Sharbel Weidner Maluf

Evaluation of Sinos River water genotoxicity using the comet assay in fish

The Sinos River, in southern Brazil, is polluted by industrial discharges and untreated urban wastes. Fish genotoxicity biomarkers are valuable parameters for environmental risk assessment. In this study, we used the comet assay to detect genotoxicity due to multiple sources of pollution in the peripheral blood of a native fish species (Hyphessobrycon luetkenii). In addition, we analysed possible DNA damage from aluminum, lead, chromium, copper, nickel, iron and zinc contamination. Water samples were collected seasonally from three sampling sites and the fish were assessed under laboratory conditions. Water chemical analysis showed an increased level of aluminum and iron in most of the samples at sites 2 and 3, located in the middle and lower river course, respectively. The index of DNA damage assessed by the comet assay demonstrated no significant differences in different seasons or at the different sampling sites, while the frequency of cells with DNA damage was higher in water samples collected at sites 1 and 2 during the spring season. None of the metals studied seems to be associated with the increase in the frequency of cells with DNA damage observed during the spring season. The results of this study indicate that the Sinos River is contaminated with substances that are genotoxic to fish, including the waters near the river spring.

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

OBJECTIVESnFree 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.nnnDESIGN AND METHODSn110 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.nnnRESULTSnMDA 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).nnnCONCLUSIONSnExercise 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.

The influence of the S19W SNP of the APOA5 gene on triglyceride levels in southern Brazil: Interactions with the APOE gene, sex and menopause status

BACKGROUND AND AIMSnHypertriglyceridemia is an important independent risk factor for coronary artery diseases and is determined by a wide range of factors, both genetic and exogenous. The A5 apolipoprotein, which is associated with the synthesis and removal of triglycerides (TG), is encoded by the APOA5 gene. One of the polymorphisms of this gene that has been the focus of a large number of studies, and which appears to be associated with increased TG, is S19W (rs 3135506). In this study, we examined the influence of this single nucleotide polymorphism (SNP) on TG levels of a sample of southern Brazilians.nnnMETHODS AND RESULTSnSamples obtained from 567 people of European descent were genotyped; interactions between this variant and anthropometric variables were analyzed, and the effects of lifestyle, sex, menopause, and variations of the APOE gene were evaluated. We found that the 19W allele is associated with increased TG (p = 0.025) and that this influence was modulated by sex (p = 0.003), menopause (p = 0.022) and the presence of the E*4 allele (p = 0.027).nnnCONCLUSIONnOur data showed, for the first time, the importance and magnitude of the influence of the S19W variant in a southern Brazilian population.

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

OBJECTIVEnThis 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.nnnMETHODSnThe 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.nnnRESULTSnAccording 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.nnnCONCLUSIONnAn intake of iron ≥ 15 mg/d can decrease DNA damage in young subjects.

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.

Length of treatment and dose as determinants of mutagenicity in sickle cell disease patients treated with hydroxyurea.

Hydroxyurea (HU) is an antineoplastic drug widely used in the clinical management of patients with sickle cell disease (SCD), and many questions related with its use remain unresolved. Given the severity of SCD, HU benefits, although not thoroughly confirmed, seem to outweigh its potential carcinogenicity. This study aimed to assess the genotoxicity associated with HU dose and treatment length by evaluating mutagenicity in patients with SCD treated with HU (SCHU) using the cytokinesis-block micronucleus assay (CBMN) in white cells. The study was conducted with 35 individuals in the SCHU group and 34 controls matched according to age, sex and smoking habit. CBMN results showed an increase (p=0.032) in the number of micronuclei (MN), but not of nucleoplasmic bridges (NPB) or nuclear buds (NBUD) in the SCHU group. The increased frequency of MN in the SCHU group was significantly correlated with treatment length and final HU dose, which confirms that patients with SCD treated with HU should be carefully monitored to reduce the risk of carcinogenicity.

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.