Cell growth on 3D microstructured surfaces.

Chinese Hamster Ovary (CHO) cell cultures were grown on surfaces lithographed with periodic 3D hexagonal microcavity array morphology. The range of microcavity size (inscribed circle diameter) was from 12 µm to 560 µm. CHO cells were grown also on flat surfaces. The characterization was performed with respect to cell growth density (number of nuclei per unit area) by fluorescence optical microscopy and evaluated by correlation function analysis. We found that optimum microcavity radius was 80 µm, concerning to the maximum cell growth density, being even greater than the growth density on a flat (unstructured) substrate of the same material. This finding can be important for optimization of biotechnological processes and devices.

Gene polymorphisms and increased DNA damage in morbidly obese women.

Obesity is characterized by increased adipose tissue mass resulting from a chronic imbalance between energy intake and expenditure. Furthermore, there is a clearly defined relationship among fat mass expansion, chronic low-grade systemic inflammation and reactive oxygen species (ROS) generation; leading to ROS-related pathological events. In the past years, genome-wide association studies have generated convincing evidence associating genetic variation at multiple regions of the genome with traits that reflect obesity. Therefore, the present study aimed to evaluate the relationships among the gene polymorphisms ghrelin (GHRL-rs26802), ghrelin receptor (GHSR-rs572169), leptin (LEP-rs7799039), leptin receptor (LEPR-rs1137101) and fat mass and obesity-associated (FTO-rs9939609) and obesity. The relationships among these gene variants and the amount of DNA damage were also investigated. Three hundred Caucasian morbidly obese and 300 eutrophic (controls) women were recruited. In summary, the results demonstrated that the frequencies of the GHRL, GHSR, LEP and LEPR polymorphisms were not different between Brazilian white morbidly obese and eutrophic women. Exceptions were the AA-FTO genotype and allele A, which were significantly more frequent in obese women than in the controls (0.23% vs. 0.10%; 0.46 vs. 0.36, respectively), and the TT-FTO genotype and the T allele, which were less frequent in morbidly obese women (p<0.01). Furthermore, significant differences in the amount of genetic lesions associated with FTO variants were observed only in obese women. In conclusion, this study demonstrated that the analyzed SNPs were not closely associated with morbid obesity, suggesting they are not the major contributors to obesity. Therefore, our data indicated that these gene variants are not good biomarkers for predicting risk susceptibility for obesity, whereas ROS generated by the inflammatory status might be one of the causes of DNA damage in obese women, favoring genetically related diseases as obesity comorbidities.

Association among XRCC1, XRCC3, and BLHX gene polymorphisms and chromosome instability in lymphocytes from patients with endometriosis and ovarian cancer.

Endometriosis is a complex disease that has both benign and malignant characteristics. It affects 5-10% of women of reproductive age. Studies have demonstrated the existence of common genetic changes in endometriosis and ovarian cancer, suggesting a possible association between these 2 diseases. However, the mechanisms that lead to the development of cancer from endometriosis remain unknown. In this study, we evaluated 3 groups of women: 72 patients with endometriosis, 70 with ovarian cancer, and 70 healthy individuals (controls). Repair (XRCC1 codons 194 and 399, XPD codons 312 and 751, and XRCC3 codon 241)- and metabolism (BLHX codon 443)-related gene polymorphisms were analyzed using the polymerase chain reaction-restriction fragment length polymorphism technique; the efficiency of DNA damage repair was analyzed in vitro in lymphocytes exposed to bleomycin. The logistic regression model was used to evaluate key associations. The results showed an increased average of chromosome breakage in bleomycin-treated lymphocytes from patients with endometriosis and ovarian cancer compared with healthy women. We also detected significant association between XRCC1, XRCC3, and BLHX polymorphisms and a high frequency of chromosomal damage. Women with endometriosis or ovarian cancer may have an altered mechanism of DNA repair, and these defects may be related to a higher incidence of ovarian cancer.

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.

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.

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.

Analysis of DNA damage induced by aflatoxin B1 in Dunkin-Hartley guinea pigs.

Aflatoxin B1 (AFB1) is among the most potent naturally occurring carcinogens and classified as a group I carcinogen. Since the ingestion of aflatoxin-contaminated food is associated with several liver diseases, the aim of the present study was to evaluate the effect of 2, 20, and 200 ppb of AFB1 on DNA damage in peripheral blood lymphocytes and liver cells in Dunkin-Hartley guinea pigs. The animals were divided into four groups according to the given diet. After the treatment the lymphocytes and liver cells were isolated and DNA damage determined by Comet assay. The levels of DNA damage in lymphocytes were higher animals treated with 200 ppb of AFB1-enriched diet (P = 0.02). In the liver cells there were a relationship between the levels of DNA damage and the consumption of AFB1 in all studied groups. These results suggest that Comet assay performed on lymphocytes is a valuable genotoxic marker for high levels of exposure to AFB1 in guinea pig. Additionally our results indicate that the exposure to this toxin increases significantly and increases the level of DNA damage in liver cells, which is a key step on liver cancer development. We also suggest that the Comet assay is an useful tool for monitoring the genotoxicity of AFB1 in liver.

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.

Biocompatibility of glass-ionomer cements using mouse lymphoma cells in vitro.

Glass-ionomer cements are widely used in dentistry as restorative materials and adhesives for composite restorations. A number of genotoxicity studies have been conducted using these materials with results conflicting so far. Thus, the approach was aimed to look at the genotoxic and cytotoxic potential of three different glass-ionomer cements available commercially (Ketac Cem, Ketac Molar and Vitrebond) by the single cell gel (comet) assay and trypan blue exclusion test, respectively. For this, such materials were exposed to mouse lymphoma cells in vitro for 1 h at 37 degrees C. Data were assessed by Kruskall-Wallis non-parametric test. The results showed that all powders assayed did not show genotoxic effects. On the other hand, the liquid from Vitrebond at 0.1% dilution caused an increase of DNA injury. Significant statistically differences (P < 0.05) in cytotoxicity provoked by all powders tested were observed for exposure at 1,000 micro g mL(-1) concentration and 100 micro g mL(-1) for Ketac Molar. With respect to liquids of glass-ionomer cements evaluated, the major toxic effect on cell viability was produced at 1%, beginning at the dilution of 0.5% for Vitrebond. Taken together, these results support the notion that some components of glass-ionomer cements show both genotoxic and cytotoxic effects in higher concentrations.

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.

Evaluation of genetic damage in human peripheral lymphocytes exposed to mineral trioxide aggregate and Portland cements.

summary Mineral trioxide aggregate (MTA) and Portland cement are being used in dentistry as root-end-filling material for periapical surgery and for the sealing of communications between the root canal system and the surrounding tissues. However, genotoxicity tests for complete risk assessment of these compounds have not been conducted up to now. In the present study, the genotoxic effects of MTA and Portland cements were evaluated in peripheral lymphocytes from 10 volunteers by the alkaline single cell gel (comet) assay. The results pointed out that the single cell gel (comet) assay failed to detect the presence of DNA damage after a treatment of peripheral lymphocytes by MTA and Portland cements for concentrations up to 1000 mug mL(-1). In summary, our results indicate that exposure to MTA or Portland cements may not be a factor that increases the level of DNA lesions in human peripheral lymphocytes as detected by single cell gel (comet) assay.

Ex vivo biocompatibility tests of regular and white forms of mineral trioxide aggregate.

AIM: To examine the genotoxicity and cytotoxicity of regular and white mineral trioxide aggregate (MTA) ex vivo by the single-cell gel (comet) assay and trypan blue exclusion test, respectively. METHODOLOGY: Aliquots of 1 x 10(4) Chinese hamster ovary cells were incubated at 37 degrees C for 3 h with grey and white forms of MTA at final concentrations ranging from 1 to 1,000 microg mL(-1). The negative control group was treated with vehicle control phosphate buffer solution for 3 h at 37 degrees C and the positive control group was treated with methyl metasulfonate (at 1 microg mL(-1)) for 1 h at 37 degrees C. After incubation, the cells were centrifuged at 180 g for 5 min and washed twice with fresh medium and resuspended with fresh medium. Each individual treatment was repeated three times consecutively to ensure reproducibility. Parameters from single-cell gel (comet) and cytotoxicity assays were assessed by the Kruskal-Wallis nonparametric test. RESULTS: Neither compounds produced genotoxic effects with respect to the single-cell gel (comet) assay in all concentrations evaluated. In the same way, the dose-response relationships of all compounds tested at concentrations ranging from 1 to 1,000 microg mL(-1) on cell viability assessed by the trypan blue assay displayed no statistically significant differences (P > 0.05) for either endodontic material. CONCLUSIONS: Regular (grey) and white MTA are not genotoxins and do not induce cellular death.

Assessment of genetic damage induced by dental bleaching agents on mouse lymphoma cells by single cell gel (comet) assay.

Dental bleaching is a simple and conservative procedure for aesthetic restoration of vital discoloured teeth. However, dental bleaching agents may represent a hazard to human health, especially by causing DNA strand breaks. Genotoxicity tests form an important part of cancer research and risk assessment of potential carcinogens. In the current study, the genotoxic potential associated with exposure to dental bleaching agents was assessed by the single cell gel (comet) assay in vitro. Six commercial dental bleaching agents (Clarigel Gold - Dentsply; Whitespeed - Discus Dental; Nite White - Discus Dental; Magic Bleaching - Vigodent; Whiteness HP - FGM and Lase Peroxide - DMC) were exposed to mouse lymphoma cells in vitro. The results pointed out that all dental bleaching agents tested contributed to the DNA damage as depicted by the mean tail moment. Clear concentration-related effects were obtained for DNA damaging, being the strongest effect observed at the highest dose of the hydrogen peroxide (Whiteness HP and Lase Peroxide, at 35% concentration). On the contrary, Whitespeed (Discus Dental) induced the lowest level of DNA breakage. Taken together, these results suggest that dental bleaching agents may be a factor that increases the level of DNA damage as detected by the single cell gel (comet) assay.

No relationship between subchronic fluoride intake and DNA damage in Wistar rats.

Fluoride has been widely used in dentistry because it is an effective caries prophylactic agent. However, excess fluoride may represent a hazard to human health, especially by causing injury on the genetic apparatus. Genotoxicity tests form an important part of cancer research and risk assessment of potential carcinogens. In the current study, the potential DNA damage associated with exposure to fluoride was assessed by the single cell gel (comet) assay in peripheral blood, oral mucosa and brain cells in vivo. Male Wistar rats were exposed to sodium fluoride (NaF) at a 0, 7 and 100 ppm dose for drinking water during 6 weeks. The results pointed out that NaF did not contribute to the DNA damage in all cellular types evaluated as depicted by the mean tail moment and tail intensity. These findings are clinically important since they represent an important contribution to the correct evaluation of the potential health risk associated with dental agents exposure.

Absence of carcinogenic and anticarcinogenic effects of annatto in the rat liver medium-term assay.

Annatto (Bixa orellana L.) is a natural food colorant extensively used in many processed foods, especially dairy products. The lower cost of production and the low toxicity, make annatto a very attractive and convenient pigment in substitution to the many synthetic colorants. In the present study we investigate the carcinogenic and anticarcinogenic effects of dietary annatto in Wistar rat liver using the preneoplastic glutathione S-transferase (GST-P) foci and DNA damage biomarkers. Annatto, containing 5% bixin, was administered in the diet at concentrations of 20, 200, and 1000 ppm (0.07; 0.80 and 4.23 bixin/kg body wt/day, respectively), continuously during 2 weeks before, or 8 weeks after DEN treatment (200 mg/kg body wt, i.p.), to evaluate its effect on the liver-carcinogenesis medium-term bioassay. The comet assay was used to investigate the modifying potential of annatto on DEN (20 mg/kg body wt)-induced DNA damage. The results showed that annatto was neither genotoxic nor carcinogenic at the highest concentration tested (1000 ppm). No protective effects were also observed in both GST-P foci development and comet assays. In conclusion, in such experimental conditions, annatto shows no hepatocarcinogenic effect or modifying potential against DEN-induced DNA damage and preneoplastic foci in the rat liver.

Acute bacterial cystitis does not cause deoxyribonucleic acid damage detectable by the alkaline comet assay in urothelial cells of dogs.

Considering the high incidence of dogs with acute bacterial cystitis (BC) and the relationship among inflammation, genotoxicity, and carcinogenesis, we conducted a case-control study comparing the frequency of deoxyribonucleic acid (DNA) lesions assessed by the comet assay between disease-free animals (13 males and 13 females) and cytology-confirmed cases of acute BC (12 males and 12 females), which was mainly caused by Staphylococcus sp. (40%) and Escherichia coli (35%). The results show no increase in DNA damage in cells obtained by bladder washings and no influence of age, sex, and breed due to acute BC. In conclusion, DNA damage was seemingly not associated with the infection by specific bacteria.

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.

Black bean (Phaseolus vulgaris L.) as a protective agent against DNA damage in mice.

This study was designed to evaluate the toxicogenetic or protective effect of cooked and dehydrated black beans (Phaseolus vulgaris L.) in bone marrow and peripheral blood cells of exposed mice. The frequency of micronuclei detected using the bone marrow erythrocyte micronucleus test and level of DNA lesions detected by the comet assay were chosen as end-points reflecting mutagenic and genotoxic damage, respectively. Initially, Swiss male mice were fed with a 20% black bean diet in order to detect mutagenic and genotoxic activity. However, no increase in the frequency of bone marrow micronucleated polychromatic erythrocytes (MN PCEs) or DNA lesion in leukocytes was observed. In contrast, received diets containing 1, 10 or 20% of black beans, a clear, but not dose-dependent reduction in the frequency of MN PCEs were observed in animals simultaneously treated with cyclophosphamide, an indirect acting mutagen. Similar results were observed in leukocytes by the comet assay. Commercial anthocyanin was also tested in an attempt to identify the bean components responsible for this protective effect. However, instead of being protective, the flavonoid, at the highest dose administered (50 mg/kg bw), induced primary DNA lesion, as detected by the comet assay. These data indicate the importance of food components in preventing genetic damage induced by chemical mutagens, and also reinforce the role of toxicogenetic techniques in protecting human health.

Antimutagenic effect of Lentinula edodes (BERK.) Pegler mushroom and possible variation among lineages.

This study was performed to evaluate the efficiency of four different lineages (95/01, L1, 96/22 and JABK) of Lentinula edodes (BERK.) Pegler mushroom (shiitake) for inhibiting the N-ethyl-N-nitrosourea (ENU) clastogenicity in vivo. Male Swiss mice (10 animals/group) were treated during 15 consecutive days with dried mushroom added to basal diet under three different concentrations (1, 5 and 10%). At day 15, mice were intraperitoneally injected with ENU (50 mg/kg body weight) and sacrificed 24 h later for evaluation of micronucleated bone marrow polychromatic erythrocytes (MNPCE). Negative and positive controls (10 animals each), receiving basal diet and saline or ENU ip injection, respectively, were also evaluated. Results showed that pretreatments with diets containing the lineages 95/01, L1 and 96/22 reduce the frequencies of MNPCE induced by ENU. The absence of an antimutagenic activity for the lineage JABK might be related to intrinsic differences among the lineages such as biochemical composition. Taken together, our data show that the differences in protective activities of the mushrooms need to be clarified in further studies and the mechanisms for such activities need to be investigated.