DNA damage in peripheral blood mononuclear cells of patients undergoing anti-tuberculosis treatment

Tuberculosis (TB), a chronic infectious disease, is a major cause of morbidity and mortality worldwide. Expression of iNOS and consequent production of NO during the inflammatory process is an important defense mechanism against TB bacteria. We have tested whether pulmonary TB patients undergoing anti-tuberculosis treatment present DNA damage, and whether this damage is related to oxidative stress, by evaluating total hydrophilic antioxidant capacity and iNOS expression. DNA damage in peripheral blood mononuclear cells from patients and healthy tuberculin test (PPD) positive controls was evaluated by single-cell gel electrophoresis (comet assay), and iNOS expression was measured by qPCR. We also evaluated total hydrophilic antioxidant capacity in plasma from patients and controls. Compared to controls, pulmonary TB patients under treatment presented increased DNA damage, which diminished during treatment. Also, the antioxidant capacity of these individuals was increased at the start of treatment, and reduced during treatment. TB patients showed lower iNOS expression, but expression tended to increase during treatment. Our results indicate that pulmonary TB patients under anti-TB treatment exhibit elevated DNA damage in peripheral blood mononuclear cells. This damage was not related to nitric oxide but may be due to other free radicals. (C) 2012 Elsevier B.V. All rights reserved.

DNA damage and nitric oxide production in mice following infection with L. chagasi

Leishmania chagasi, which causes visceral leishmaniasis in South America, is an obligate intracellular protozoan. Production of nitric oxide by macrophages during the inflammatory response is one of the main microbicidal mechanisms against this parasite. The goal of this study was to evaluate whether L. chagasi infection causes DNA damage in peripheral blood and spleen cells of Balb/c mice and whether such damage may be related to NO production. Balb/c mice were either infected with L chagasi or maintained as controls. The single-cell gel electrophoresis (comet) assay was used to measure DNA damage in peripheral blood and spleen cells, and the Griess reaction was used to measure NO production in the spleen. L chagasi infection induced DNA damage in peripheral blood and spleen cells of infected mice. Macrophages from the control group, challenged with L. chagasi, showed significantly (p < 0.05) greater NO production, compared to non-challenged cells. Treatment of spleen cells with N(G)-monomethyl-L-arginine (LNMMA) caused a significant reduction of NO production and DNA damage (p < 0.05). Our results indicate that L. chagasi induces DNA damage in the peripheral blood and spleen cells and that NO not only causes killing of the parasite but also induces DNA damage in adjacent cells. (C) 2011 Elsevier B.V. All rights reserved.

Evaluation of level of DNA damage in blood leukocytes of non-diabetic and diabetic rat exposed to cigarette smoke

The objective of the present study was to use the comet assay to evaluate the steady-state level of DNA damage in peripheral blood leukocytes from diabetic and non-diabetic female Wistar rats exposed to air or to cigarette smoke. A total of 20 rats were distributed into four experimental groups (n= 5 rats/group): non-diabetic (control) and diabetic exposed to filtered air; non-diabetic and diabetic exposed to cigarette smoke. A pancreatic beta (beta)-cytotoxic agent, streptozotocin (40 mg/kg b.w.) was used to induce experimental diabetes in rats. Rats placed into whole-body exposure chambers were exposed for 30 min to filtered air (control) or to tobacco smoke generated from 10 cigarettes, twice a day, for 2 months. At the end of the 2-month exposure period, each rat was anesthetized and humanely killed to obtain blood samples for genotoxicity analysis using the alkaline comet assay. Blood wleukocytes sampled from diabetic rats presented higher DNA damage values (tail moment =0.57 +/- 0.05; tail length =19.92 +/- 0.41, p < 0.05) compared to control rats (tail moment =0.34 +/- 0.02; tail length= 17.42 +/- 0.33). Non-diabetic (tail moment =0.43 +/- 0.04, p > 0.05) and diabetic rats (tail moment= 0.41 +/- 0.03, p > 0.05) exposed to cigarette smoke presented non-significant increases in DNA damage levels compared to control group. In conclusion, our data show that the exposure of diabetic rats to cigarette smoke produced no additional genotoxicity in peripheral blood cells of female Wistar rats. (c) 2007 Elsevier B.V. All rights reserved.

Levels of DNA damage in blood leukocyte samples from non-diabetic and diabetic female rats and their fetuses exposed to air or cigarette smoke

dThe objective of the present study was to evaluate DNA damage level in blood leukocytes from diabetic and non-diabetic female Wistar rats exposed to air or to cigarette smoke, and to correlate the findings with levels of DNA damage detected in blood leukocyte samples from their fetuses. A total of 20 rats were distributed into four experimental groups: non-diabetic (control; G1) and diabetic exposed to filtered air (G2): non-diabetic (G3) and diabetic (G4) exposed to cigarette smoke. Rats placed into whole-body exposure chambers were exposed for 30 min to filtered air (control) or to tobacco smoke generated from 10 cigarettes, twice a day, for 2 months. Diabetes was induced by a pancreatic beta-cytotoxic agent, streptozotocin (40 mg/kg b.w.). At day 21 of pregnancy, each rat was anesthetized and humanely killed to obtain maternal and fetal blood samples for genotoxicity analysis using the alkaline comet assay. G2, G3 and G4 dams presented higher DNA damage values in tail moment and tail length as compared to G1 group. There was a significant positive correlation between DNA damage levels in blood leukocyte samples from G2 and G3 groups (tail moment); G3 and G4 groups (tail length) and G3 group (tail intensity) and their fetuses. Thus, this study showed the association of severe diabetes and tobacco cigarette smoke exposure did not exacerbate levels of maternal and fetal DNA damages related with only diabetes or cigarette smoke exposure. Based on the results obtained and taking into account other published data, maternal diabetes requires rigid clinical control and public health and education campaigns should be increased to encourage individuals, especially pregnant women, to stop smoking. (C) 2008 Elsevier B.V. All rights reserved.

Large nuclear vacuoles are indicative of abnormal chromatin packaging in human spermatozoa

The aim of this investigation was to determine the presence of abnormal sperm chromatin packaging in spermatozoa with large nuclear vacuoles (LNV) selected via high magnification by analysing the pattern of chromomycin A3 (CMA3) staining. A prospective observational study was designed to analyse semen samples obtained from 66 men undergoing infertility diagnosis and treatment. The numbers of cells with normal (dull yellow staining of the sperm head/CMA3-negative) and abnormal (bright yellow fluorescence of the sperm head/CMA3-positive) chromatin packaging were determined on slides with normal and LNV spermatozoa. The presence of bright yellow fluorescence (CMA3-positive) was significantly higher (p < 0.0001) in spermatozoa with LNV than in normal spermatozoa (719/1351; 53.2% vs. 337/835; 40.3%, respectively), reflecting a higher percentage of abnormal chromatin packaging in spermatozoa with large LNV. Our data support the hypothesis that the presence of LNV reflects the presence of abnormal chromatin packaging, which may facilitate sperm DNA damage. As sperm nuclear vacuoles are evaluated more precisely at high magnifications using motile sperm organelle morphology examination (MSOME), the present results support the use of high-magnification sperm selection for intracytoplasmic sperm injection (ICSI).

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. (C) 2003 Elsevier Ltd. All rights reserved.

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.

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.

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.

Mate attenuates DNA damage and carcinogenesis induced by diethylnitrosamine and thermal injury in rat esophagus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Protective effects of lemongrass (Cymbopogon citratus STAPF) essential oil on DNA damage and carcinogenesis in female Balb/C mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

DNA methylation in the CTCF-binding site I and the expression pattern of the H19 gene: Does positive expression predict poor prognosis in early stage head and neck carcinomas?

Loss of allele-specific expression by the imprinted genes IGF2 and H19 has been correlated with a differentially methylated region (DMR) upstream to the H19 gene. The H19-DMR contains seven potential CCCTC-binding factor (CTCF) binding sites. CTCF is a chromatin insulator and a multifunctional transcription factor whose binding to the H19-DMR is suppressed by DNA methylation. Our study included a group of 41 head and neck squamous cell carcinoma (HNSCC) samples. The imprinting status of the H19 gene was analyzed in 11 out of 35 positive cases for H19 gene expression, and only 1 of them showed loss of imprinting. We detected a significant correlation (P=0.041, Fisher's exact test) between H19 expression and tumor recurrence. Among H19 positive cases, six were T2, in which five developed recurrence and/or metastasis. Inversely, in the group of tumors that showed no H19 gene expression, 5 out of 24 were T2 and only I presented regional recurrence. These data support the hypothesis that H19 expression could be used as a prognostic marker to indicate recurrence in early stage tumors. We also examined the methylation of the CTCF binding site 1 in a subgroup of these samples. The H19 gene silencing and loss of imprinting were not correlated with the methylation pattern of the CTCF binding site 1. However, the significant correlation between H19 expression and tumor recurrence suggest that this transcript could be a marker for the progression of HNSCC. (c) 2005 Wiley-Liss, Inc.

LaTBP1: A Leishmania amazonensis DNA-binding protein that associates in vivo with telomeres and GT-rich DNA using a Myb-like domain

Different species of Leishmania can cause a variety of medically important diseases, whose control and treatment are still health problems. Telomere binding proteins (TBPs) have potential as targets for anti-parasitic chemotherapy because of their importance for genome stability and cell viability. Here, we describe LaTBP1 a protein that has a Myb-like DNA-binding domain, a feature shared by most double-stranded telomeric proteins. Binding assays using full-length and truncated LaTBP1 combined with spectroscopy analysis were used to map the boundaries of the Myb-like domain near to the protein only tryptophan residue. The Myb-like domain of LaTBP1 contains a conserved hydrophobic cavity implicated in DNA-binding activity. A hypothetical model helped to visualize that it shares structural homology with domains of other Myb-containing proteins. Competition assays and chromatin immunoprecipitation confirmed the specificity of LaTBP1 for telomeric and GT-rich DNAs, suggesting that LaTBP1 is a new TBP. (C) 2007 Elsevier B.V. All rights reserved.

Evidence of epigenetic regulation of the tumor suppressor gene cluster flanking RASSF1 in breast cancer cell lines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)