 expression in bone cells.

Lycopene activity against chemically induced DNA damage in Chinese hamster ovary cells.

Lycopene is a natural pigment synthesized by plants and microorganisms, and it is mainly found in tomatoes. It is an acyclic isomer of beta-carotene and one of the most potent antioxidants. Several studies have demonstrated the ability of lycopene to prevent chemically induced DNA damage; however, the mechanisms involved are still not clear. In the present study, we investigated the antigenotoxic/antimutagenic effects of lycopene in Chinese Hamster Ovary Cells (CHO) treated with hydrogen peroxide, methylmethanesulphonate (MMS), or 4-nitroquinoline-1-oxide (4-NQO). Lycopene (97%), at final concentrations of 10, 25, and 50 microM, was tested under three different protocols: before, simultaneously, and after the treatment with the mutagens. Comet and cytokinesis-block micronucleus assays were used to evaluate the level of DNA damage. Data showed that lycopene reduced the frequency of micronucleated cells induced by the three mutagens. However, this chemopreventive activity was dependent on the concentrations and treatment schedules used. Similar results were observed in the comet assay, although some enhancements of primary DNA damage were detected when the carotenoid was administered after the mutagens. In conclusion, our findings confirmed the chemopreventive activity of lycopene, and showed that this effect occurs under different mechanisms.

Lack of effect of prior treatment with fluoride on genotoxicity of two chemical agents in vitro.

The goal of this study was to investigate the ability of fluoride to modulate the genotoxic effects induced by the oxidative agent hydrogen peroxide (H2O2) and the alkylating agent methyl methanesulfonate (MMS) in vitro by the single-cell gel (comet) assay. Chinese hamster ovary cells were exposed in culture for 1 h at 37 degrees C to sodium fluoride at 7-100 microg/ml. NaF-treated and control cells were then incubated with 0-10 microM MMS in phosphate-buffered saline (PBS) for 15 min at 37 degrees C, or 0-100 microM H2O2 in distilled water for 5 min on ice. Negative control cells were treated with PBS for 1 h at 37 degrees C. Clear concentration-related effects were observed for the two genotoxins. Increase of DNA damage induced by either MMS or H2O2 was not significantly altered by pretreatment with NaF. The data indicate that NaF does not modulate alkylation-induced genotoxicity or oxidative DNA damage as measured by the single-cell gel (comet) assay.

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.

Brazilian natural dietary components (annatto, propolis and mushrooms) protecting against mutation and cancer.

Considering the high number of new cancer cases in Brazil (approximately 470000 cases in 2005) and the remarkable differences in the incidence of this disease around the world, the development of chemopreventive strategies using foods widely consumed would have a huge impact, both medically and economically. This review summarizes some of our studies conducted to verify the anti-mutagenic and anti-carcinogenic potential of some Brazilian natural dietary constituents (annatto, mushrooms, and propolis). Overall data have shown a clear role for these compounds in preventing mutation and specific preneoplastic lesions. Taken together, these agents indicate a favorable side-effect profile and may prove to be a promising alternative for cancer prevention strategies, although more investigation is needed to fully explore this issue.

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.

Does toxoplasmosis cause DNA damage? An evaluation in isogenic mice under normal diet or dietary restriction.

Toxoplasmosis is an anthropozoonotic widespread disease, caused by the coccidian protozoan parasite Toxoplasma gondii. Since there are no data regarding the genotoxicity of the parasite in vivo, this study was designed to evaluate the genotoxic potential of the toxoplasmosis on isogenic mice with normal diet or under dietary restriction and submitted to a treatment with sulfonamide (375 microg/kg per day). DNA damage was assessed in peripheral blood, liver and brain cells using the comet assay (tail moment). The results for leucocytes showed increases in the mean tail moment in mice under dietary restriction; in infected mice under normal diet; in infected, sulfonamide-treated mice under normal diet; in infected mice under dietary restriction and in infected sulfonamide-treated mice under dietary restriction. In liver and brain cells, no statistically significant difference was observed for the tail moment. These results indicated that dietary restriction and T. gondii were able to induce DNA damage in peripheral blood cells, as detected by the comet assay.