DNA damage and aberrant crypt foci as putative biomarkers to evaluate the chemopreventive effect of annatto (Bixa orellana L.) in rat colon carcinogenesis

Chemoprevention opens new perspectives in the prevention of cancer and other degenerative diseases. Use of target-organ biological models at the histological and genetic levels can markedly facilitate the identification of such potential chemopreventive agents. Colon cancer is one of the highest incidence rates throughout the world and some evidences have indicated carotenoids as possible agents that decrease the risk of colorectal cancer. In the present study, we evaluate the activity of annatto (Bixa orellaria L.), a natural food colorant rich in carotenoid, on the formation of aberrant crypt foci (ACF) induced by dimethy1hydrazine (DMH) in rat colon. Further, we investigate, the effect of annatto on DMH-induced DNA damage, by the comet assay. Male Wistar rats were given s.c. injections of DMH (40 mg/kg body wt.) twice a week for 2 weeks to induce ACE They also received experimental diets containing annatto at 20, 200 or 1000 ppm for five 5 weeks before (pre-treatment), or 10 weeks after (post-treatment) DMH treatment. In both protocols the rats were sacrificed on week 15th. For the comet assay, the animals were fed with the same experimental diets for 2 weeks. Four hours before the sacrifice, the animals received an s.c. injection of DMH (40 mg/kg body wt.). Under such conditions, dietary administration of 1000 ppm annatto neither induce DNA damage in blood and colon cells nor aberrant crypt foci in rat distal colon. Conversely, annatto was successful in inhibiting the number of crypts/colon (animal), but not in the incidence of DMH-induced ACF, mainly when administered after DMH. However, no antigenotoxic effect was observed in colon cells. These findings suggest possible chemopreventive effects of annatto through their modulation of the cryptal cell proliferation but not at the initiation stage of colon carcinogenesis. (c) 2005 Elsevier B.V. All rights reserved.

Genotoxicity of corrosion eluates obtained from endosseous implants

Purpose: Commercially pure titanium alloys are currently used as metallic biomaterials in implantology. Corrosion phenomena appear to play a decisive role in metallic implant long-term behavior. Thus, the goal of this study was to examine the genotoxic potential of corrosion eluates obtained from dental implants using Chinese ovary hamster cells in vitro by the single-cell gel (comet) assay. This technique detects deoxyribonucleic acid strand breaks in individual cells in alkaline conditions.Materials and Methods: the materials tested included 3 dental implants commercially available. Each of the tested materials was corroded in a solution consisting of equal amounts of acetic acid and sodium chloride (0.1 M) for 1, 3, 7, 14, and 21 days. The Chinese ovary hamster cultures were then exposed to all corrosion eluates obtained from endosseous dental implants for 30 minutes at 37 degrees C.Results: None of the eluates was found to exhibit genotoxicity, regardless of the type of dental implant used.Conclusion: the results suggest that all dental implants tested in this study did not induce deoxyribonucleic acid breakage as depicted by the single-cell gel (comet) assay.

Chlorhexidine induces DNA damage in rat peripheral leukocytes and oral mucosal cells

Objective: Chlorhexidine digluconate is widely used in dental practice for decreasing plaque control, controlling gingivitis and disinfecting root canals. However, the undesirable effects of chlorhexidine digluconate regarding its genotoxicity are conflicting in the literature. Thus, the aim of this study was to investigate the genotoxicity of chlorhexidine digluconate in rat peripheral blood and oral mucosal cells by the single cell gel (comet) assay and micronucleus assay.Methods: Thirty male Wistar rats were distributed into three groups: negative control; experimental group orally treated with 0.5 ml of 0.12% chlorhexidine digluconate, twice daily, during 8 days; and positive control, which received 4-nitroquinoline 1-oxide at 0.5 g/l by drinking water.Results: A statistically significant increase of DNA damage was observed in leukocytes and oral mucosal cells of the chlorhexidine digluconate treated group, as assessed by the comet assay. However, no increase of micronucleated cells was detected in reticulocytes from peripheral blood cells.Conclusions: Taken together, the data indicate that chlorhexidine digluconate is able to induce primary DNA damage in leukocytes and in oral mucosal cells, but no chromosome breakage or loss in erythrocytes.

Genotoxicity and cytotoxicity of glass ionomer cements on Chinese hamster ovary (CHO) cells

Glass ionomer cements are widely used in dentistry as restorative materials and adhesives for composite restorations. However, the results of genotoxicity studies using these materials are inconclusive in literature. The goal of this study was to examine 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 Chinese hamster ovary (CHO) cells in vitro for 1 h at 37 degrees C. Data were assessed by Kruskall-Wallis nonparametric test. The results showed that the powder from Ketac Molar displayed genotoxicity only in the maximum concentration evaluated (100 mu g/mL). In the same way, the liquid from Vitrebond at 0.1% dilution caused an increase of DNA injury. Significant differences (P < 0.05) in cytotoxicity provoked by all powders tested of glass ionomer cements were observed for exposure at 1000 mu g/mL concentration. With respect to liquids of glass ionomer cements evaluated, the major toxic effect on cell viability was produced at 10%, beginning at the dilution of 0.5% for Vitrebond. Taken together, we conclude that some components of glass ionomer cements show both genotoxic and cytotoxic effects.

Protective action of propolis on the rat colon carcinogenesis

Propolis is a honeybee product with several biological and therapeutical properties. Its effect on the process of colon carcinogenesis and DNA damage were evaluated in the male Wistar rats using the aberrant crypt foci (ACF) assay and the comet assay, respectively. For both tests, animals were treated with the colon carcinogen 1,2 dimethylhydrazine (DMH, 40 mg/kg, s.c.) for 2 weeks (two injections/week) in order to induce both DNA damage and ACF. The animals were divided into groups that received propolis (ethanolic extract) at three different doses (10, 30, and 90 mg/kg b.w., by gavage), either simultaneously or after DMH treatment. For the comet assay, peripheral blood samples were collected 4 h after the last DMH treatment. All animals were sacrificed at the 5th week for evaluation of ACF. The results show that only the intermediate dose (30 mg/kg) of propolis, administered after DMH initiation, is significantly associated to a smaller number of aberrant crypts in the distal colon. No effect on DNA damage in peripheral blood cells, however, was verified by the comet assay. These data suggest that propolis has a protective influence on the process of colon carcinogenesis, suppressing the development of preneoplastic lesions, and probably exerts no protection against the initiation of carcinogenesis.

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 mu g/ml. NaF-treated and control cells were then incubated with 0-10 mu M MMS in phosphate-buffered saline (PBS) for 15 min at 37 degrees C, or 7-100 mu M 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. Copyright (c) 2007 S. Karger AG, Basel

Tomato-oleoresin supplement prevents doxorubicin-induced cardiac myocyte oxidative DNA damage in rats

Doxorubicin (DOX) is an efficient chemotherapeutic agent used against several types of tumors; however, its use is limited due to severe cardiotoxicity. Since it is accepted that reactive oxygen species are involved in DOX-induced cardiotoxicity, antioxidant agents have been used to attenuate its side effects. To determine tomato-oleoresin protection against cardiac oxidative DNA damage induced by DOX, we distributed Wistar male rats in control (C), lycopene (L), DOX (D) and DOX+lycopene (DL) groups. They received corn oil (C, D) or tomato-oleoresin (5 mg/kg body wt. day) (L, DL) by gavage for a 7-week period. They also received saline (C, L) or DOX (4 ma/kg body wt.) (D, DL) intraperitoneally at the 3rd, 4th, 5th, and at 6th week. Lycopene absorption was checked by HPLC. Cardiac oxidative DNA damage was evaluated by the alkaline Comet assay using formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (endo 111). Cardiomyocyte levels of SBs, SBs FPG and SBs Endo III were higher in rats from D when compared to other groups. DNA damage levels in cardiomyocytes from DL were not different when compared to C and L groups. The viability of cardiomyocytes from D or DL was lower than C or L groups (p < 0.01). Lycopene levels (mean +/- S.D. nmol/kg) in saponified hearts were similar between L (47.43 +/- 11.78) and DL (49.85 +/- 16.24) groups. Our results showed: (1) lycopene absorption was confirmed by its cardiac levels; (2) DOX-induced oxidative DNA damage in cardiomyocyte; (3) tomato-oleoresin supplementation protected against cardiomyocyte oxidative DNA damage. (c) 2007 Elsevier B.V. All rights reserved.

Effects of Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] on the urinary bladder of male Wistar rats

Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a substituted urea herbicide widely used on agricultural crops such as soy, cotton and sugar cane. In a previous long-term study this herbicide exerted carcinogenic activity on the urinary bladder mucosa of male Wistar rats. In general, the genotoxic and mutagenic potentials of Diuron are considered to be negative. The present study aimed to evaluate the mode of action of Diuron on the urinary bladder mucosa of male Wistar rats. Six-week old male Wistar rats were fed pelleted Nuvilab diet mixed with Diuron at 125, 500 and 2500 ppm. As a positive control, 8.3% sodium saccharin (NaS) was fed in the diet. Preceding the sacrifice of the animals at the 20th week, urinary pH was measured and the genotoxic potential of Diuron was evaluated by the comet assay. Histological urothelial lesions in the urinary bladder and in the renal pelvis mucosa, cell proliferation/apoptosis evaluations, and scanning electron microscopy (SEM) of the urinary bladder mucosa were also performed. No DNA changes were found in urothelial or peripheral blood cells, and urinary pH was comparable to controls in all Diuron groups. In the urinary bladder urothelium, the incidence of simple hyperplasia (SH) by light microscopy was significantly increased (7/10; p < 0.005) in the 2500 ppm Diuron group but not at the lower doses. By SEM, three of five animals treated with 2500 ppm Diuron showed urothelial cell necrosis and hyperplasia. In the renal pelvis, the incidence of SH was significantly increased in the Diuron 500 and 2500 ppm and in the NaS 8.3% groups. Cell proliferation was significantly increased in the Diuron 2500 ppm, (p < 0.05) and NaS 8.3% (p < 0.05) groups. The results indicate that a high dietary concentration of Diuron is associated with urothelial necrosis and continuous regenerative cell proliferation that leads to urothelial hyperplasia. (c) 2006 Elsevier B.V.. All rights reserved.

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 1000 mu g mL(-1) concentration and 100 mu 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.

Variation of the antimutagenicity effects of water extracts of Agaricus blazei Murrill in vitro

Agaricus blazei Murill, popularly known as Sun Mushroom or Himematsutake, is native to Brazil. Nowadays, this mushroom has been target of great scientific interest due to its medical power and because it has shown antitumoral and immune modulatory properties. This work evaluated the mutagenic and antimutagenic potential from aqueous extracts prepared in different temperatures (4 degreesC, 25 degreesC and 60 degreesC) from the lineage AB 97/29 in two basidiocarp phases (young and sporulated) and from A. blazei commercialized in Londrina- PR - Brazil, named here as AB PR, and in Piedade- SP- Brazil, named as AB SP. Both micronucleus (MN) as comet assays were used. Chinese hamster lung V79 cells were treated in three antimutagenic experimental protocols: pre-, post- and simultaneous treatments, with the aqueous extracts of the A. blazei Murill and methyl methanesulfonate (MMS). The results suggested that under these circumstances of treatment, aqueous extracts of the A. blazei in both assays did not show any genotoxic potential. However, by the MN test, an antigenotoxic effect was shown against mutagenicity inducted by MMS for aqueous extracts at 60 degreesC of mushroom commercialized in Piedade- SP, in pre-, post- and simultaneous treatments and for AB PR only when used in pre-treatment. on the other hand, with comet assay, the results showed no protective effect in any case. The numbers indicated that different results can be get from A. blazei teas, and that not all of them seemed to be an efficient antimutagen against the induction of micronuclei by MMS. (C) 2003 Elsevier Ltd. All rights reserved.

Antimutagenic effects of the mushroom Agaricus blazei Murrill extracts on V79 cells

Agaricus blazei Murrill, a native mushroom in Brazil, has been widely consumed in different parts of the world due to its medicinal power. Its anticarcinogenic activity has been shown in experimental animals, and antimutagenic activity has been demonstrated only in Salmonella. In this work, the multagenic and antimutagenic activities of mushroom teas of strains AB96/07, AB96/09 and AB97/11 were evaluated in Chinese hamster V79 cells, using the comet assay and the micronucleus test. The cells were treated with three different concentrations (0.05, 0.1 and 0.15) of teas prepared from a 2.5% aqueous solution, under three different temperatures: (1) room (20-25 degreesC); (2) ice-cold (2-8 degreesC); and (3) warm (60 degreesC). The teas were applied in co-, pre- and post-treatments in combination with the mutagen methyl methanesulfonate (MMS; 1.6 x 10(-4) and 4 x 10(-4) M). The duration of the treatment was 1 h in the comet assay and 2 h in the micronucleus test. The results showed that the mushroom was not mutagenic itself. Nevertheless, the mushroom is an efficient antimutagen against the induction of micronuclei by MMS in all concentrations and preparations tested. The observed reductions in the frequencies of micronuclei ranged from 61.5 (room temperature 0.1% tea in post-treatment) to 110.3% (co-treatment with warm and ice-cold 0.15% tea). In the comet assay, the antimutagenic activity was detected only when the cells were pre-treated with the following teas: warm 0.1 and 0.15%, room temperature 0.05% and ice-cold 0.1%. The results indicate that the mushroom A. blazei extracts are antimutagenic when tested in V79 cells. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Letinula edodes (Berk.) Pegler (Shiitake) modulates genotoxic and mutagenic effects induced by alkylating agents in vivo

We evaluated the antimutagenic effect of Letinula edodes (Berk.) Pegler (Shiitake) on the frequency of micronuclei in mice treated with N-ethyl-N-nitrosourea (ENU) or cyclophosphamide (Cl?). Mice were orally (gavage) pretreated for 15 consecutive days with solutions of Shiitake (0.6 ml per day, gavage) prepared at three different temperatures: 4, 21 (RT), and 60 degreesC. Then, the animals were intraperitoneally injected on day 15 with CP (25 or 50 mg/kg) or ENU (50 mg/kg) and killed 24 or 48 h after treatment for evaluation of micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow and micronucleated reticulocytes (MNRETs). A mixture of L. edodes lineages (LE 95/016, 96/14, 96/17, 96/22, 96/23, 97/27, and 97/28) significantly decreased the frequencies of MNPCEs and MNRETs induced by CP (25 and 50 mg/kg). When a single lineage from the mixture (LE 96/17) was tested we also found a significant reduction in the frequencies of MNPCEs and MNRETs induced by both CP or ENU (50 mg/kg). The comet assay was also performed 3 h after ENU treatment using mice pretreated with the single lineage (LE 96/17) of L. edodes. The results showed a high degree of variability with some indications of an antigenotoxic effect. Taken together, our data show that solutions from Shiitake inhibit in vivo mutagenicity of CP and ENU. (C) 2001 Elsevier B.V. B.V. All rights reserved.

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. Copyright (C) 2004 S. Karger AG, Basel.

Absence of DNA damage in multiple organs (blood, liver, kidney, thyroid gland and urinary bladder) after acute fluoride exposure in rats

Fluoride has been widely used in dentistry as a caries prophylactic agent. However, there has been some speculation that excess fluoride could cause an impact on genome integrity. In the current study, the potential DNA damage associated with exposure to fluoride was assessed in cells of blood, liver, kidney, thyroid gland and urinary bladder by the single cell gel (comet) assay. Male Wistar rats aging 75 days were distributed into seven groups: Groups 1 (control), 2, 3, 4, 5, 6 and 7 received 0 (deionized water), 10, 20, 40, 60, 80 and 100 mgF/Kg body weight from sodium fluoride (NaF), respectively, by gastrogavage. These groups were killed at 2 h after the administration of the fluoride doses. The level of DNA strand breaks did not increase in all organs evaluated and at all doses of NaF tested, as depicted by the mean tail moment. Taken together, our results suggest that oral exposure to NaF did not result in systemic genotoxic effect in multiple organs related to fluoride toxicity. Since 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 risk associated with chemical exposure.

Genomic instability in non-neoplastic oral mucosa cells can predict risk during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis

4-Nitroquinotine 1-oxide (4NQO)-induced rat tongue carcinogenesis is a useful model for studying oral squamous cell carcinoma. The aim of this study was to investigate the level of DNA damage induced by 4NQO in oral mucosa cells by the single cell get (comet) assay. Mate Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution by drinking water for 4, 12 or 20 weeks. Ten animals were used as negative control. Statistically significant increase of DNA damage was observed in non-neoplastic oral cells at four weeks of 4NQO administration when compared with control (P < 0.05). The level of DNA damage was directly associated with the severity of histological changes. The results suggest that histologically normal tissue is able to harbor genetically unstable cells contributing to the initiation of oral carcinogenesis. Genomic instability appears to be associated with the risk and progression of oral cancer. (C) 2004 Elsevier Ltd. All rights reserved.