Grape Seed Components as Protectors of Inflammation, DNA Damage, and Cancer.

PURPOSE OF REVIEW: Oxidative stress is related to the pathogenesis of several chronic diseases, including inflammatory processes. Free radicals excess increase not only oxidative stress but also genomic instability. Polyphenols are non-enzymatic antioxidants that act as a defense barrier against free radicals and non-radical oxidants. The purpose of this article was to review published articles relating dietary polyphenols contained in grape seed proanthocyanidin extracts with its potential for reversing DNA damage. RECENT FINDINGS: Proanthocyanidin components exert pleiotropic actions having several biological, biochemical, and significant pharmacological effects and showed the ability to reduce cytotoxicity and genotoxicity. Grape seed proanthocyanidin extracts showed the ability to reduce cytotoxicity and genotoxicity through the comet assay and the micronucleus technique.

DNA damage analysis in newborns and their mothers related to pregnancy and delivery characteristics.

INTRODUCTION: Increased DNA damage is associated with early events in carcinogenesis. The foetus may be more susceptible to effects of environment by transplacental exposure. We aimed to evaluate DNA damage in cells from umbilical cord (arteries and vein) and maternal blood from pregnant women. METHODS: Fifty eight pregnant women and their offspring were included in this study. They were submitted to an interview to obtain information about personal history, clinical history, and lifestyle habits. Other Information was obtained from medical records. The samples were prepared for Single Cell Gel/Comet assay and Cytokinesis-block Micronucleus Cytome (CBMN-Cyt) assay. RESULTS: Correlation between DNA damage frequency by Comet assay from newborns and their mothers was statistically significant and was significantly associated with nulliparity and more than 1 h of second stage of labour (umbilical vein and maternal blood). A positive MNi relationship was noticed for age (mother's blood) and inappropriate birth weight for gestational age (maternal blood). When multivariate statistical analyses were applied to measure the degree of association between variables that influenced DNA damage markers in the first evaluation, inadequate birth weight and pregnant weight gain were associated with MNi frequency in maternal and newborns blood, respectively. DISCUSSION: Significant associations between DNA damage in newborns and pregnant women, and birth and pregnancy events suggest molecular evidence of transplacental genotoxic effects. However, a potentially increased risk of degenerative diseases, such as cancers, in this population should be carefully investigated by further prospective cohort studies.

DNA damage, oxidative stress, and inflammation in children with celiac disease.

The objective of this study was to evaluate the level of genomic instability in patients with celiac disease and to establish a relationship between inflammation, oxidative stress, and DNA damage in these patients. Myeloperoxidase (MPO) activity, adenosine deaminase, nitric oxide (NOx), thiobarbituric acid, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and DNA damage were evaluated in peripheral blood samples from 47 celiac disease patients and 31 controls. Patients with celiac disease presented higher levels of DNA damage in comparison to controls (p=0.023). This difference was also observed for markers of oxidative stress, such as CAT (p=0.011) and SOD (p=0.013), and inflammatory markers such as MPO (p < 0.001) and NOx (p=0.009). Positive correlations were found between DNA damage levels and the values of CAT (r=0.405; p=0.009) and SOD (r=0.516; p < 0.001). Positive correlations were also found between GPx and NOx (r=0.349; p=0.030) and MPO and NOx (r=0.239; p=0.039). CAT and NOx showed a negative correlation (r= -0.315; p=0.042). In conclusion, intestinal inflammation can have systemic effects, causing an imbalance between oxidant and antioxidant markers, which may promote increased levels of DNA damage.

DNA damage and repair in individuals with ataxia-telangiectasia and their parents.

Ataxi A-T elangiectasia (AT) is a multisystem, complex and rare disease inherited in an autosomal recessive manner. Homozygous individuals have a variety of pathological manifestations, however, heterozygotes only present a higher risk of developing cancer. We evaluated the background levels of DNA damage (basal damage) and cell response to bleomycin or ionizing radiation using Comet assay and the cytokinesis-block micronucleus (CBMN) test in individuals with AT, their parents and controls. To evaluate DNA repair, the challenge experiment with ionizing radiation was performed using Comet assay, and different recovery times were evaluated. Results showed that basal MN frequencies differ between patients, parents and controls. Meanwhile, using the Comet assay, the results from the basal analysis do not differ between the groups, but monitoring the kinetics of DNA repair, we verified that the group of patients showed a delay in repair, compared to controls. Another finding was the nuclear bud (NBUD) frequency: spontaneous and induced cell cultures (with bleomycin and radiation) showed clear differences between patients, parents and controls. The CBMN assay and repair measurement with the Comet assay can help in the diagnosis of AT patients and ATM gene carriers, as complementary methods. The use of genomic instability evaluation techniques for the identification of the heterozygotes in families, where at least one member is affected, may be of great clinical importance.

DNA damage response in patients with pediatric Acute Lymphoid Leukemia during induction therapy.

Predicting the individual response to chemotherapy is a crucial challenge in cancer treatment. DNA damage caused by antitumor therapies evokes different repair mechanisms responses, such as Nucleotide Excision Repair (NER), whose components are being studied as prognosis biomarkers and target therapies. However, few reports have addressed DNA damages in pediatric Acute Lymphoid Leukemia (ALL). Hence, we conducted an observational follow-up study with pediatric patients to assess DNA damage (by Comet Assay) and gene expression from NER pathway during chemotherapy induction. Bone marrow samples from diagnosis, 15th(D15) and 35th (D35) days of the treatment were collected from 28 patients with ALL. There was no increase in damage index. However, there was a reduction of cells with low damages on D35 compared with diagnosis. NER pathway expression remained the same, however, in a single patient, a significant decrease was observed, maybe due to silencing or downregulation of repair pathways. DNA damage levels and repair may influence the clinical outcome, being involved in drug resistance and risk of relapse. In pediatric ALL, we analyzed for the first time DNA damage and repair behavior in BM samples. Monitoring patient's outcomes will help to access the implication of our findings in survival and relapse rates.

Frequencies of polymorphisms of the Rh, Kell, Kidd, Duffy and Diego systems of Santa Catarina, Southern Brazil.

BACKGROUND: Red blood cell genes are highly polymorphic with the distribution of alleles varying between different populations and ethnic groups. The objective of this study was to investigate gene polymorphisms of blood groups in the state of Santa Catarina, Southern Brazil. METHODS: Three hundred and seventy-three unrelated blood donors and 31 transfusion-dependent patients were evaluated to investigate polymorphisms of the Rh, Kell, Duffy, Kidd, and Diego blood group systems in a population from the state of Santa Catarina. The subjects, from seven regions that comprise the blood-banking network of the state, were assessed between August 2011 and March 2014. The genotypes of the Rh, Kell, Duffy, Kidd, and Diego systems were determined using the restriction fragment length polymorphism-polymerase chain reaction and allele-specific polymerase chain reaction techniques. RESULTS: The genotype frequencies in this study were significantly different when populations from different regions of Santa Catarina were compared. Furthermore, there were also significant differences in the genetic frequencies compared to other Brazilian states. The genotype frequencies of the Kell and Kidd blood groups are similar to European populations from Naples, Italy and Zurich, Switzerland. CONCLUSION: This article reports for the first time the frequency of polymorphisms of blood group systems in blood donors from Santa Catarina, Southern Brazil.

Novel synthetic chalcones induces apoptosis in human glioblastoma cells.

Glioblastoma multiforme is the main and most frequent tumor in adults' central nervous system. With a survival average of 5% two years after diagnosis, this type of cancer is a main health problem. Substances like the chalcones have been tested in order to develop new treatments. Here, we studied the effects of three synthetic chalcones (A23, C31 and J11) on A172 and surgery obtained-glioma cells. All chalcones showed a decrease in cell viability, mainly C31. An increase in apoptosis levels with no further increase of necrosis was observed. This augmentation may be linked to the high oxidative effect found, caused by the increased presence of reactive oxygen species and nitric oxide production. Cell cycle distribution showed an arrest at G0/G1 and S phases, suggesting that C31 interferes in cell cycle control. Our results shall aid in directing future research with this substance and its antitumor effect.

Genomic instability in human lymphocytes from male users of crack cocaine.

Recent research suggests that crack cocaine use alters systemic biochemical markers, like oxidative damage and inflammation markers, but very few studies have assessed the potential effects of crack cocaine at the cellular level. We assessed genome instability by means of the comet assay and the cytokinesis-block micronucleus technique in crack cocaine users at the time of admission to a rehabilitation clinic and at two times after the beginning of withdrawal. Thirty one active users of crack cocaine and forty control subjects were evaluated. Comparison between controls and crack cocaine users at the first analysis showed significant differences in the rates of DNA damage (p = 0.037). The frequency of micronuclei (MN) (p < 0.001) and nuclear buds (NBUDs) (p < 0.001) was increased, but not the frequency of nucleoplasmic bridges (NPBs) (p = 0.089). DNA damage decreased only after the end of treatment (p < 0.001). Micronuclei frequency did not decrease after treatment, and nuclear buds increased substantially. The results of this study reveal the genotoxic and mutagenic effects of crack cocaine use in human lymphocytes and pave the way for further research on cellular responses and the possible consequences of DNA damage, such as induction of irreversible neurological disease and cancer.

Chromosome instability and oxidative stress markers in patients with ataxia telangiectasia and their parents.

Ataxia telangiectasia (AT) is a rare neurodegenerative disorder, inherited in an autosomal recessive manner. Total blood samples were collected from 20 patients with AT, 13 parents of patients, and 17 healthy volunteers. This study aimed at evaluating the frequency of chromosomal breaks in spontaneous cultures, induced by bleomycin and ionizing radiation, and further evaluated the rates of oxidative stress in AT patients and in their parents, compared to a control group. Three cell cultures were performed to each individual: the first culture did not receive induction to chromosomal instability, the second was exposed to bleomycin, and the last culture was exposed to ionizing radiation. To evaluate the rates of oxidative stress, the markers superoxide dismutase (SOD), catalase (CAT), and thiobarbituric acid (TBARS) were utilized. Significant differences were observed between the three kinds of culture treatments (spontaneous, bleomycin, and radiation induced) and the breaks and chromosomal aberrations in the different groups. The oxidative stress showed no significant differences between the markers. This study showed that techniques of chromosomal instability after the induction of ionizing radiation and bleomycin are efficient in the identification of syndrome patients, with the ionizing radiation being the most effective.

Vitamin C intake reduces the cytotoxicity associated with hyperglycemia in prediabetes and type 2 diabetes.

Hyperglycemia leads to the formation of free radicals and advanced glycation end-products (AGEs). Antioxidants can reduce the level of protein glycation and DNA damage. In this study, we compared the levels of vitamin C intake, which is among the most abundant antioxidants obtained from diet, with the levels of fasting plasma glucose (FPG), glycated hemoglobin (A1C), DNA damage, and cytotoxicity in prediabetic subjects and type 2 diabetic subjects. Our results indicated that there was no significant correlation between FPG or A1C and DNA damage parameters (micronuclei, nucleoplasmic bridges, and nuclear buds). FPG and A1C correlated with necrosis (r = 0.294; P = 0.013 and r = 0.401; P = 0.001, resp.). Vitamin C intake correlated negatively with necrosis and apoptosis (r = -0.246; P = 0.040, and r = -0.276; P = 0.021, resp.). The lack of a correlation between the FPG and A1C and DNA damage could be explained, at least in part, by the elimination of cells with DNA damage by either necrosis or apoptosis (cytotoxicity). Vitamin C appeared to improve cell survival by reducing cytotoxicity. Therefore, the present results indicate the need for clinical studies to evaluate the effect of low-dose vitamin C supplementation in type 2 diabetes.

The influence on DNA damage of glycaemic parameters, oral antidiabetic drugs and polymorphisms of genes involved in the DNA repair system.

The hyperglycaemia seen in type 2 diabetes mellitus (DM2) is associated with increased oxidative stress and production of reactive oxygen species, both of which are factors that can provoke DNA damage. Notwithstanding, other factors, including medications and individual susceptibility, can also induce this type of DNA lesion. The objective of this study was, therefore, to investigate the influence of glycaemic control, oral antidiabetic drugs (metformin and glibenclamide) and polymorphisms of the XRCC1 and XRCC3 genes on the frequency of DNA damage in DM2 patients, which was accessed by the cytokinesis-block micronucleus cytome and the comet assays on the ex vivo mitogenically stimulated lymphocytes. The 53 people recruited to take part in the study were already on treatment with metformin and were followed for 5 months. Ten of these patients were put on combined treatment with the addition of glibenclamide. It was observed that the greater the plasma metformin concentration, the lower the frequency of micronuclei (MN) in the sample total (P = 0.009) and also that the subset of patients using combined treatment including glibenclamide had a significantly higher MN rate 90 days after starting combined treatment (P = 0.024). In the subset who only took metformin, the rate of MN was significantly higher among carriers of the 399Gln allele on the XRCC1 gene (P = 0.008). In addition, homozygotes for the 241Thr allele exhibited a significant increase in MN in the combined treatment group (P = 0.008). Our results suggest that different combinations of oral antidiabetic drugs and polymorphisms on genes involved in the DNA damage repair system could influence the frequency of this type of chromosome lesion, which can be a useful biomarker for assessing the risk of developing cancer.

DNA damage and cytotoxicity in adult subjects with prediabetes.

Prediabetes (intermediate hyperglycemia) is a high-risk state for diabetes that is defined by higher than normal glycemic levels that are below the level required for a diagnosis of diabetes. Prediabetes is characterized by oxidative stress, yet the associated DNA damage and cytotoxicity remain unknown to date. Therefore, we evaluated the relationship between glycemic alterations, DNA damage and cytotoxicity in the lymphocytes of individuals with pre-diabetes. Fasting plasma glucose (FPG) and glycated hemoglobin (A1C) levels were quantified and used as inclusion criteria. Anthropometric parameters were also evaluated. The cytokinesis-block micronucleus cytome assay (CBMN Cyt) was used to evaluate DNA damage and cytotoxicity. FPG correlated with A1C (r=0.562, p=0.002). Because A1C is the best predictor of diabetes complications, the association between A1C and the evaluated variables was assessed. The waist-hip ratio correlated with A1C (p<0.01). Regarding DNA damage, the frequency of nucleoplasmic bridges correlated with A1C (p<0.05). Both apoptosis and necrosis correlated with A1C (p<0.05). The overall frequency of DNA damage and cytotoxicity also correlated with A1C (p<0.01). Additional studies evaluating cell cycle and cell death patterns in prediabetes are necessary.

Genomic instability in patients with type 2 diabetes mellitus on hemodialysis.

OBJECTIVE: A previous study by our research group evaluated the levels of DNA damage using the comet assay in hemodialysis patients with type 2 diabetes mellitus. The same blood samples were also evaluated using the cytochalasin B micronucleus assay. A comparison of the results of the two assays is presented here. METHODS: Whole blood samples were collected from 22 type 2 diabetes mellitus patients on hemodialysis and from 22 control subjects. Samples were collected from patients early in the morning on Mondays, before the first weekly hemodialysis session. The cytokinesis-block micronucleus assay (CBMN) was used to evaluate genomic instability. RESULTS: The frequencies of micronuclei and nuclear buds were higher in patients than in controls (p-value = 0.001 and p-value < 0.001, respectively). There was a correlation between the frequency of micronuclei and DNA damage with the results of the comet assay (p-value < 0.001). The difference in the frequency of micronuclei and nuclear buds between patients and controls was more pronounced in the group with higher median comet values than in the group with lower comet values. CONCLUSION: Our results suggest that the increased rates of DNA damage as measured by the comet assay and influenced by the weekly routine therapy of these patients has a mutagenic effect, thereby increasing the risk of cancer in this group.

Genomic instability in patients with type 2 diabetes mellitus on hemodialysis

OBJECTIVE: A previous study by our research group evaluated the levels of DNA damage using the comet assay in hemodialysis patients with type 2 diabetes mellitus. The same blood samples were also evaluated using the cytochalasin B micronucleus assay. A comparison of the results of the two assays is presented here. METHODS: Whole blood samples were collected from 22 type 2 diabetes mellitus patients on hemodialysis and from 22 control subjects. Samples were collected from patients early in the morning on Mondays, before the first weekly hemodialysis session. The cytokinesis-block micronucleus assay (CBMN) was used to evaluate genomic instability. RESULTS: The frequencies of micronuclei and nuclear buds were higher in patients than in controls (p-value = 0.001 and p-value < 0.001, respectively). There was a correlation between the frequency of micronuclei and DNA damage with the results of the comet assay (p-value < 0.001). The difference in the frequency of micronuclei and nuclear buds between patients and controls was more pronounced in the group with higher median comet values than in the group with lower comet values. CONCLUSIONS: Our results suggest that the increased rates of DNA damage as measured by the comet assay and influenced by the weekly routine therapy of these patients has a mutagenic effect, thereby increasing the risk of cancer in this group.

Are MPS II heterozygotes actually asymptomatic? A study based on clinical and biochemical data, X-inactivation analysis and imaging evaluations.

For some X-linked disorders the expressivity and penetrance in females are almost similar to those ones found in males. For mucopolysaccharidosis type II (MPS II), there are no studies in the literature trying to identify subtle signs and symptoms of this disease in heterozygotes. The objective of this study was to compare heterozygotes and non-heterozygotes for MPS II, in order to test the hypothesis that heterozygotes may present subtle manifestations of the disease. In this observational and transversal study we collected data on 40 Brazilian women with a positive familial history for MPS II that included clinical and physical exam, karyotype, pattern of X-inactivation, iduronate-2-sulfatase (IDS) activity in leukocytes and plasma, urinary glycosaminoglycans levels, computerized tomography scans (CT) of abdomen and spine, and brain magnetic resonance imaging. The Results showed the following: According to DNA analysis, 22 women were classified as heterozygote and 18 as non-heterozygotes. We did not find any abnormality on physical examination, karyotype, or spine CT. Also the pattern of X-inactivation was not different between the groups. Applying the Bonferroni's correction, both groups were found to differ only in relation to IDS activity in plasma and in leukocyte, which were lower in heterozygotes. In our investigation we did not find any evidence of subtle clinical manifestations of MPS II in heterozygotes. Our findings suggest there is no relation between the absence of clinical signs in these women and the occurrence of a favorable skewing pattern of X-inactivation.

Iron intake, red cell indicators of iron status, and DNA damage in young subjects.

OBJECTIVE: This study evaluated the association between primary DNA damage and chromosomal damage with iron intake and red blood cell parameters of iron status in a sample of healthy children and adolescents from a low-socioeconomic community. METHODS: The level of primary DNA damage was assessed using an alkaline comet assay and the level of chromosomal damage was assessed using the cytokinesis-block micronucleus assay. A automated complete blood count was used to evaluate red blood cell status. The intake of iron was measured using a food-recall questionnaire. RESULTS: According to hemoglobin levels, only 1 of the 30 subjects evaluated was anemic. Nevertheless, 43% of the sampled subjects showed decreased mean corpuscular volume in addition to an increased amount of primary DNA damage (P < 0.05). Mean corpuscular volume was negatively correlated with primary DNA damage (r = -0.429, P = 0.020) but not with chromosomal damage. The association between iron and primary DNA damage showed a U-shaped curve, indicating that an intake of approximately 15 mg of iron per day (up to two-fold of the dietary recommended intake) could minimize primary DNA damage in this age group. The frequency of micronuclei and nucleoplasmic bridges, indicators of chromosomal breakage/loss and chromosomal end-fusions, respectively, showed a negative correlation with iron intake. These results indicate that an intake of iron >15 mg/d could increase genomic stability in binucleated lymphocytes of the same group. CONCLUSION: An intake of iron ≥ 15 mg/d can decrease DNA damage in young subjects.

Oxidative stress and DNA damage in older adults that do exercises regularly.

OBJECTIVES: Free radicals may damage lipids, proteins and DNA, which may lead to critical diseases in the aging. This work evaluated levels of malondialdehyde (MDA), glutathione peroxidase (GPx) and DNA damage by comet assay (SCGE) in older adults that do exercises regularly. DESIGN AND METHODS: 110 females, aged 66.3+/-8 years were divided into sedentary (n=54), walking (n=36) and muscle building (n=20) groups. Levels of MDA, GPx and SCGE were measured in venous blood before and after exercise. RESULTS: MDA levels were higher (P<0.005) and GPx levels were lower (P<0.005) in active groups than in sedentary group. SCGE index after physical activity was greater than at baseline (muscle building: P=0.004; walking: P=0.002). CONCLUSIONS: Exercise reduces the diseases risk, but may promote the production of free radicals. It remains unclear whether cell adaptations responsible for health benefits are associated with such events. However we may suggest the existence of a different biochemical pattern for older adults that do exercise regularly.