Roberta Passos Palazzo

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.

Induction and removal of DNA damage in blood leukocytes of patients with type 2 diabetes mellitus undergoing hemodialysis

Type 2 diabetes mellitus (T2DM) is associated with a high production of reactive oxygen species, which may cause oxidative DNA damage. High levels of genomic damage have been associated with renal failure and hemodialysis. However, no information is available in the literature concerning the levels of DNA damage in T2DM individuals who are dependent on hemodialysis. This study used the comet assay to assess the levels of DNA damage before, immediately after and 48 h after the hemodialysis session in 25 patients with T2DM and in a group of 20 healthy individuals, selected according to mean age, sex and smoking habit. Our results showed increased levels of DNA damage in hemodialysis-dependent T2DM individuals (12.36+/-8.04) when compared with healthy individuals (7.35+/-7.41) (p=0.014). Damage levels increased immediately after the hemodialysis session (19.76+/-12.40) (p=0.04), which suggests a possible action of pro-oxidative factors related to the therapy, with a genotoxic effect on cells. Results obtained 48 h after hemodialysis (6.44+/-5.99) evidenced damage removal (p=0.001), which may be suggestive of DNA repair.

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.

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 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.

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 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.