Evaluation of hydroxyurea genotoxicity in patients with sickle cell disease.

OBJECTIVE: To evaluate the induction of DNA damage in peripheral blood mononuclear cells of patients with sickle cell disease, SS and SC genotypes, treated with hydroxyurea. METHODS: The study subjects were divided into two groups: one group of 22 patients with sickle cell disease, SS and SC genotypes, treated with hydroxyurea, and a Control Group composed of 24 patients with sickle cell disease who were not treated with hydroxyurea. Peripheral blood samples were submitted to peripheral blood mononuclear cell isolation to assess genotoxicity by the cytokinesis-block micronucleus cytome assay, in which DNA damage biomarkers - micronuclei, nucleoplasmic bridges and nuclear buds - were counted. RESULTS: Patients with sickle cell disease treated with hydroxyurea had a mean age of 25.4 years, whereas patients with sickle cell disease not treated with hydroxyurea had a mean age of 17.6 years. The mean dose of hydroxyurea used by the patients was 12.8mg/kg/day, for a mean period of 44 months. The mean micronucleus frequency per 1,000 cells of 8.591±1.568 was observed in the Hydroxyurea Group and 10.040±1.003 in the Control Group. The mean frequency of nucleoplasmic bridges per 1,000 cells and nuclear buds per 1,000 cells for the hydroxyurea and Control Groups were 0.4545±0.1707 versus 0.5833±0.2078, and 0.8182±0.2430 versus 0.9583±0.1853, respectively. There was no statistically significant difference between groups. CONCLUSION: In the study population, patients with sickle cell disease treated with the standard dose of hydroxyurea treatment did not show evidence of DNA damage induction.

Vitamin D supplementation alters the expression of genes associated with hypertension and did not induce DNA damage in rats.

Vitamin D3 deficiency has been correlated with altered expression of genes associated with increased blood pressure (BP); however, the role of vitamin D3 supplementation in the genetic mechanisms underlying hypertension remains unclear. Thus, the aim of this study was investigate the consequences of vitamin D3 supplemented (10,000 IU/kg) or deficient (0 IU/kg) diets on regulation of expression of genes related to hypertension pathways in heart cells of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) controls. An additional aim was to assess the impact of vitamin D3 on DNA damage and oxidative stress markers. The gene expression profiles were determined by PCR array, DNA damage was assessed by an alkaline comet assay, and oxidative stress markers by measurement of thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) levels. In SHR rats data showed that the groups of genes most differentially affected by supplemented and deficient diets were involved in BP regulation and renin-angiotensin system. In normotensive WKY controls, the profile of gene expression was similar between the two diets. SHR rats were more sensitive to changes in gene expression induced by dietary vitamin D3 than normotensive WKY animals. In addition to gene expression profile, vitamin D3 supplemented diet did not markedly affect DNA or levels of TBARS and GSH levels in both experimental groups. Vitamin D3 deficient diet produced lipid peroxidation in SHR rats. The results of this study contribute to a better understanding of the role of vitamin D3 in the genetic mechanisms underlying hypertension. Abbreviations: AIN, American Institute of Nutrition; EDTA, disodium ethylenediaminetetraacetic acid; GSH, glutathione; PBS, phosphate buffer solution; SHR, spontaneously hypertensive rats; TBARS, thiobarbituric acid reactive substances; WKY, Wistar Kyoto.

Evaluation of hydroxyurea genotoxicity in patients with sickle cell disease / Avaliação da indução de genotoxicidade pela hidroxiureia em pacientes com doença falciforme

ABSTRACT Objective To evaluate the induction of DNA damage in peripheral blood mononuclear cells of patients with sickle cell disease, SS and SC genotypes, treated with hydroxyurea. Methods The study subjects were divided into two groups: one group of 22 patients with sickle cell disease, SS and SC genotypes, treated with hydroxyurea, and a Control Group composed of 24 patients with sickle cell disease who were not treated with hydroxyurea. Peripheral blood samples were submitted to peripheral blood mononuclear cell isolation to assess genotoxicity by the cytokinesis-block micronucleus cytome assay, in which DNA damage biomarkers - micronuclei, nucleoplasmic bridges and nuclear buds - were counted. Results Patients with sickle cell disease treated with hydroxyurea had a mean age of 25.4 years, whereas patients with sickle cell disease not treated with hydroxyurea had a mean age of 17.6 years. The mean dose of hydroxyurea used by the patients was 12.8mg/kg/day, for a mean period of 44 months. The mean micronucleus frequency per 1,000 cells of 8.591±1.568 was observed in the Hydroxyurea Group and 10.040±1.003 in the Control Group. The mean frequency of nucleoplasmic bridges per 1,000 cells and nuclear buds per 1,000 cells for the hydroxyurea and Control Groups were 0.4545±0.1707 versus 0.5833±0.2078, and 0.8182±0.2430 versus 0.9583±0.1853, respectively. There was no statistically significant difference between groups. Conclusion In the study population, patients with sickle cell disease treated with the standard dose of hydroxyurea treatment did not show evidence of DNA damage induction.

RESUMO Objetivo Avaliar o efeito da indução de danos ao DNA em células monocelulares do sangue periférico de pacientes com doença falciforme, genótipos SS e SC, tratados com hidroxiureia. Métodos Os sujeitos da pesquisa foram divididos em dois grupos: um de 22 pacientes com doença falciforme genótipos SS e SC tratados com hidroxiureia, e o outro controle, composto por 24 pacientes com doença falciforme que não eram tratados com o fármaco. As amostras de sangue periférico foram submetidas ao isolamento de células mononucleares do sangue periférico para avaliação da genotoxicidade pelo ensaio de micronúcleo citoma com bloqueio da citocinese, tendo sido quantificados os biomarcadores de danos ao DNA - micronúcleos, pontes nucleoplasmáticas e brotamento nuclear. Resultados Os pacientes com doença falciforme tratados com hidroxiureia apresentaram média de idade de 25,4 anos, enquanto aqueles com doença falciforme não tratados com hidroxiureia tiveram média de idade de 17,6 anos. A dose média de hidroxiureia utilizada pelos pacientes foi de 12,8mg/kg/dia, por período médio de 44 meses. A frequência média de micronúcleos por 1.000 células de 8,591±1,568 foi observada no Grupo Hidroxiureia e de 10,040±1,003 no Grupo Controle. Adicionalmente, a frequência média de pontes nucleoplasmáticas por 1.000 células e brotamento nuclear por 1.000 células para o Grupo Hidroxiureia e Controle foi de 0,4545±0,1707 versus 0,5833±0,2078, e de 0,8182±0,2430 versus 0,9583±0,1853, respectivamente. Não houve diferença estatisticamente significativa entre os grupos. Conclusão Na população estudada de pacientes com doença falciforme com tratamento em dose padrão de hidroxiureia, não houve evidência de indução de danos ao DNA.

Methionine-supplemented diet affects the expression of cardiovascular disease-related genes and increases inflammatory cytokines in mice heart and liver.

Some important environmental factors that influence the development of cardiovascular diseases (CVD) include tobacco, excess alcohol, and unhealthy diet. Methionine obtained from the diet participates in the synthesis of DNA, proteins, lipids and affects homocysteine levels, which is associated with the elevated risk for CVD development. Therefore, the aim of this study was to investigate the manner in which dietary methionine might affect cellular mechanisms underlying CVD occurrence. Swiss albino mice were fed either control (0.3% DL-methionine), methionine-supplemented (2% DL-methionine), or a methionine-deprived diet (0% DL-methionine) over a 10-week period. The parameters measured included plasma homocysteine concentrations, oxidative stress by reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, levels of inflammatory cytokines IL-1ß, TNF-α, and IL-6, as well as expression of genes associated with CVD. The levels of apolipoprotein A5 (APOA5), a regulator of plasma triglycerides, were measured. The methionine-supplemented diet increased oxidative stress by lowering the GSH/GSSG ratio in heart tissues and decreased expression of the genes Apob, Ctgf, Serpinb2, Spp1, Il1b, and Sell, but elevated expression of Thbs4, Tgfb2, Ccr1, and Vegfa. Methionine-deprived diet reduced expression of Col3a1, Cdh5, Fabp3, Bax, and Hbegf and increased expression of Sell, Ccl5, Itga2, Birc3, Msr1, Bcl2a1a, Il1r2, and Selp. Methionine-deprived diet exerted pro-inflammatory consequences as evidenced by elevated levels of cytokines IL-1ß, TNF-α, and IL-6 noted in liver. Methionine-supplemented diet increased hepatic IL-6 and cardiac TNF-α. Both methionine supplementation and deprivation lowered hepatic levels of APOA5. In conclusion, data demonstrated that a methionine-supplemented diet modulated important biological processes associated with high risk of CVD development.

Vitamin D3 deficiency increases DNA damage and the oxidative burst of neutrophils in a hypertensive rat model.

Deficiency of vitamin D3, a lipophilic micronutrient, plays a role in the development of some chronic diseases. Vitamin D3 deficiency affects 25-50% of the human population and has been associated with increased risk for development of hypertension. DNA damage induced by reactive oxygen species (ROS) occurs more often in hypertensive than in normotensive individuals, and vitamin D3 status can influence this relationship. The aim of this study was to evaluate whether a diet supplemented with (10,000 IU/kg) or deficient in (0 IU/kg) vitamin D3, compared to a vitamin D3 control diet (1000 IU/kg), would modulate DNA damage and ROS production in spontaneously hypertensive rats (SHR) and normotensive control Wistar-Kyoto (WKY) rats after 12 weeks of treatment. ROS production was assessed by measuring the oxidative burst of neutrophils. DNA damage was evaluated using the comet assay in peripheral blood and the micronucleus test in bone marrow and peripheral blood. Vitamin D3 supplementation did not induce DNA damage and did not change neutrophil ROS production in SHR and WKY rats. Vitamin D3 deficiency induced neutrophil ROS production and a high frequency of micronucleus formation in the bone marrow and peripheral blood of SHR rats only, and induced DNA damage (comet) in peripheral blood of both SHR and WKY rats. In conclusion, vitamin D3 deficiency showed a more pronounced effect on hypertensive animals. Population studies are needed to test whether this relationship also exists in humans.

Comparative study of curcumin and curcumin formulated in a solid dispersion: Evaluation of their antigenotoxic effects.

Curcumin (CMN) is the principal active component derived from the rhizome of Curcuma longa (Curcuma longa L.). It is a liposoluble polyphenolic compound that possesses great therapeutic potential. Its clinical application is, however, limited by the low concentrations detected following oral administration. One key strategy for improving the solubility and bioavailability of poorly water-soluble drugs is solid dispersion, though it is not known whether this technique might influence the pharmacological effects of CMN. Thus, in this study, we aimed to evaluate the antioxidant and antigenotoxic effects of CMN formulated in a solid dispersion (CMN SD) compared to unmodified CMN delivered to Wistar rats. Cisplatin (cDDP) was used as the damage-inducing agent in these evaluations. The comet assay results showed that CMN SD was not able to reduce the formation of cDDP-DNA crosslinks, but it decreased the formation of micronuclei induced by cDDP and attenuated cDDP-induced oxidative stress. Furthermore, at a dose of 50 mg/kg b.w. both CMN SD and unmodified CMN increased the expression of Tp53 mRNA. Our results showed that CMN SD did not alter the antigenotoxic effects observed for unmodified CMN and showed effects similar to those of unmodified CMN for all of the parameters evaluated. In conclusion, CMN SD maintained the protective effects of unmodified CMN with the advantage of being chemically water soluble, with maximization of absorption in the gastrointestinal tract. Thus, the optimization of the physical and chemical properties of CMN SD may increase the potential for the therapeutic use of curcumin.