RESUMO
OBJECTIVES: This study aimed to determine the relationship between oxidative stress, DNA damage, inflammation, and metabolic biomarkers as the mediating factor between Islamic Sunnah intermittent fasting (IF) practice and cognitive function among older adults with mild cognitive impairment (MCI). DESIGN: This study was a 36 months prospective cohort study. SETTING: Community-dwelling older participants recruited through a stratified random sampling method from four states representing Malaysia's central, north-west, northeast and southern regions. PARTICIPANTS: Ninety-nine Malay Muslim older adults (n= 99) aged 60 and above with MCI and no known critical illnesses were included in the current analysis. The participants were divided into regularly practicing IF (r-IF), irregularly practicing IF (i-IF) and not practicing IF (n-IF) groups. MEASUREMENTS: Fasting venous blood was collected and used to determine the levels of oxidative stress, DNA damage, inflammatory and metabolic biomarkers. Mini-Mental State Examination, Montreal Cognitive Assessment, Rey Auditory Verbal Learning Test, Digit Span and Digit symbol were used to evaluate the cognitive function. Then, the mediation analysis was conducted using a multistep regression model to determine the mediating role of various biomarkers between IF practice and cognitive function. RESULTS: When comparing the r-IF and n-IF groups, higher SOD activity, lower DNA damage (percentage of DNA in tail), lower CRP levels and higher HDL-cholesterol levels established partial mediation while lower insulin levels established complete mediation between IF practice and better cognitive function. Meanwhile, when comparing the r-IF and i-IF groups, higher SOD activity and lower CRP levels completely mediated the effects of IF practice on better cognitive function. CONCLUSION: It can be concluded that changes in antioxidant function, DNA damage, inflammation and a limited set of metabolic biomarkers (insulin and HDL cholesterol) may mediate improvements in cognitive function among older participants with MCI who practice Islamic Sunnah IF.
Assuntos
Disfunção Cognitiva , Insulinas , Idoso , Antioxidantes , Biomarcadores , Cognição , Dano ao DNA , Jejum , Controle Glicêmico , Humanos , Inflamação , Islamismo , Metabolismo dos Lipídeos , Análise de Mediação , Estudos Prospectivos , Superóxido DismutaseRESUMO
Benzene is a known hematotoxic and leukemogenic agent with hematopoietic stem cells (HSCs) niche being the potential target. Occupational and environmental exposure to benzene has been linked to the incidences of hematological disorders and malignancies. Previous studies have shown that benzene may act via multiple modes of action targeting HSCs niche, which include induction of chromosomal and micro RNA aberrations, leading to genetic and epigenetic modification of stem cells and probable carcinogenesis. However, understanding the mechanism linking benzene to the HSCs niche dysregulation is challenging due to complexity of its microenvironment. The niche is known to comprise of cell populations accounted for HSCs and their committed progenitors of lymphoid, erythroid, and myeloid lineages. Thus, it is fundamental to address novel approaches via lineage-directed strategy to elucidate precise mechanism involved in benzene-induced toxicity targeting HSCs and progenitors of different lineages. Here, we review the key genetic and epigenetic factors that mediate hematotoxicological effects by benzene and its metabolites in targeting HSCs niche. Overall, the use of combined genetic, epigenetic, and lineage-directed strategies targeting the HSCs niche is fundamental to uncover the key mechanisms in benzene-induced hematological disorders and malignancies.
Assuntos
Benzeno/toxicidade , Carcinógenos/toxicidade , Células-Tronco Hematopoéticas/efeitos dos fármacos , Neoplasias/induzido quimicamente , Animais , Benzeno/farmacocinética , Carcinógenos/farmacocinética , Epigênese Genética , Hematopoese/efeitos dos fármacos , Humanos , Neoplasias/genética , Nicho de Células-Tronco/efeitos dos fármacosRESUMO
Malaysian locally processed raw food products are widely used as main ingredients in local cooking. Previous studies showed that these food products have a positive correlation with the incidence of cancer. The cytotoxicity effect was evaluated using MTT assay (3-(4,5-dimetil-2-thiazolil)-2,5-diphenyl-2H-tetrazolium bromide) against Chang liver cells at 2000 microg/ml following 72 h incubation. Findings showed all methanol extracts caused a tremendous drop in the percentage of cell viability at 2000 microg/ml (shrimp paste - 41.69+/-3.36%, salted fish - 37.2+/-1.06%, dried shrimp - 40.32+/-1.8%, p<0.05). To detect DNA damage in a single cell, alkaline Comet Assay was used. None of the extracts caused DNA damage to the Chang liver cells at 62.5 microg/ml following 24 h incubation, as compared to the positive control, hydrogen peroxide (tail moment - 9.50+/-1.50; tail intensity - 30.50+/-2.50). Proximate analysis which was used for the evaluation of macronutrients in food showed that shrimp paste did not comply with the protein requirement (<25%) as in Food Act 1983. Salt was found in every sample with the highest percentage being detected in shrimp paste which exceeded 20%. Following heavy metal analysis (arsenic, cadmium, lead and mercury), arsenic was found in every sample with dried shrimps showing the highest value as compared to the other samples (6.16 mg/kg). In conclusion, several food extracts showed cytotoxic effect but did not cause DNA damage against Chang liver cells. Salt was found as the main additive and arsenic was present in every sample, which could be the probable cause of the toxicity effects observed.
Assuntos
Citotoxinas/toxicidade , Alimentos/toxicidade , Mutagênicos/toxicidade , Alimentos Marinhos/toxicidade , Análise de Variância , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Dano ao DNA , Fermentação , Peixes , Análise de Alimentos , Contaminação de Alimentos/análise , Conservantes de Alimentos/análise , Conservantes de Alimentos/toxicidade , Hepatócitos/efeitos dos fármacos , Humanos , Fígado/citologia , Fígado/metabolismo , Malásia , Metais Pesados/análise , Metais Pesados/toxicidade , Alimentos Marinhos/análise , Sais de Tetrazólio , TiazóisRESUMO
BACKGROUND: A cross sectional study was conducted in a group of 317 subjects older than 60 in Malaysia, aimed to determine risk factors associated with cognitive impairment in older adults, focusing on trace elements and DNA damage. METHOD: Cognitive decline was determined by Montreal Cognitive Assessment (MoCA). Oxidative stress markers (malondialdehyde-MDA and superoxide dismutase-SOD) were determined and DNA damage was assayed using Alkaline Comet Assay. Toenail samples were taken and analyzed using ICP-MS to determine trace element levels. RESULTS: A total of 62.1 % of subjects had cognitive impairment. Subjects with cognitive impairment had significantly higher levels of MDA and DNA damage as compared to the group with normal cognitive function; MDA (2.07 ± 0.05 nmol/L vs 1.85 ± 0.06 nmol/L) (p<0.05) and DNA damage (% Tail Density, 14.52 ± 0.32 vs 10.31 ± 0.42; Tail Moment, 1.79 ± 0.06 vs 1.28 ± 0.06) (p<0.05 for all parameters). However, the level of SOD among subjects with cognitive impairment (6.67 ± 0.33 u.e/min/mg protein) was lower than the level among those with normal cognitive functions (11.36 ± 0.65 u.e/min/mg protein) (p<0.05). Multiple logistic regression revealed the predictors for cognitive impairment among the subjects were DNA damage (Adjusted odd ratio [OR], 1.37; 95% confidence interval [CI], 1.18-1.59), level of trace elements in toenails namely, lead (OR, 2.471; CI, 1.535-3.980) and copper (OR, 1.275; CI, 1.047-1.552) (p<0.05). CONCLUSION: High levels of lead and copper can lead to increase in oxidative stress levels and are associated with DNA damage that eventually could be associated with cognitive decline.
Assuntos
Cognição/efeitos dos fármacos , Disfunção Cognitiva/etiologia , Cobre/efeitos adversos , Dano ao DNA , Chumbo/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Idoso , Biomarcadores/sangue , Transtornos Cognitivos/etiologia , Ensaio Cometa , Cobre/administração & dosagem , Cobre/metabolismo , Estudos Transversais , Feminino , Humanos , Chumbo/administração & dosagem , Chumbo/metabolismo , Modelos Logísticos , Malásia , Masculino , Malondialdeído/sangue , Pessoa de Meia-Idade , Unhas/metabolismo , Razão de Chances , Fatores de Risco , Superóxido Dismutase/sangue , Oligoelementos/administração & dosagem , Oligoelementos/efeitos adversos , Oligoelementos/metabolismoRESUMO
The essential oils of Litsea elliptica, Piper aduncum, and Piper sarmentosum were prepared as repellents in gel formulation, and their repellent properties against Aedes aegypti were experimentally investigated. The lowest effective doses against adult mosquitoes were 0.8%, 0.5%, and 0.4% for Lit. elliptica, P. sarmentosum and P. aduncum, respectively. In laboratory testing with human subjects, all three gels provided over 90.0% repellency at one hour after application and over 80.0% repellency at four hours, compared with 100% and 95.8% protection after one and four hours, respectively, by DEET. In the field, gels with ED95 concentrations of Lit. elliptica, P. aduncum, and P. sarmentosum essential oils provided 99.3%, 97.5%, and 100% protection, respectively, at two hours. The physical properties and biological stability of the three repellents after storage in hot and cold conditions were also compared. In conclusion, all three gels have the potential for development as repellents against Ae. aegypti.
RESUMO
Restenosis represents a major impediment to the success of coronary angioplasty. Abnormal proliferation of vascular smooth muscle cells (VSMCs) has been shown to be an important process in the pathogenesis of restenosis. A number of agents, particularly rapamycin and paclitaxel, have been shown to impact on this process. This study was carried out to determine the mechanisms of cytotoxicity of goniothalamin (GN) on VSMCs. Results from MTT cytotoxicity assay showed that the IC(50) for GN was 4.4 microg/ml (22 microM), which was lower compared to the clinically used rapamycin (IC(50) of 25 microg/ml [27.346 microM]). This was achieved primarily via apoptosis where up to 25.83 +/- 0.44% of apoptotic cells were detected after 72 h treatment with GN. In addition, GN demonstrated similar effects as rapamycin in inhibiting VSMCs proliferation using bromodeoxyuridine (BrdU) cell proliferation assay after 72 h treatment at IC(50) concentration (p > 0.05). In order to understand the mechanisms of GN, DNA damage detection using comet assay was determined at 2h post-treatment with GN. Our results showed that there was a concentration-dependent increase in DNA damage in VSMCs prior to cytotoxicity. Moreover, GN effects were comparable to rapamycin. In conclusion, our data show that GN initially induces DNA damage which subsequently leads to cytotoxicity primarily via apoptosis in VSMCs.
Assuntos
Apoptose/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Pironas/farmacologia , Bromodesoxiuridina , Divisão Celular , Células Cultivadas , Ensaio Cometa , Humanos , Músculo Liso Vascular/citologiaRESUMO
Cognitive impairments are often related to aging and micronutrient deficiencies. Various essential micronutrients in the diet are involved in age-altered biological functions such as, zinc, copper, iron, and selenium that play pivotal roles either in maintaining and reinforcing the antioxidant performances or in affecting the complex network of genes (nutrigenomic approach) involved in encoding proteins for biological functions. Genomic stability is one of the leading causes of cognitive decline and deficiencies or excess in trace elements are two of the factors relating to it. In this review, we report and discuss the role of micronutrients in cognitive impairment in relation to genomic stability in an aging population. Telomere integrity will also be discussed in relation to aging and cognitive impairment, as well as, the micronutrients related to these events. This review will provide an understanding on how these three aspects can relate with each other and why it is important to keep a homeostasis of micronutrients in relation to healthy aging. Micronutrient deficiencies and aging process can lead to genomic instability.
Assuntos
Transtornos Cognitivos/genética , Transtornos Cognitivos/metabolismo , Instabilidade Genômica/genética , Oligoelementos/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Antioxidantes/metabolismo , Transtornos Cognitivos/etiologia , Dano ao DNA , Dieta , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Nutrigenômica , Estresse Oxidativo , Telômero/genética , Telômero/metabolismo , Oligoelementos/administração & dosagem , Oligoelementos/deficiênciaRESUMO
Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.
Assuntos
Materiais Biocompatíveis/toxicidade , Substitutos Ósseos/toxicidade , Dano ao DNA , Hidroxiapatitas/toxicidade , Testes de Mutagenicidade , Próteses e Implantes , Animais , Sobrevivência Celular/efeitos dos fármacos , Células L , CamundongosRESUMO
Biomaterials intended for end-use application as bone-graft substitutes have to undergo safety evaluation. In this study, we investigated the in vitro cytotoxic effects especially to determine the mode of death of two hydroxyapatite compounds (HA2, HA3) which were synthesized locally. The methods used for cytotoxicity was the standard MTT assay whereas AO/PI staining was performed to determine the mode of cell death in HA treated L929 fibroblasts. Our results demonstrated that both HA2 and HA3 were not significantly cytotoxic as more than 75% cells after 72 hours treatment were viable. Furthermore, we found that the major mode of cell death in HA treated cells was apoptosis. In conclusion, our results demonstrated that these hydroxyapatite compounds are not cytotoxic where the mode of death was primarily via apoptosis.
Assuntos
Materiais Biocompatíveis/toxicidade , Substitutos Ósseos/toxicidade , Morte Celular/efeitos dos fármacos , Durapatita/toxicidade , Próteses e Implantes , Animais , Apoptose/efeitos dos fármacos , Células L , CamundongosRESUMO
Ficus deltoidea is traditionally consumed by Malay woman to augment labour and hastening parturition. This study was to investigate the phytochemical present and uterotonic activity of F. deltoidea var. Deltoidea (FDD) and F. deltoidea var. Angustifolia (FDA) leaves aqueous extract. FDD and FDA were qualitatively analysed. In uterine contraction activity, adult female Sprague Dawley rats were pretreated with 0.2 mg kg(-1) diethylstilbestrol 24 h to induce oestrus phase. The rats then killed and uterine horns were taken out, cut into two centimetres length and put into organ bath that connected to Powerlab instrument. The uterus separately tested with cumulative concentrations of FDD (10-1280 µg mL(-1)), FDA (10-1280 µg mL(-1)), oxytocin (0.02-0.64 µg mL(-1)) and combination of oxytocin (0.08 µg mL(-1)) with FDD and FDA (10-1280 µg mL(-1)). FDD showed presence of flavonoid, saponin and tannin meanwhile FDA consist of flavonoid, tannin and terpenoid. Result showed FDD, FDA and oxytocin induced a dose-related increase in force of contraction of isolated rat uterus. The maximum uterine contraction (Emax) produced by FDD, FDA and oxytocin were at the concentration 640 µg mL(-1) (EC50, 5.903 ± 0.529 µg mL), 20 µg mL(-1) (EC50, 290.5 ± 0.158 µg mL(-1)) and 0.4 µg mL(-1) (EC50, 0.060 ± 0.011 µg mL(-1)) respectively. Combination effects of oxytocin with FDD and FDA produced Emax at the concentration 80 µg mL(-1) (EC50, 270.3 ± 0.643 µg mL(-1)) and 1280 µg mL(-1) (EC50, 26.83 ± 0.727 µg mL(-1)), respectively. Study indicated F. deltoidea possess contractile effect on uterine contraction. This plant has great potential to develop as natural uterotonic agent in inducing labour and treatment for post-partum haemorrhage.
Assuntos
Ficus/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Útero/efeitos dos fármacos , Animais , Feminino , Ratos , Ratos Sprague-Dawley , Água/químicaRESUMO
UNLABELLED: Studies on the discovery of new cancer treatment by using metal-based compounds such as tin (Sn) has now greatly being synthesized and evaluated to identify their effectiveness and suitability to be developed as a new anticancer drug. APPROACH: This study was carried out to evaluate the cytotoxicity of triphenyltin(lV) methylisopropyldithiocarbamate (compound 1) and triphenyltin(IV) ethylisopropyldithiocarbamate (compound (2) on chronic myelogenus leukemia cells. The determination of their cytotoxicity (IC50) at different time of exposure and concentration was carried out through the employment of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. RESULTS: The IC50 values obtained for compound 1 and 2 following treatment at 24, 48 and 72 h were 0.660, 0.223, 0.370 microM and 0.677, 0.306, 0.360 microM, respectively. Cell morphological changes such as apoptotic and necrotic features were also been observed. CONCLUSION: The compounds tested were found to give cytotoxic effect against chronic myelogenus leukemia (K-562) cell at a micromolar dose. Thus, further study on their specific mechanism of actions in the human cells should be carried out to elucidate their potential as an anticancer agent.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Compostos Orgânicos de Estanho/farmacologia , Tiocarbamatos/farmacologia , Apoptose/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Células K562 , Necrose , Fatores de TempoRESUMO
Goniothalamin (GTN) isolated from Goniothalamus sp. has been demonstrated to induce apoptosis in a variety of cancer cell lines including Jurkat T leukemia cells. However, the mechanism of GTN-induced apoptosis upstream of mitochondria is still poorly defined. In this study, GTN caused a decrease in GSH with an elevation of reactive oxygen species as early as 30 min and DNA damage as assessed by Comet assay. Analysis using topoisomerase II processing of supercoiled pBR 322 DNA showed that GTN caused DNA damage via a topoisomerase II-independent pathway suggesting that cellular oxidative stress may contribute to genotoxicity. A 12-fold increase of caspase-2 activity was observed in GTN-treated Jurkat cells after 4h treatment and this was confirmed using Western blotting. Although the caspase-2 inhibitor Z-VDVAD-FMK inhibited the proteolytic activity of caspase-2, apoptosis ensued confirming that caspase-2 activity was not crucial for GTN-induced apoptosis. However, GTN-induced apoptosis was completely abrogated by N-acetylcysteine further confirming the role of oxidative stress. Since cytochrome c release was observed as early as 1h without any appreciable change in Bcl-2 protein expression, we further investigated whether overexpression of Bcl-2 confers resistance in GTN-induced cytotoxicity. Using a panel of Jurkat Bcl-2 transfectants, GTN cytotoxicity was not abrogated in these cells. In conclusion, GTN induces DNA damage and oxidative stress resulting in apoptosis which is independent of both caspase-2 and Bcl-2.
Assuntos
Antineoplásicos Fitogênicos/toxicidade , Apoptose/efeitos dos fármacos , Caspase 2/fisiologia , Dano ao DNA , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Pironas/toxicidade , Western Blotting , Caspase 2/metabolismo , Inibidores de Caspase , Ensaio Cometa , Citocromos c/metabolismo , DNA Topoisomerases Tipo II/química , Inibidores Enzimáticos , Citometria de Fluxo , Glutationa/metabolismo , Goniothalamus/química , Humanos , Células Jurkat , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio , Transdução de Sinais/efeitos dos fármacos , Sais de Tetrazólio , TiazóisRESUMO
Goniothalamin, a styryllactone, has been shown to induce cytotoxicity via apoptosis in several tumor cell lines. In this study, we have examined the potential role of several genes, which were stably transfected into T-cell lines and which regulate apoptosis in different ways, on goniothalamin-induced cell death. Overexpression of full-length receptor for activated protein C-kinase 1 (RACK-1) and pc3n3, which up-regulates endogenous RACK-1, in both Jurkat and W7.2 T cells resulted in inhibition of goniothalamin-induced cell death as assessed by MTT and clonogenic assays. However, overexpression of rFau (antisense sequence to Finkel-Biskis-Reilly murine sarcoma virus-associated ubiquitously expressed gene) in W7.2 cells did not confer resistance to goniothalamin-induced cell death. Etoposide, a clinically used cytotoxic agent, was equipotent in causing cytotoxicity in all the stable transfectants. Assessment of DNA damage by Comet assay revealed goniothalamin-induced DNA strand breaks as early as 1 h in vector control but this effect was inhibited in RACK-1 and pc3n3 stably transfected W7.2 cells. This data demonstrate that RACK-1 plays a crucial role in regulating cell death signalling pathways induced by goniothalamin.
Assuntos
Neuropeptídeos/fisiologia , Pironas/toxicidade , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Ensaio de Unidades Formadoras de Colônias , Corantes , Ensaio Cometa , Meios de Cultura , Dano ao DNA , Humanos , Células Jurkat , Camundongos , Neuropeptídeos/biossíntese , Neuropeptídeos/genética , Receptores de Quinase C Ativada , Sais de Tetrazólio , TiazóisRESUMO
ABSTRACT I: Management of invasive transitional cell human bladder carcinoma. The two main treatment options for invasive transitional cell bladder carcinoma are radiotherapy or primary cystectomy with urinary diversion or bladder substitution. Approximately 50% of patients fail to respond to radiotherapy and such patients so treated are disadvantaged by the absence of predictive information regarding their radiosensitivity, since the tumour gains additional time for metastatic spread before cystectomy is performed. The SF2 clonogenic assay, which measures the surviving fraction of tumour cells after 2 Gy X-ray irradiation, is regarded as a good measure of radiosensitivity. However, the assay is time consuming and provides results for only approximately 70% of human tumours. In this paper three bladder transitional cell carcinoma cell lines (HT1376, UMUC-3 and RT112) were exposed to X-irradiation (0-10 Gy). We have compared the responses obtained using a clonogenic assay and a more clinically feasible alkaline single cell gel electrophoresis (Comet) assay. A very good inverse correlation was obtained between cell survival (clonogenic assay) and mean tail moment (Comet assay) for the three cell lines, indicating that the Comet assay can be used to predict the radio-responsiveness of individual cell lines. The clinical usefulness of the assay for predicting response to radiotherapy in bladder cancer patients is currently being investigated. ABSTRACT II: Fluorescent in situ hybridization (FISH) Comets for the identification of damaged and repaired DNA sequences in individual cells. In mammalian cells the extent of DNA damage is partly and the rate of DNA repair very considerably dependent on DNA position and transcription. This has been established by biochemical techniques which are labour intensive and require large numbers of cells. The Comet assay for overall DNA damage and repair is relatively simple and allows individual cells to be examined. Here we present a protocol for combination of the Comet assay with fluorescent in situ hybridization (FISH) using a p53 gene probe which allows specific observation of p53 sequences within DNA comets. Chromosome-specific probes can also be used. Optimization of the FISH/Comet protocol to include automation of the analysis is currently underway to facilitate future application of the technique to study selective DNA damage and repair in defined sequences in single mammalian cells.