Influence of endogenous and synthetic female sex hormones on human blood cells in vitro studied with comet assay

The comet assay has been conducted with numerous cell lines to assess in vitro genotoxicity. In order to use the comet assay as part of an in vitro test for evaluating genotoxicity, however, there are cell-specific factors that need to be better understood. In this present study we have evaluated some factors that may impact upon the DNA damage detected in whole blood (WB) cells and lymphocytes (ILs). Experiments were conducted comparing responses of both cells, and investigating the effects of the female hormonal cycle, and oral contraceptive (OC) use on DNA damage detection in the in vitro comet assay, at three sampling time. No significant differences were detected in the basal levels of DNA damage detected in ILs and WB cells from women OC users and non-users and from men. Basal DNA damage in ILs was unaffected by gender and stage of the menstrual cycle or the stage of the treatment schedule. Our results also indicated that the H2O2 induces DNA damage in human lymphocytes independently of gender, low-dose OC use and hormonal fluctuation. However, data showed that in 3rd sampling of menstrual cycle, lymphocytes were more resistant to H2O2-induced DNA damage than those from OC users and men. © 2007 Elsevier Ltd. All rights reserved.

Amifostine reduces the seminiferous epithelium damage in doxorubicin-treated prepubertal rats without improving the fertility status

Background: Amifostine is an efficient cytoprotector against toxicity caused by some chemotherapeutic drugs. Doxorubicin, a potent anticancer anthracycline, is known to produce spermatogenic damage even in low doses. Although some studies have suggested that amifostine does not confer protection to doxorubicin-induced testicular damage, schedules and age of treatment have different approach depending on the protocol. Thus, we proposed to investigate the potential cytoprotective action of amifostine against the damage provoked by doxorubicin to prepubertal rat testes (30-day-old) by assessing some macro and microscopic morphometric parameters 15, 30 and 60 days after the treatment; for fertility evaluation, quantitative analyses of sperm parameters and reproductive competence in the adult phase were also carried out.Methods: Thirty-day-old male rats were distributed into four groups: Doxorubicin (5 mg/kg), Amifostine (400 mg/kg), Amifostine/Doxorubicin (amifostine 15 minutes before doxorubicin) and Sham Control (0.9% saline solution). Standard One Way Anova parametric and Anova on Ranks non-parametric tests were applied according to the behavior of the obtained data; significant differences were considered when p < 0.05.Results: The rats killed 30 and 60 days after doxorubicin treatment showed diminution of seminiferous epithelium height and reduction on the frequency of tubular sections containing at least one type of differentiated spermatogonia; reduction of sperm concentration and motility and an increase of sperm anomalous forms where observed in doxorubicin-treated animals. All these parameters were improved in the Amifostine/Doxorubicin group only when compared to Doxorubicin group. Such reduction, however, still remained below the values obtained from the Sham Control group. Nevertheless, the reproductive competence of doxorubicin-treated rats was not improved by amifostine pre-administration.Conclusions: These results suggest that amifostine promotes a significant reduction of the doxorubicin long-term side effects on the seminiferous epithelium of prepubertal rats, which is reflected in the epidydimal fluid parameters in the adult phase. However, fertility status results suggest that such protection may not be effective against sperm DNA content damage. Further investigation of sperm DNA integrity must be carried out using amifostine and doxorubicin-treated experimental models. © 2010 Vendramini et al; licensee BioMed Central Ltd.

DNA repair gene polymorphism is associated with the genetic basis of atherosclerotic coronary artery disease

Background: Atherosclerotic coronary artery disease (CAD) is a multifactorial process that appears to be caused by the interaction of environmental risk factors with multiple predisposing genes. It is nowadays accepted that increased levels of DNA damage induced by xenobiotics play an important role in the early phases of atherogenesis. Therefore, in this study, we focus on determining whether genetic variations in xenobiotic-metabolizing [glutathione-S-transferase theta 1 (GSTT1), glutathione-S-transferase mu 1 (GSTM1), cytochrome P450 IIEI (CYP2E1)] and DNA repair [X-ray cross-complementing group 1 (XRCC1)] genes might be associated with increased risk for CAD. Methods: A case-control study was conducted with 400 individuals who underwent subjected to coronary angiography. A total of 299 were patients diagnosed with effective coronary atherosclerosis (case group; >20% obstructive lesion), and 101 (control group) were individuals diagnosed as negative for CAD (<20% obstructive lesions). The polymorphism identifications for GSTM1 and GSTT1, and for CYP2E1 and XRCC1 genes were performed by polymerase chain reaction (PCR) amplification and by PCR-RFLP, respectively. Results and conclusions: The XRCC1 homozygous wild-type genotype Arg/Arg for codon 399 was statistically less pronounced in the case subjects (21.4%) than in controls (38.5%); individuals with the variant XRCC1 genotype had a 2.3-fold increased risk for coronary atherosclerosis than individuals with the wild-type genotype (OR=2.3, 95% CI=1.13-4.69). Conversely, no association between GSTM1, GSTT1, and CYP2E1gene polymorphisms and coronary atherosclerosis was detected. The results provide evidence of the role of DNA damage and repair in cardiovascular disease. © 2011 Elsevier Inc. All rights reserved.

Genetic and modifying factors that determine the risk of brain tumors

Some modifying factors may determine the risk of brain tumors. Until now, it could not be attempted to identify people at risk and also to improve significantly disease progression. Current therapy consists of surgical resection, followed by radiation therapy and chemotherapy. Despite of these treatments, the prognosis for patients is poor. In this review, we highlight general aspects concerning genetic alterations in brain tumors, namely astrocytomas, glioblastomas, oligodendrogliomas, medulloblastomas and ependymomas. The influence of these genetic alterations in patients' prognosis is discussed. Mutagen sensivity is associated with cancer risk. The convincing studies that linked DNA damages and DNA repair alterations with brain tumors are also described. Another important modifying factor is immunity. General immune response against cancer, tumor microenvironment and immune response, mechanisms of tumor escape, CNS tumor immunology, immune defects that impair anti-tumor systemic immunity in brain tumor patients and local immunosuppressive factors within CNS are also reviewed. New hope to treatment perspectives, as dendritic-cell-based vaccines is summarized too. Concluding, it seems well established that there is association between brain tumor risk and mutagen sensivity, which is highly heritable. Primary brain tumors cause depression in systemic host immunity; local immunosuppressive factors and immunological characteristics of tumor cells may explain the poor prognosis and DNA damages responses can alert immune system. However, it is necessary to clarify if individuals with both constitutional defects in immune functions and genetic instability have higher risk of developing brain tumors. Cytogenetic prospective studies and gene copy number variations analysis also must be performed in peripheral lymphocytes from brain tumor patients. © 2011 Bentham Science Publishers Ltd.

Evaluation of Agaricus blazei in vivo for antigenotoxic, anticarcinogenic, phagocytic and immunomodulatory activities

The development of various types of cancer results from the interaction among endogenous, environmental and hormonal factors, where the most notable of these factors is diet. The aim of the present study was to determine the antigenotoxic, anticarcinogenic, phagocytic and immunomodulatory activities of Agaricus blazei. The test antigenotoxicity (Comet Assay) and anticarcinogenic (Test of Aberrant Crypt Foci) assess changes in DNA and/or intestinal mucosa that correlate to cancer development. Tests of phagocytosis in the spleen and differential count in blood cells allow the inference of modulation of the immune system as well as to propose a way of eliminating cells with DNA damage. Supplementation with the mushroom was carried out under pre-treatment, simultaneous treatment, post-treatment and pre-treatment. +. continuous conditions. Statistical analysis demonstrated that the mushroom did not have genotoxic activity but showed antigenotoxic activity. Supplementation caused an increase in the number of monocytes and in phagocytic activity, suggesting that supplementation increases a proliferation of monocytes, consequently increasing phagocytic capacity especially in the groups pre-treatment, simultaneous and pre-treatment. +. continuous. The data suggest that A. blazei could act as a functional food capable of promoting immunomodulation which can account for the destruction of cells with DNA alterations that correlate with the development of cancer, since this mushroom was demonstrated to have a preventive effect against pre-neoplastic colorectal lesions evaluated by the aberrant crypt foci assay. According to these results and the literature, it is believed that supplementation with A. blazei can be an efficient method for the prevention of cancer as well as possibly being an important coadjuvant treatment in chemotherapy. © 2011 Elsevier Inc.

Tethered DNA scaffolds on optical sensor platforms for detection of hipO gene from Campylobacter jejuni

DNA biosensors have gained increased attention over traditional diagnostic methods due to their fast and responsive operation and cost-effective design. The specificity of DNA biosensors relies on single-stranded oligonucleotide probes immobilized to a transduction platform. Here, we report the development of biosensors to detect the hippuricase gene (hipO) from Campylobacter jejuni using direct covalent coupling of thiol- and biotin-labeled single-stranded DNA (ssDNA) on both surface plasmon resonance (SPR) and diffraction optics technology (DOT, dotLab) transduction platforms. This is the first known report of the dotLab to detect targeted DNA. Application of 6-mercapto-1-hexanol as a spacer thiol for SPR gold surface created a self-assembled monolayer that removed unbound ssDNA and minimized non-specific detection. The detection limit of SPR sensors was shown to be 2.5 nM DNA while dotLab sensors demonstrated a slightly decreased detection limit of 5.0 nM (0.005 μM). It was possible to reuse the SPR sensor due to the negligible changes in sensor sensitivity (∼9.7 × 10 -7 ΔRU) and minimal damage to immobilized probes following use, whereas dotLab sensors could not be reused. Results indicated feasibility of optical biosensors for rapid and sensitive detection of the hipO gene of Campylobacter jejuni using specific ssDNA as a probe. © 2011 Elsevier B.V.

The Effects of Refrigeration Temperature and Storage Time on Apoptotic Markers in Equine Semen

Evaluation of the damage caused by the sperm preservation process is crucial to improving fertilization rates. The objective of this study was to evaluate the effects of refrigeration temperature (5°C and 15°C) and storage time (0, 12, 24, 48, and 72 hours) on apoptotic markers in equine semen. Membrane phosphatidylserine translocation index, caspase activation index, and DNA fragmentation index were analyzed using epifluorescence microscopy. Analysis of variance was used for statistical analysis, and Tukey test was used to compare means. The significance level was set at P < .05. The results demonstrated that for transport duration shorter than 24 hours, semen quality was maintained when stored at either 5°C or 15°C. A storage temperature of 5°C should be used when it is necessary to transport semen for longer than 24 hours. There was a significant decrease in semen quality after 48 hours of refrigeration. © 2013 Elsevier Inc.

Population Fluctuation and Damage Caused by Phytophagous Mites on Three Rubber Tree Clones

Our aim was to investigate the population fluctuation and the damage caused by the phytophagous mites Calacarus heveae Feres, Tenuipalpus heveae Baker, and Eutetranychus banksi (McGregor) on clones FX 2784, FX 3864, and MDF 180 in rubber tree crops from southeastern Bahia, Brazil. Moreover, we tested for the influence of climatic variables on occurrence patterns of these species throughout weekly samples performed from October to April. The infestation peaks was between mid-January and late February. The clones FX 2784 and FX 3864 had the highest infestations and more severe damage possibly caused by C. heveae, which was the most frequent and abundant species in all clones. We found that sunlight duration and rainfall were the most important factors for C. heveae while T. heveae was affected by rainfall and temperature. Eutetranychus banksi was only affected by sunlight duration. However, the best models had low goodness of fit. We concluded that the clones FX 2784 and FX 3864 had a higher susceptibility to mite attack, and the association between climatic variables and favorable physiological conditions were determinant for the population increase of the species from January to April. © 2012 Sociedade Entomológica do Brasil.

Chemoprotective activity of the isoflavones, genistein and daidzein on mutagenicity induced by direct and indirect mutagens in cultured HTC cells

Isoflavones are phenolic compounds widely distributed in plants and found in a high percentage in soybeans. They have important biological properties and are regarded as potential chemopreventive agents. The aim of this study was to verify the preventive effect of two soy isoflavones (genistein and daidzein) by a micronucleus assay, analysis of GST activity, and real-time RT-PCR analysis of GSTa2 gene expression. Mutagens of direct (doxorubicin) and indirect (2-aminoanthracene) DNA damage were used. Hepatoma cells (HTC) were treated with genistein or daidzein for 26 h at noncytotoxic concentrations; 10 μM when alone, and 0.1, 1.0 and 10 μM when combined with genotoxic agents. The micronucleus test demonstrated that both isoflavones alone had no genotoxic effect. Genistein showed antimutagenic effects at 10 μM with both direct and indirect DNA damage agents. On phase II enzyme regulation, the current study indicated an increase in total cytoplasmic GST activity in response to genistein and daidzein at 10 μM supplementation. However, the mRNA levels of GSTa2 isozymes were not differentially modulated by genistein or daidzein. The results point to an in vitro antimutagenic activity of genistein against direct and indirect DNA damage-induced mutagenicity. © 2012 Springer Science+Business Media B.V.

Assessment of the chemopreventive effect of casearin B, a clerodane diterpene extracted from Casearia sylvestris (Salicaceae)

Studies have shown that Casearia sylvestris compounds protect DNA from damage both in vitro and in vivo. Complementarily, the aim of the present study was to assess the chemopreventive effect of casearin B (CASB) against DNA damage using the Ames test, the comet assay and the DCFDA antioxidant assay. The genotoxicity was assessed by the comet assay in HepG2 cells. CASB was genotoxic at concentrations higher than 0.30μM when incubated with the FPG (formamidopyrimidine-DNA glycosylase) enzyme. For the antigenotoxicity comet assay, CASB protected the DNA from damage caused by H2O2 in the HepG2 cell line in concentrations above 0.04μM with post-treatment, and above 0.08μM with pre-treatment. CASB was not mutagenic (Ames test) in TA 98 and TA 102. In the antimutagenicity assays, the compound was a strong inhibitor against aflatoxin B1 (AFB) in TA 98 (>88.8%), whereas it was moderate (42.7-59.4%) inhibitor against mytomicin C (MMC) in TA 102. Additionally, in the antioxidant assay using DCFDA, CASB reduced reactive oxygen species (ROS) generated by H2O2. In conclusion, CASB was genotoxic to HepG2 cells at high concentrations; was protective of DNA at low concentrations, as shown by the Ames test and comet assay; and was also antioxidant. © 2012 Elsevier Ltd.

Silver colloidal nanoparticles: Effect on matrix composition and structure of Candida albicans and Candida glabrata biofilms

Aim: The aim of this study was to assess the effect of different silver nanoparticles (SN) concentrations on the matrix composition and structure of Candida albicans and Candida glabrata biofilms. Methods and Results: Candida biofilms were developed in 6-well microtiter plates during 48 h. After, these biofilms were exposed to 13·5 or 54 μg SN ml-1 for 24 h. Then, extracellular matrices were extracted from biofilms and analysed chemically in terms of proteins, carbohydrates and DNA. To investigate the biofilm structure, scanning electron microscopy (SEM) and epifluorescence microscopy were used. SN interfered with the matrix composition of Candida biofilms tested in terms of protein, carbohydrate and DNA, except for the protein content of C. albicans biofilm. By SEM, Candida biofilms treated with SN revealed structural differences, when compared with the control groups. Further, SN showed a trend of agglomeration within the biofilms. Epifluorescence microscopy images suggest that SN induced damage on cell walls of the Candida isolates tested. Conclusions: In general, irrespective of concentration, SN affected the matrix composition and structure of Candida biofilms and these findings may be related to the mechanisms of biocide action of SN. Significance and Impact of the Study: This study reveals new insights about the behaviour of SN when in contact with Candida biofilms. SN may contribute to the development of therapies to prevent or control Candida infections. © 2012 The Society for Applied Microbiology.

Antimutagenic and anticarcinogenic effects of wheat bran in vivo

Previous studies in rodents treated with the pro-carcinogen 1,2-dimethylhydrazine suggested that the consumption of wheat bran protected against DNA damage in the colon and rectum. Based on this information, we evaluated wheat bran as a functional food in the prevention and treatment of colon cancer. We used the aberrant crypt focus assay to evaluate the anticarcinogenic potential of wheat bran (Triticum aestivum variety CD-104), the comet assay to evaluate its antigenotoxicity potential, and the micronucleus assay to evaluate its antimutagenic potential. The wheat bran gave good antimutagenic and anticarcinogenic responses; the DNA damage decreased from 90.30 to 26.37% and from 63.35 to 28.73%, respectively. However, the wheat bran did not significantly reduce genotoxicity. Further tests will be necessary, including tests in human beings, before this functional food can be recommended as an adjunct in the prevention and treatment of colon cancer. © FUNPEC-RP.

Effects of β-glucan extracted from Agaricus blazei on the expression of ERCC5, CASP9, and CYP1A1 genes and metabolic profile in HepG2 cells

The polysaccharide β-glucan has biological properties that stimulate the immune system and can prevent chronic pathologies, including cancer. It has been shown to prevent damage to DNA caused by the chemical and physical agents to which humans are exposed. However, the mechanism of β-glucan remains poorly understood. The objective of the present study was to verify the protective effect of β-glucan on the expression of the genes ERCC5 (involved in excision repair of DNA damage), CASP9 (involved in apoptosis), and CYP1A1 (involved in the metabolism of xenobiotics) using real-time polymerase chain reaction and perform metabolic profile measurements on the HepG2 cells. Cells were exposed to only benzo[a]pyrene (B[a]P), β-glucan, or a combination of B[a]P with β-glucan. The results demonstrated that 50 μg/mL β-glucan significantly repressed the expression of the ERCC5 gene when compared with the untreated control cells in these conditions. No change was found in the CASP9 transcript level. However, the CYP1A1 gene expression was also induced by HepG2 cells exposed to B[a]P only or in association with β-glucan, showing its effective protector against damage caused by B[a]P, while HepG2 cells exposed to only β-glucan did not show CYP1A1 modulation. The metabolic profiles showed moderate bioenergetic metabolism with an increase in the metabolites involved in bioenergetic metabolism (alanine, glutamate, creatine and phosphocholine) in cells treated with β-glucan and to a lesser extent treated with B[a]P. Thus, these results demonstrate that the chemopreventive activity of β-glucan may modulate bioenergetic metabolism and gene expression. © 2013 The Author(s).

Evaluation of the antimutagenic and anticarcinogenic effects of inulin in vivo

The incidence of colorectal cancer is growing worldwide. The characterization of compounds present in the human diet that can prevent the occurrence of colorectal tumors is vital. The oligosaccharide inulin is such a compound. The aim of this study was to evaluate the antigenotoxic, antimutagenic and anticarcinogenic effects of inulin in vivo. Our study is based on 3 assays that are widely used to evaluate chemoprevention (comet assay, micronucleus assay, and aberrant crypt focus assay) and tests 4 protocols of treatment with inulin (pre-treatment, simultaneous, post-treatment, and pre + continuous). Experiments were carried out in Swiss male mice of reproductive age. In order to induce DNA damage, we used the pro-carcinogenic agent 1,2-dimethylhydrazine. Inulin was administered orally at a concentration of 50 mg/kg body weight following the protocols mentioned above. Inulin was not administered to the control groups. Our data from the micronucleus assay reveal antimutagenic effects of inulin in all protocols. The percentage of inulin-induced damage reduction ranged from 47.25 to 141.75% across protocols. These data suggest that inulin could act through desmutagenic and bio-antimutagenic mechanisms. The anticarcinogenic activity (aberrant crypt focus assay) of inulin was observed in all protocols and the percentages of damage reduction ranged from 55.78 to 87.56% across protocols. Further tests, including human trials, will be necessary before this functional food can be proven to be effective in the prevention and treatment of colon cancer. © FUNPEC-RP.

Genotoxic evaluation of the antimalarial drugs artemisinin and artesunate in human HepG2 cells and effects on CASP3 and SOD1 gene expressions

The malaria treatment recommended by the World Health Organization involves medicines derived from artemisinin, an active compound extracted from the plant Artemisia annua, and some of its derivatives, such as artesunate. Considering the lack of data regarding the genotoxic effects of these compounds in human cells, the objective of this study was to evaluate the cytotoxicity and genotoxicity, and expressions of the CASP3 and SOD1 genes in a cultured human hepatocellular liver carcinoma cell line (HepG2 cells) treated with artemisinin and artesunate. We tested concentrations of 2.5, 5, 7.5, 10, and 20 μg/mL of both substances with a resazurin cytotoxicity assay, and the concentrations used in the genotoxicity experiments (2.5, 5, and 10 μg/mL) and gene expression analysis (5 mg/mL) were determined. The results of the comet assay in cells treated with artemisinin and artesunate showed a significant dosedependent increase (P < 0.001) in the number of cells with DNA damage at all concentrations tested. However, the gene expression analysis revealed no significant change in expression of CASP3 or SOD1. Our data showed that although artemisinin and artesunate exhibited genotoxic effects in cultured HepG2 cells, they did not significantly alter expression of the CASP3 and SOD1 genes at the doses tested. ©FUNPEC-RP.