Formamidopyrimidine Glycosylase Research Articles

Pitavastatin induces caspase-mediated apoptotic death through oxidative stress and DNA damage in combined with cisplatin in human cervical cancer cell line.

Pitavastatin (PITA) is a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor to treat hypercholesterolemia and in recent studies is focused that its potential anti-cancer effect. This study was aimed to elucidate the effect of PITA alone and in combination with cisplatin on cervical cancer cells (HeLa) in vitro. Cytotoxicity of PITA (5-200 μM) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake (NRU) assays for 24, 48, and 72 h. Cell apoptosis and cell cycle analyses were performed in flow cytometry (0.1-100 μM). The evaluation of genotoxic effects and oxidative DNA damage of PITA (2-200 μM) were performed with standard comet assay, formamidopyrimidine glycosylase (fpg)-modified comet assay, and reactive oxygen species (ROS) activation in HeLa cells. PITA alone reduced cell viability in a dose-dependent manner (20-200, 20-200, and 5-200 μM for 24, 48, and 72 h, respectively, in MTT). The combined treatment of PITA with cisplatin resulted in significantly greater inhibition of cell viability. ROS and DNA damage increased significantly at 100 μM for 4 h and 20 μM for 24 h, respectively. PITA-induced apoptosis, an increased proportion of sub G1 cells, was monitored, and also, it increased the expression of active caspase-9 and caspase-3 and upregulated cleaved poly adenosine diphosphate ribose polymerase (PARP) by western blotting and caspase 3/8/9 multiple assay kit. We conclude that PITA can be used to efficiently cervical cancer studies, and promising findings have been obtained for further studies.

Evaluation of Systemic Genotoxic/Oxidative and Proinflammatory Effects in Workers of a Titanium Dioxide Production Plant.

This study is aimed at evaluating whether the occupational exposure to TiO2 during the industrial production process is able to induce genotoxic, oxidative, and inflammatory effects on blood, biomonitoring the same workers that showed micronucleus induction in the exfoliated buccal cells, as previous published. The final aim was to find sensitive and suitable biomarkers to evaluate potential early toxicity of occupational exposure to TiO2. On the same 40 workers involved in the manufacture of TiO2 pigment, 5 office workers, and 18 controls previously studied, we used formamidopyrimidine glycosylase- (Fpg-) comet assay on lymphocytes to evaluate genotoxic/oxidative effects and detected cytokine (IL-6, IL-8, and TNFα) release by ELISA to evaluate proinflammation. Moreover, we studied the possible influence of single nucleotide polymorphisms of XRCC1 and hOGG1 DNA repair genes and of GST metabolism-related genes (GSTT1 and GSTM1) on the evaluated effects. We did not find statistically significant differences in the mean values of the analysed Fpg-comet assay parameters; only the percentage of DNA damaged cells appearing in the test as comets (% comets) resulted higher in the exposed workers compared to controls. Also, the data analysed taking into account the specific task (bagging, industrial cleaning, mobile operations, maintaining, and production) showed differences only for % comets which resulted higher in industrial cleaners compared to controls. We found variations of IL-6 and IL-8 levels in the exposed workers with concentrations that were lower for IL-6 and higher for IL-8 compared to the control group. XRCC1, hOGG1, and GSTT1 polymorphisms did not influence neither comet parameters nor cytokine release. These findings demonstrate that TiO2 production process is able to induce slight proinflammatory effects in terms of IL-8 increased release but not significant genotoxic/oxidative effects on lymphocytes, which do not seem to be a target of TiO2, prevalently inhalable particles, generated in the studied production site.

Lack of mutagenicity of TiO2 nanoparticles in vitro despite cellular and nuclear uptake.

The potential genotoxicity of titanium dioxide (TiO2) nanoparticles (NPs) is a conflictive topic because both positive and negative findings have been reported. To add clarity, we have carried out a study with two cell lines (V79-4 and A549) to evaluate the effects of TiO2 NPs (NM-101), with a diameter ranging from 15 to 60nm, at concentrations 1-75μg/cm2. Using two different dispersion procedures, cell uptake was determined by Transmission Electron Microscopy (TEM). Mutagenicity was evaluated using the Hprt gene mutation test, while genotoxicity was determined with the comet assay, detecting both DNA breaks and oxidized DNA bases (with formamidopyrimidine glycosylase - Fpg). Cell internalization, as determined by TEM, shows TiO2 NM-101 in cytoplasmic vesicles, as well as close to and inside the nucleus. Such internalization did not depend on the state of agglomeration, nor the dispersion used. In spite of such internalization, no cytotoxicity was detected in V79-4 cells (relative growth activity and plating efficiency assays) or in A549 cells (AlamarBlue assay) after exposure lasting for 24h. However, a significant decrease in the relative growth activity was detected at longer exposure times (48 and 72h) and at the highest concentration 75µg/cm2. When the modified enzyme-linked alkaline comet assay was performed on A549 cells, although no significant induction of DNA damage was detected, a positive concentration-effects relationship was observed (Spearman's correlation = 0.9, p0.0001). Furthermore, no significant increase of DNA oxidized purine bases was observed. When the frequency of Hprt gene mutants was determined in V79-4 cells, no increase was observed in the exposed cells, relative to the unexposed cultures. Our general conclusion is that, under our experimental conditions, TiO2 NM-101 exposure does not exert mutagenic effects despite the evidence of NP uptake by V79-4 cells.

Cardioprotective Effect of Maternal Supplementation with Resveratrol on Toxicity Induced by Doxorubicin in Offspring Cardiomyocytes.

ResumoFundamentoA doxorrubicina (DOX) é frequentemente usada para tratar muitos tipos de cânceres, apesar da cardiotoxicidade dose-dependente. Como alternativa, o resveratrol é um polifenol que tem demonstrado efeitos cardioprotetores em vários modelos de disfunção cardíaca.ObjetivoEste estudo investigou se o tratamento com resveratrol em ratas gestantes protege contra toxicidade induzida por doxorrubicina em cardiomiócitos da ninhada.MétodosRatas Wistar (n-8) receberam sresveratrol como suplemento alimentar durante a gestação. No nascimento da ninhada, os corações (9-11) foram usados para se obter a cultura primária de cardiomiócitos. A cardiotoxicidade induzida por DOX e os efeitos da suplementação com resveratrol foram avaliados por marcadores de stress oxidativo, tais como oxidação da diclorofluoresceína diacetato, diminuição da atividade de enzimas antioxidantes, e oxidação do teor total de grupos sulfidrila, além da avaliação da viabilidade celular, geração de danos ao DNA, bem como a resposta de reparo aos danos ao DNA. Um valor de p <0,05 foi considerado estatisticamente significativo.ResultadosOs cardiomiócitos de neonatos de ratas que receberam suplemento resveratrol apresentaram um aumento (p <0,01) na viabilidade das células, e diminuição (p <0,0001) de células apoptóticas/necróticas após o tratamento com DOX, o que está correlacionado às atividades de enzimas antioxidantes e produção de diclorofluoresceína. Além disso, o resveratrol protegeu os cardiomiócitos de danos ao DNA induzidos por DOX, apresentando uma diminuição (p <0,05) nas quebras de DNA induzidas por stress oxidativo, avaliadas pela atividade de enzimas reparadoras do DNA endonuclease III e formamidopirimidina glicosilase. A suplementação com resveratrol aumentou (p <0,05) a expressão da proteína reparadora Sirt6 nos cardiomiócitos dos filhotes.ConclusãoEssa pesquisa indica que a suplementação com resveratrol durante o período gestacional tem um efeito cardioprotetor no coração da ninhada contra a toxicidade induzida por DOX, o que pode se dever a sua função antioxidante, e o aumento na resposta de danos ao DNA.

Melanin, lipofuscin and the effects of visible light in the skin

Photoexcitation of endogenous photosensitizers (ePS) in the human skin by sunlight culminates with the formation of electronic excited states, such as triplet excited states, singlet oxygen, and a variety of reactive oxygen and nitrogen species, either free radicals or two-electron oxidants. Considering the endogenous skin photosensitizers, melanin is the most abundant and is involved in photoprotection mechanisms, while lipofuscin is an unintentionally pigment accumulated during cell aging or oxidative stress. Both pigments severely increase the phototoxicity of visible light (VL) to skin cells. In the presence of these pigments, VL induces significant oxidative damage in nucleic acids, lipids and proteins, triggering regulated and unregulated cell death mechanisms. Besides, there is accumulation of premutagenic DNA lesions, indicated by the formamidopyrimidine glycosylase (Fpg)- and endonuclease III (Endo III)-sensitive sites in the nuclear DNA. As consequence of the photoinduced oxidative damage, skin cells, under the stimulus of VL release pro-inflammatory cytokines and metalloproteinases, prompting further cell death and skin aging. In this review we describe the photochemical properties of both melanin and lipofuscin pigments and discuss the consequences of their accumulation in terms of the phototoxicity of VL to the human skin.

Oxidative Stress and DNA Damage of Zebrafish Sperm at Different Stages of the Cryopreservation Process

Although gamete cryopreservation has facilitated advancement of reproduction research by allowing the storage of cells over prolonged periods of time, during freezing-thawing cycles, cells inevitably suffer from cryoinjuries. Here, we evaluate oxidative stress and DNA damage of zebrafish sperm at different stages of the cryopreservation process. It was generally observed that the freezing and thawing of the samples led to an increase in the generation of reactive oxygen species and the activity of the catalase enzyme and a reduction in the generation of sulfhydryl groups and superoxide dismutase activity. The alkaline comet assay demonstrated that DNA damage increased after equilibration time, with an even greater increase after freezing and thawing. The comet assay modified with the enzyme formamidopyrimidine glycosylase, and Endonuclease III demonstrated greater DNA damage than the standard comet assay, demonstrating a high degree of oxidation of purines and pyrimidines at all stages of cryopreservation. Our results show that the freeze and thaw processes cause greater oxidative stress and DNA damage than cryoprotectant toxicity during exposure at the equilibrium stage.

Protective effect of Trigonella foenum-graecum and Cinnamomum zeylanicum against diabetes induced oxidative DNA damage in rats

Diabetes mellitus has become a global epidemic as its prevalence is steadily increasing everywhere at an alarming rate, most markedly in the India. Traditional Indian system of medicine based on herbal therapies is being practiced since ancient times and has been proved very effective against number of diseases. Trigonella foenum-graecum (Tfg) and Cinnamomum zeylanicum (Cz), the two spices, possess multiple medicinal properties. Hence present study is aimed to evaluate the protective effects of these plants on the oxidative DNA damage in diabetic rats. The presence of DNA base oxidation was investigated by modified comet assay which uses bacterial repair enzymes, formamidopyrimidine glycosylase and Endonuclease III which can measure oxidatively damaged purines and pyrimidines, respectively. Diabetes induced alteration in total antioxidant capacity (TAC), generation of superoxide anion and the levels of GSH, GSSG, NADH, NAD+, NADPH and NADP+ were monitored. Diabetes was induced by intravenous injection of alloxan, followed by oral administration of aqueous suspension of Tfg seeds and bark of Cz. Treatment of Tfg and Cz significantly restored the altered level of superoxide anion, TAC, ratios of GSH/GSSG, NAD(P)H/NAD(P)+ and NADH/NAD+ in the diabetic group. Furthermore, both the treatment effectively decreased the level of oxidatively damaged purine and pyrimidine bases.

The Comet Assay as a Tool to Detect the Genotoxic Potential of Nanomaterials.

The interesting physicochemical characteristics of nanomaterials (NMs) has brought about their increasing use and, consequently, their increasing presence in the environment. As emergent contaminants, there is an urgent need for new data about their potential side-effects on human health. Among their potential effects, the potential for DNA damage is of paramount relevance. Thus, in the context of the EU project NANoREG, the establishment of common robust protocols for detecting genotoxicity of NMs became an important aim. One of the developed protocols refers to the use of the comet assay, as a tool to detect the induction of DNA strand breaks. In this study, eight different NMs—TiO2NP (2), SiO2NP (2), ZnONP, CeO2NP, AgNP, and multi-walled carbon nanotubes (MWCNT)—were tested using two different human lung epithelial cell lines (A549 and BEAS-2B). The comet assay was carried out with and without the use of the formamidopyrimidine glycosylase (FPG) enzyme to detect the induction of oxidatively damaged DNA bases. As a high throughput approach, we have used GelBond films (GBF) instead of glass slides, allowing the fitting of 48 microgels on the same GBF. The results confirmed the suitability of the comet assay as a powerful tool to detect the genotoxic potential of NMs. Specifically, our results indicate that most of the selected nanomaterials showed mild to significant genotoxic effects, at least in the A549 cell line, reflecting the relevance of the cell line used to determine the genotoxic ability of a defined NM.

542 Xeroderma Pigmentosum C mutations increase oxidative DNA damages by inhibiting Base Excision Repair System’s expression and activity

Xeroderma Pigmentosum C (XPC) is a rare genodermatosis which manifests clinically as pronounced photosensitivity, abnormal pigmentation and numerous skin cancers. It is caused by mutations in XPC gene that initiates global-genome nucleotide excision repair (GG-NER), a repair system responsible for bulky DNA adducts removal. Interestingly, development of internal and skin cancers in non-photo-exposed areas in parallel to an increase in oxidative DNA damages are observed upon these mutations. Therefore, we propose in our work to study XPC role in the modulation of BER, a major route for repairing oxidative DNA damage, at transcriptomic and proteomic levels along with repair activity using normal and XPC-mutated primary fibroblasts. To do so we used real time q-PCR and western blot to check effect of different XPC mutations on BER enzymes (OGG1, MYH in particular) at basal level and upon ultraviolet radiation type B (UVB) stress. Meanwhile, for studying the effect on BER’s activity, we measured the kinetic of repair of 7, 8-dihydro-8-oxoguanine (8-oxoguanine) using comet assay associated with formamidopyrimidine glycosylase (Fpg). A decrease in basal level and stress-induced level of BER was observed at gene and protein expression levels. Interestingly OGG1 and MYH, both of which are involved in 8-oxoguanine repair, were significantly downregulated. Finally, upon comet-fpg, a higher prolonged 8-oxoguanine oxidative lesions, persisting for more than 24 hours, were seen in XPC-mutated fibroblasts compared to the control especially after UVB-stress. Upon this, XPC deficient cell lines exhibit a reduction in 8-oxoguanine excision capacity, in agreement with the reduced genomic and proteomic levels BER enzymes. This may explain the higher incidence of skin cancer, especially in non-photo-exposed areas where impaired BER leads to an accumulation of lesions in the genome upon an increased induction and poorer repair of oxidative lesions.

Investigation of DNA damage of glycidol and glycidol fatty acid esters using Fpg-modified comet assay

Glycidol fatty acid esters (GEs) are food process contaminants detected in edible oils. It has been thought that glycidol is released from GEs by lipase in vivo, and shows genotoxicity. While DNA damage from glycidol has been reported, there is very little information on the DNA damaging potency of GEs in vivo. Therefore, we estimated DNA damage of glycidol and glycidyl oleate, which is one type of GEs, using the standard comet assay and a formamidopyrimidine glycosylase(Fpg)-modified comet assay. ICR male mice were orally administrated glycidol and glycidyl oleate (1.0 and 2.0 mmol/kg body weight) at 24 and 3 hr prior to dissection. In the standard comet assay, DNA damage (tail length and % tail DNA) in liver, kidney and blood samples of glycidol-treated groups were increased in a concentration-dependent manner. In Fpg-modified comet assay, glycidol showed DNA damage with higher sensitivity compared with the standard comet assay. DNA damage was not observed in the administration group of glycidyloleate in the standard comet assay. However, in Fpg-modified comet assay, glycidyl oleate showed significant DNA damage in the liver, kidney and blood samples compared with the standard comet assay. In this study, it was revealed that glycidol and glycidyl oleate induce DNA damage, such as oxidative and alkylation damage, recognized by Fpg protein.

Targeting Base Excision Repair Glycosylases with DNA Containing Transition State Mimics Prepared via Click Chemistry.

DNA glycosylases of the base excision repair (BER) pathway are front-line defenders in removing compromising modifications of the DNA nucleobases. Aberrantly modified nucleobases mediate genomic mutations and inhibit DNA replication leading to adverse health consequences such as cancer, neurological diseases, and aging. In an effort to develop high-affinity transition state (TS) analogues as chemical biology probes for DNA glycosylases, oligonucleotides containing a propargyl-modified pyrrolidine TS mimic nucleotide were synthesized. A small library of TS mimic-containing oligonucleotides was generated using a structurally diverse set of five azides via copper(I)-catalyzed azide-alkyne cycloaddition "click" chemistry. The relative affinity ( Kd) was evaluated for BER glycosylases Escherichia coli MutY, bacterial formamidopyrimidine glycosylase (Fpg), and human OG glycosylase 1 (hOGG1) with the library of TS mimic DNA duplexes. All of the BER glycosylases were found to exhibit extremely high affinities (approximately picomolar Kd values) for the TS mimics. However, binding preferences, distinct for each glycosylase, for the TS mimic library members were observed, suggesting different modes of binding and transition state stabilization among the three glycosylases. Fpg bound all of the TS mimics with exceptionally high affinities, while the MutY binding affinity correlated inversely with the size of the appended moiety. Of note, we identified one member of the small TS mimic library that exhibited a particularly high affinity for hOGG1. These results strongly support the use of the propargyl-TS mimic oligonucleotides and elaboration via click chemistry in screening and identification of high-affinity ligands for BER glycosylases of interest.

Increased TFAM binding to mtDNA damage hot spots is associated with mtDNA loss in aged rat heart

The well-known age-related mitochondrial dysfunction deeply affects heart because of the tissue's large dependence on mitochondrial ATP provision. Our study revealed in aged rat heart a significant 25% decrease in mtDNA relative content, a significant 29% increase in the 4.8 Kb mtDNA deletion relative content, and a significant inverse correlation between such contents as well as a significant 38% decrease in TFAM protein amount. The TFAM-binding activity to specific mtDNA regions increased at those encompassing the mtDNA replication origins, D-loop and Ori-L. The same mtDNA regions were screened for different kinds of oxidative damage, namely Single Strand Breaks (SSBs), Double Strand Breaks (DSBs), abasic sites (AP sites) and oxidized bases as 7,8-dihydro-8-oxoguanine (8oxoG). A marked increase in the relative content of mtDNA strand damage (SSBs, DSBs and AP sites) was found in the D-loop and Ori-L regions in the aged animals, unveiling for the first time in vivo an age-related, non-stochastic accumulation of oxidative lesions in these two regions that appear as hot spots of mtDNA damage. The use of Formamidopyrimidine glycosylase (Fpg) demonstrated also a significant age-related accumulation of oxidized purines particularly in the D-loop and Ori-L regions. The detected increased binding of TFAM to the mtDNA damage hot spots in aged heart suggests a link between TFAM binding to mtDNA and loss of mitochondrial genome likely through hindrance of repair processes.

Quantification of glucosylshingosine (lyso-Gb1) for the diagnosis and monitoring of Gaucher disease

Miltefosine was developed to treat skin cancer; further studies showed that the drug also has activity against Leishmania. Miltefosine is the first oral agent for treating leishmaniasis. However, its mechanism of action is not completely understood. We have evaluated the induction of DNA damage by miltefosine. Cytotoxicity and genotoxicity (comet assay) tests were performed on human leukocytes exposed to the drug in vitro. Apoptosis and necrosis were also evaluated. In vivo tests were conducted in Swiss male mice (Mus musculus) treated orally with miltefosine. Oxidation of DNA bases in peripheral blood cells was measured using the comet assay followed by digestion with formamidopyrimidine glycosylase (FPG), which removes oxidized guanine bases. The micronucleus test was performed on bone marrow erythrocytes. Miltefosine caused DNA damage, apoptosis, and necrosis in vitro. Mice treated with miltefosine showed an increase in the DNA damage score, which was further increased following FPG digestion. The micronucleus test was also positive.

Identification of PMN-released mutagenic factors in a co-culture model for colitis-associated cancer.

Microsatellite instability (MSI) is present in ulcerative colitis (UC) and colitis-associated colorectal cancers (CAC). Certain factors released by polymorphonuclear cells (PMNs) may drive mucosal frameshift mutations resulting in MSI and cancer. Here, we applied a co-culture system with PMNs and colon epithelial cells to identify such culprit factors. Subjecting HCT116 + chr3 and human colonic epithelial cells (HCEC)-1CT MSI-reporter cell lines harboring mono-, di- or tetranucleotide DNA repeats linked to enhanced green fluorescent protein (EGFP) to activated PMNs induced frameshift mutations within all repeats, as quantified by flow cytometry. Activated PMNs released superoxide and hydrogen peroxide (H2O2), as measured by lucigenin-amplified chemiluminescence and fluorometry, respectively. Catalase, which scavenges H2O2, reduced such PMN-induced MSI. The NADPH-oxidase inhibitor apocynin, which blocks the oxidative burst in PMNs, similarly inhibited PMN-induced MSI. A bead-based multiplex assay revealed that PMNs release a wide range of cytokines such as interleukin (IL)-8, IL-6 and tumor necrosis factor-α (TNF-α). In vitro, these cytokines increased MSI in colon epithelial cells, and the Janus kinase (JAK) inhibitor tofacitinib abolished IL-6-induced or PMN-induced MSI. Intracellular reactive oxygen species (ROS) formation, as measured by 2’,7’–dichlorofluorescein diacetate (DCFDA) assay, was induced upon cytokine treatment. DNA oxidation upon IL-6 was present, as detected by formamidopyrimidine glycosylase (FPG)-modified comet assay. In conclusion, activated PMNs induce frameshift mutations in colon epithelial cells resulting in MSI. Both oxidative burst with release of ROS and PMN-secreted cytokines, such as IL-8, IL-6 or TNF-α, contribute to MSI. ROS scavengers and/or specific inhibitors of cytokine signaling may delay or prevent cancer development in the setting of colitis.

Assessment of DNA damage in a group of professional dancers during a 10-month dancing season

ABSTRACTDespite the numerous health benefits of physical activity, some studies reported that increased intensity and duration may induce oxidative stress in several cellular components including DNA. The aim of this study was to assess the level of basal DNA damage as well as oxidative DNA damage in a group of professional dancers before and after a 10-month dancing season. A group of individuals from general population was also assessed as a control. The alkaline version of the comet assay was the method selected to measure both basal DNA damage and oxidative stress, since this method quantifies both endpoints. In order to measure oxidative stress, the comet assay was coupled with a lesion-specific endonuclease (formamidopyrimidine glycosylase) to detect oxidized purines. The levels of oxidative DNA damage in dancers were significantly increased after the dancing season. Pre-season levels of oxidative DNA damage were lower in dancers than those obtained from the general population, suggesting an adaptation of antioxidant system in dancers. Results of the present biomonitoring study indicate the need for more effective measures to protect ballet dancers from potentially occupational health risks related to regular intensive physical exercise.

Mechanism of Discrimination of 8-Oxoguanosine versus Guanosine by Escherichia coli Fpg.

The mutagenic 8-oxoguanosine monophosphate, the predominant product of DNA oxidation, is excised by formamidopyrimidine glycosylase (Fpg) in bacteria. The mechanism of recognition of 8-oxodG, which differs subtly from its normal counterpart, guanosine monophosphate (dG), by Escherichia coli Fpg remains elusive due to the lack of structural data of E. coli Fpg bound to 8-oxodG. Here, we present solution-state structure of 8-oxodG oligomer bound to E. coli E3Q Fpg using UV resonance Raman (UVRR) spectroscopy. The vibrational spectra report on the π-stacking and hydrogen bonding interactions established by 8-oxodG with E. coli E3Q Fpg. Furthermore, we report on the interactions of E. coli E3Q Fpg with the normal, undamaged nucleotide, dG. We show that E. coli Fpg recognizes 8-oxodG and dG through their C2-amino group but only 8-oxodG forms extensive contacts with E. coli Fpg. Our findings provide a basis for mechanism of lesion recognition by E. coli Fpg.

The antileishmanial drug miltefosine (Impavido®) causes oxidation of DNA bases, apoptosis, and necrosis in mammalian cells

Miltefosine was developed to treat skin cancer; further studies showed that the drug also has activity against Leishmania. Miltefosine is the first oral agent for treating leishmaniasis. However, its mechanism of action is not completely understood. We have evaluated the induction of DNA damage by miltefosine. Cytotoxicity and genotoxicity (comet assay) tests were performed on human leukocytes exposed to the drug in vitro. Apoptosis and necrosis were also evaluated. In vivo tests were conducted in Swiss male mice (Mus musculus) treated orally with miltefosine. Oxidation of DNA bases in peripheral blood cells was measured using the comet assay followed by digestion with formamidopyrimidine glycosylase (FPG), which removes oxidized guanine bases. The micronucleus test was performed on bone marrow erythrocytes. Miltefosine caused DNA damage, apoptosis, and necrosis in vitro. Mice treated with miltefosine showed an increase in the DNA damage score, which was further increased following FPG digestion. The micronucleus test was also positive.

Gas-Phase Studies of Formamidopyrimidine Glycosylase (Fpg) Substrates.

Gas-phase thermochemical properties (tautomerism, acidity, and proton affinity) have been measured and calculated for a series of nucleobase derivatives that have not heretofore been examined under vacuum. The studied species are substrates for the enzyme formamidopyrimidine glycosylase (Fpg), which cleaves damaged nucleobases from DNA. The gas-phase results are compared and contrasted to solution-phase data, to afford insight into the Fpg mechanism. Calculations are also used to probe the energetics of various possible mechanisms and to predict isotope effects that could potentially allow for discrimination between different mechanisms. Specifically, (18) O substitution at the ribose O4' is predicted to result in a normal kinetic isotope effect (KIE) for a ring-opening "endocyclic" mechanism and an inverse KIE for a direct base excision "exocyclic" pathway.

Selective enzymatic cleavage and labeling for sensitive capillary electrophoresis laser-induced fluorescence analysis of oxidized DNA bases

Oxidatively generated DNA damage is considered to be a significant contributing factor to cancer, aging, and age-related human diseases. It is important to detect oxidatively generated DNA damage to understand and clinically diagnosis diseases caused by oxidative damage. In this study, using selective enzymatic cleavage and quantum dot (QD) labeling, we developed a novel capillary electrophoresis-laser induced fluorescence method for the sensitive detection of oxidized DNA bases. First, oxidized DNA bases are recognized and removed by one DNA base excision repair glycosylase, leaving apurinic and apyrimidinic sites (AP sites) at the oxidized positions. The AP sites are further excised by the AP nicking activity of the chosen glycosylase, generating a nucleotide gap with 5′- and 3′- phosphate groups. After dephosphorylation with one alkaline phosphatase, a biotinylated ddNTP is introduced into the nucleotide space within the DNA strand by DNA polymerase I. The biotin-tagged DNA is further labeled with a QD-streptavidin conjugate via non-covalent interactions. The DNA-bound QD is well-separated from excess DNA-unbound QD by highly efficient capillary electrophoresis and is sensitively detected by online coupled laser-induced fluorescence analysis. Using this method, we can assess the trace levels of oxidized DNA bases induced by the Fenton reaction and UV irradiation. Interestingly, the use of the formamidopyrimidine glycosylase (FPG) protein and endonuclease VIII enables the detection of oxidized purine and pyrimidine bases, respectively. Using the synthesized standard DNA, the approach has low limits of detection of 1.1×10−19mol in mass and 2.9pM in concentration.

Protective effects of melatonin-loaded lipid-core nanocapsules on paraquat-induced cytotoxicity and genotoxicity in a pulmonary cell line

Many acute poisonings lack effective and specific antidotes. Due to both intentional and accidental exposures, paraquat (PQ) causes thousands of deaths annually, especially by pulmonary fibrosis. Melatonin (Mel), when incorporated into lipid-core nanocapsules (Mel-LNC), has enhanced antioxidant properties. The effects of such a formulation have not yet been studied with respect to mitigation of PQ- induced cytotoxicity and DNA damage. Here, we have tested whether Mel-LNC can ameliorate PQ-induced toxicity in the A549 alveolar epithelial cell line. Physicochemical characterization of the formulations was performed. Cellular uptake was measured using nanocapsules marked with rhodamine B. Cell viability was determined by the MTT assay and DNA damage was assessed by the comet assay. The enzyme-modified comet assay with endonuclease III (Endo III) and formamidopyrimidine glycosylase (FPG) were used to investigate oxidative DNA damage. Incubation with culture medium for 24h did not alter the granulometric profile of Mel-LNC formulations. Following treatment (3 and 24h), red fluorescence was detected around the cell nucleus, indicating internalization of the formulation. Melatonin solution (Mel), Mel-LNC, and LNC did not have significant effects on cell viability or DNA damage. Pre-treatment with Mel-LNC enhanced cell viability and showed a remarkable reduction in % DNA in tail compared to the PQ group; this was not observed in cells pre-treated with Mel. PQ induces oxidative DNA damage detected with the enzyme-modified comet assay. Mel-LNC reduced this damage more effectively than did Mel. In summary, Mel-LNC is better than Mel at protecting A549 cells from the cytotoxic and genotoxic effects of PQ.