DNA damage as a potential marker in the clinical follow-up of female patients with treated cancer / Daño del ADN como marcador potencial en el seguimiento clínico de pacientes femeninas con cáncer tratado

Abstract Objective: To identify individual differences in the basal damage (DB) of peripheral leukocyte DNA from women with cancer in remission. Methods: Cross-sectional analytical study in which 24 women with cancer in remission from different locations and 24 supposedly healthy women participated. The alkaline comet assay and the neutral variant were used to determine single-stranded breaks (DB-A), and double-stranded DNA breaks (DB-N), respectively. Results: Although there were no differences between the mean values of DNA damage in patients and controls (DB-N: p = 0.43 and DB-A: p = 0.13), 41.6% of the patients presented an increase of one type or another of DNA breaks, with respect to the corresponding cut-off points of the control women. DB-N was correlated with increasing age (r2 = 0.1833; r = 0.4281; p = 0.036) in the patients. DB-A was elevated in those who had received anticancer combination therapy (p = 0.024) and in those who were undergoing treatment with tamoxifen (p = 0.033); while it was decreased in those that consumed antioxidants (p = 0.006) and in those that combined tamoxifen and antioxidants (p = 0.020). Conclusions: Individual differences were identified in both types of DNA strand breaks that are of medical interest in the studied patients. Baseline DNA damage determined by comet assay is a potential tool in the clinical follow-up of cancer patients in remission.

Resumen Objetivo: Identificar diferencias individuales en el daño basal (DB) del ADN de leucocitos periféricos de mujeres con cáncer en remisión. Métodos: Estudio analítico de corte transversal en el que participaron 24 mujeres con cáncer en remisión de diferentes localizaciones y 24 mujeres supuestamente sanas. Se utilizó el ensayo cometa alcalino y la variante neutral para determinar roturas de simple hebra (DB-A), y roturas de doble hebra del ADN (DB-N), respectivamente. Resultados: Aunque no hubo diferencias entre los valores medios del daño del ADN de pacientes y controles (DB-N: p=0,43 y DB-A: p=0,13), el 41,6% de las pacientes presentó aumento de un tipo u otro de roturas del ADN, respecto a los correspondientes puntos de corte de las mujeres controles. El DB-N estuvo correlacionado con el incremento de la edad (r2 = 0,1833; r = 0,4281; p = 0,036) en las pacientes. El DB-A estuvo elevado en aquellas que habían recibido politerapia anticáncer (p = 0,024) y en las que estaban realizando tratamiento con tamoxifeno (p=0,033); mientras estuvo disminuido en las que consumieron antioxidantes (p=0,006) y en las que combinaron tamoxifeno y antioxidantes (p=0,020). Conclusiones: Se identificaron diferencias individuales en ambos tipos de roturas de hebra del ADN que resultan de interés médico en las pacientes estudiadas. El daño basal del ADN determinado por ensayo cometa es una herramienta potencial en el seguimiento clínico de pacientes con cáncer en remisión.

Genotoxicity assessment of three reference chemicals in rat hepatocytes by alkaline comet assay / 上海预防医学

Objective:To establish a comet test method for detection of genotoxicity of three reference chemicals in rat liver cells. Methods:6-10 week old Sprague Dawley rats were randomly divided into 4 groups， with normal saline （0.9% NaCl solution） as negative control group. Animals in three test groups were treated， respectively， with ethyl methanesulfonate （EMS） 200 mg/kg， N-methyl-N-nitrosourea （MNU） 50 mg/kg， and D-mannitol 2 000 mg/kg. There were 10 animals in each group， 5 males and 5 females. The animals received two times （21 h interval） of test compounds through intragastric administration， and their clinical symptoms and body weight changes were recorded during the experiment. The rats were sacrificed 3 h after the last exposure. The liver was weighed， then used to prepare single-cell suspensions for the alkaline comet test which determines the average tail DNA content percentage （DNA%） of hepatocytes and other comet indicators. Results:（1） D-mannitol， EMS and MNU did not show significant toxicity in the whole animal. （2） The mean values of tail DNA content percentage （DNA%） of rat hepatocytes in EMS ［（60.07±24.69）%］ and MNU ［（41.66±22.35）%］ groups were higher than that in the negative control group ［（2.32±1.39）%］ and the difference was statistically significant （P<0.05）. The difference between D-mannitol group ［（3.06±3.30）%］ and the negative control group was not significant （P>0.05）. Conclusion:This laboratory has established a comet test method using hepatocytes from treated rats. Among three testing chemicals， EMS and MNU have displayed genotoxicity by this assay， but no genotoxicity was observed in D-mannitol treated animals.

Study of Alkaline Comet Assay at Various Tissues in SD Rats with Intragastric Administration of Procarbazine Hydrochloride and Ethyl Carbamate / 中国药房

OBJECTIVE： To evaluate the DNA damage response of procarbazine hydrochloride （PCZ） and ethyl carbamate （EC） to different tissues in rats by performing alkaline comet assay， to validate the feasibility of alkaline comet assay of various tissues. METHODS： Totally 30 SD rats were randomly divided into 6 groups according to body weight， with 5 rats in each group， such as negative control group （hyperpure water）， PCZ 75 mg/kg group， PCZ 150 mg/kg group， EC 400 mg/kg group， EC 800 mg/kg group， positive control group （N-ethyl-N-nitrosourea 40 mg/kg）. Those rats were given relevant medicine intragasttrically for 4 d； clinical symptoms of rats were observed and body weight was recorded during experiment. Within 3 h after last medication， the rats were sacrificed； liver， renal and lung weight were weighed； liver， kidney， lung and peripheral blood lymphocytes were collected. The single cell suspension was prepared to perform alkaline comet assay. After lysis， unwind， electrophoresis and dying， tail DNA％ and tail distance of samples were analyzed by Komet 6.0 software. RESULTS： Compared with negative control group， body weight， liver and renal weight of rats were decreased significantly in PCZ 75 mg/kg group， PCZ 150 mg/kg group and positive control group 4 d after medication （P＜0.05 or P＜0.01）. Body weight of rats were decreased significantly in EC 800    mg/kg 4 d after medication （P＜0.05 or P＜0.01）； there was no statistical significance （P＞0.05）. Compared with negative control group， tail DNA％ and tail distance in liver， kidney and peripheral blood lymphocytes were increased significantly in PCZ 75    mg/kg group， PCZ 150 mg/kg group and positive control group （P＜0.05 or P＜0.01）； PCZ showed more significant effects on liver and lung. Tail DNA％ and tail distance of liver， kidney and peripheral blood lymphocytes were increased significantly in EC 800 mg/kg group （P＜0.05 or P＜0.01）， and tail DNA％ and tail distance of renal tissue was increased significantly in EC 400   mg/kg group （P＜0.05）. CONCLUSIONS： PCZ induced stronger DNA damage； liver and lung are the major genotoxicity target of PCZ. EC-induced DNA damage is relatively weak， and kidney is the most sensitive organ for EC-induced genotoxicity.

Hepatocytes, rather than leukocytes reverse DNA damage in vivo induced by whole body y-irradiation of mice, as shown by the alkaline comet assay

DNA damage repair was assessed in quiescent (G0) leukocytes and in hepatocytes of mice, after 1 and 2 hours recovery from a single whole body y-irradiation with 0.5, 1 or 2 Gy. Evaluation of single-strand breaks (SSB) and alkali-labile sites together were carried out by a single-cell electrophoresis at pH>13.0 (alkaline comet assay). In non-irradiated (control) mice, the constitutive, endogenous DNA damage (basal) was around 1.5 times higher in leukocytes than in hepatocytes. Irradiation immediately increased SSB frequency in both cell types, in a dose-dependent manner. Two sequential phases took place during the in vivo repair of the radio-induced DNA lesions. The earliest one, present in both hepatocytes and leukocytes, further increased the SSB frequency, making evident the processing of some primary lesions in DNA bases into the SSB repair intermediates. In a second phase, SSB frequency decreased because of their removal. In hepatocytes, such a frequency regressed to the constitutive basal level after 2 hours recovery from either 0.5 orí Gy. On the other hand, the SSB repair phase was specifically abrogated in leukocytes, at the doses and recovery times analyzed. Thus, the efficiency of in vivo repair of radio-induced DNA damage in dormant cells (lymphocytes) is quite different from that in hepatocytes whose low proliferation activity accounts only for cell renewal.

Association between Oxidative DNA Damage, Fruits and Vegetables Intake with Breast Cancer: A Preliminary Study in Klang Valley / Jurnal Sains Kesihatan Malaysia

A preliminary study was conducted to determine the level of oxidative DNA damage, fruits and vegetables intake among 50 breast cancer patients (cases) as compared to 50 healthy women (controls) with no known medical history of breast cancer in Klang Valley. Both groups were matched for age and ethnicity. Data on socio-demographic, health status and medical history, fruits and vegetables intake, and supplements intake were obtained through an interviewbased questionnaire. Anthropometry measurements included weight, height, and waist and hip circumference were also carried out on subjects. A total of 3mL fasting venous blood was drawn to assess lymphocytes oxidative DNA damage using Alkaline Comet Assay. Results indicated that the mean intake of fruits and vegetables was lower in cases (4.09 ± 1.17 servings/d) than controls (4.77 ± 0.90 servings/d)(p < 0.05) The intake of fruits and vegetables from family groups of solanaceae, myrtaceae, caricaceae, apiaceae, brinjal, rutaceae, broccoli, orange, carrot, watermelon were 0.5 - 1 servings/week significantly higher among controls as compared to cases (p < 0.05 for all parameters). However, the intake of fruits from rosaceae family and apple was higher among controls than cases (p < 0.05). The estimated intake of β-carotene, carotenoids, vitamin A, vitamin C (p < 0.001), α-carotene and lycopene (p < 0.05) from fruits and vegetables were higher among controls than cases. Mean DNA damage level of cases (4.55 ± 1.78 % DNA in tail, %TD; 0.35 ± 0.21 tail moment, TM) were 3.5 and 3.9 times higher than the value of controls (1.3 ± 0.70% TD; 0.09 ± 0.09 TM) (p < 0.001) and the damage increased with higher values of waist hip ratio (% TD, r = 0.396, p < 0.05; TM, r = 0.349, p < 0.05) and waist circumference (% TD, r = 0.334, p < 0.05; TM, r = 0.360, p < 0.05). There was an inverse relationship between oxidative DNA damage with intake of total fruits and vegetables, cauliflowers and water convolvulus and also consumption from rutaceae and solanaceae families. Similar trend was noted for estimated intake of vitamin A, carotenoids, vitamin C, β-carotene and lycopene. In conclusion, the intake of fruits and vegetables of five servings/d and the consumption of specific families and types of fruits and vegetables might protect against oxidative DNA damage and further reduce breast cancer risk.

Aplicación de dos biomarcadores para el análisis de lesiones en el DNA de bivalvos marinos

El presente trabajo describe la estandarización de las técnicas del Test de Micronúcleo (MN) y Ensayo Cometa para la evaluación del daño en el DNA de bivalvos marinos. La importancia de estas técnicas radica en que permiten evaluar la respuesta temprana al estrés ocasionado por agentes contaminantes y cambios ambientales. Tejidos branquiales de Semimytilus algosus y Aulacomya ater fueron utilizados para tratamientos in vivo e in vitro. Los tejidos fueron expuestos a los agentes mutagénicos Mitomicina C en concentraciones de 0,02; 0,04 y 0,06 x 10-6 M y H2O2 en una concentración de 100 mM con tiempos de evaluación de 1, 3, 6, 24 y 48 h. Diferentes soluciones para la colecta del tejido y aislamiento celular fueron evaluados obteniendo una mayor viabilidad celular en la solución salina CMFS, pH 7,3; en frío. El Test de Micronúcleo se modificó en la forma de obtener el disgregado celular, realizando la hipotonización en Citrato de sodio 0,9%, fijación en metanol por segundos posterior al frotis y la tinción con Giemsa 2%. La estandarización del Ensayo Cometa Alcalino, descrita por Wilson et al. (1998), se realizó modificando la suspensión celular en agarosa LMP (1% en solución Kenny, pH 7,5), la exposición a solución de lisis (pH 10 con DMSO y Tritón) con variaciones en el tiempo de 1 a 16 h, y la electroforesis en solución alcalina pH 13,7 y tinción con Bromuro de etidio y Nitrato de plata para geles de agarosa. Los cambios realizados en ambas técnicas permitieron obtener un alto número celular con morfología definida, observándose macrolesiones como la formación de MN y aberraciones nucleares, y la determinación cualitativa de microlesiones observadas por fragmentación y migración del DNA.

This work describes the standardization of Micronucleus Assay (MN) and Comet Assay techniques used for the evaluation of the DNA damage on marine bivalves. These techniques are important because they can evaluate the early stress response caused by pollutant agents and environmental changes. Branchial tissues of Semimytilus algosus and Aulacomya ater were used for in vivo and in vitro treatments. Tissues were exposed to the mutagenetic agents Mitomicine C in concentrations of 0,02; 0,04 and 0,06 x 10-6 M and H2O2 in concentration of 100 mM evaluated at 1, 3, 6, 24 and 48 h. Different solutions for the tissue collection and cell isolation were evaluated, obtaining more cell viability with cold CMFS pH 7,3 saline solution. Micronucleus Assay were modified in the obtaining of suspension cell, we used 0,9% Sodium citrate hipotonic solution, fixation in methanol for seconds after the spread and dyed with 2% Giemsa. The Alkaline Comet Assay protocol described by Wilson et al. (1998) was standardized modifying the single-cell suspension embedded in LMP Agarose (1% in Kenny solution, pH 7,5), the exposure at lysing solution (pH 10 with DMSO and Triton) with changes from 1 to 16 h, electrophoresis in alkaline solution pH 13,7 and dyed with Ethidium bromide and Silver nitrate for agarose gels. Changes made at both techniques allowed us to obtain a high cell number with a defined morphology, recognizing macrolessions like the MN formation and nuclear aberrations, and the qualitative determination of microlessions observed by the fragmentation and migration of the DNA.

Oxidative Stress of Mouse Fed with gamma -Irradiated Soybean Diet

Food irradiation has been steadily increased in many countries concomitantly with increasing international trades. Harmful contaminants naturally occurred from foods which contain high levels of unsaturated fatty acids that are easily oxidized can affect the human anti-oxidation system through the generation of free radicals. Moreover, previous studies proved that gamma-irradiation may cause production of free radicals in food. We investigated the effect of gamma-irradiated soybeans in relation to oxidative stress in mice. Oxidative index of mice was evaluated by TBARS, DNA fragmentation in various organs such as blood lymphocytes, liver and kidney. Forty male ICR mice were equally divided into 4 groups and fed control diet or gamma-irradiated diet containing 50% soybeans (5, 10, and 20 kGy, respectively )for 8 weeks. Pero-xide values of the irradiated diets were higher than that of the non-irradiated one and increased according to the storage period. There was no significant difference in weight gain as well as in TBARS value in plasma and kidney of all groups. Liver TBARS value of the group fed with irradiated diet at 20 kGy increased significantly compared with the control group (p <0.05 ). DNA oxidative damage as measured by alkaline comet assay showed that % tail DNA in the blood lymphocytes of 5 kGy and 10 kGy groups increased significantly over the control group (p <0.05 ). Also, tail moments of 5 kGy and 10 kGy groups were higher than that of the control group. Ultrastructural examination shows myeline figures and swollen mitochondria in parietal and intestinal epithelial cells of the group fed with irradiated diet. Therefore, considering unsaturated fatty acid content, consumption of soybeans gamma-irradiated with over 20 kGy or repe-atedly may decrease the body's antioxidant mechanism.

Oxidative Stress of Mouse Fed Irradiated Diet Containing High Unsaturated Fatty Acid

In this study, we investigated the in vivo effect by intake of the irradiated foods such as mackerel and sesame seed which are high in unsaturated fatty acid through TBARS (thiobarbituric acid reactive subtance) and the tissue pathological and genotoxicological test. Thirty two ICR mice are divided into four groups, one non-irradiated (control) group and three irradiated (5, 10, 20 kGy, respectively) groups. Sesame seed and pulverized mackerel in modified AIN93M diet were mixed together then divided into four identical parts. Three parts of them were irradiated by doses of 5, 10, and 20 kGy. These experimental diet were fed to each group for 4, 8 and 16 weeks. The results of the study were as follows: No significant differences in weight gain were found in each group. Peroxide value of the irradiated diet was higher than that of the non-radiated one and also increased according to the storage period. TBA values in plasma, liver, kidney and Peyer's patch were not significantly different among 4 groups. DNA% in tail, tail length (TL) and tail moment (TM) values of the blood lymphocyte in 4, 8 and 16 week groups and the liver in 16 week group were much measured over the control. DNA% in tail of kidney of 8 week group was significantly larger than the control and TL and TM of 8 week and TM of 16 week groups showed a tendency of higher values. By Peyer's patch, DNA% in tail of 8 week group, DNA% in tail and TM of 16 week groups increased significantly over the control. Ultrastructural examination shows myeline figures and swollen mitochondria in parietal cells and intestinal epithelial cells of 8 and 16 weeks groups. After this study, we need further investigations on the safety of highly consumed foods which contai high contents of unsaturated fatty acid, largely imported and which are possible to be irradiated.

Plasma, Tissue Thiobarbituric Acid Reactive Substance and Lymphocyte Oxidative DNA Damage in Mouse Fed Gamma Irradiated Diet

Food irradiation has been steadily increasing in many countries in line with increasing international trade and concerns about naturally occurring harmful contaminants in food. Although irradiation provides an excellent safeguard for the consumer by destroying almost 100% of harmful bacteria, it is necessary to ensure the safety of irradiated foods. This study was performed to investigate the effect of an irradiated diet on lipid peroxidation in the plasma, liver, small intestinal mucosa, and lymphocyte DNA damage in mice. Eight-week old ICR mice were assigned to two groups to receive either non-irradiated or irradiated (10 kGy) diets containing 20.38% fish powder and 6.06% sesame seeds for 4 weeks. The resulting changes in the degrees of lipid peroxidation were evaluated based on the level of plasma and liver thiobarbituric acid reactive substance (TBARS), transmission electron micrograph of jejunal mucosa, and free radical-induced oxidative DNA damage in lymphocytes, as measured by alkaline comet assay (single cell gel electrophoresis). The peroxide values of the gamma irradiated diet were measured every week, and the sample for comet assay was taken at the end of the four week experimental period. There was no significant difference in food efficiency ratio between the two groups. The peroxide values of the diet were immediately increased to 35.5% after gamma irradiation and kept on increasing during storage. After 4 weeks, no differences in tissue or plasma TBARS value were observed between the two groups, but epithelial cells of jejumum showed osmiophillic laminated membranous structures, considered as myelin figures,. The oxidative DNA damage expressed as tail moment (TM) increased 30% in the blood lymphocytes of the mice fed the irradiated diet. In conclusion, the comet assay sensitively detected differences in lymphocyte DNA damage after feeding with the irradiated diet for 4 weeks. However, in order to ensure the safety of irradiated foods, it would be more useful to conduct a long-term feeding regimen using an irradiated diet and examine the level of lipid peroxidation and the state of oxidative stress in a greater range of organs.