Formation Of DNA Strand Breaks Research Articles

Sensitive MALDI-TOF MS and ‘turn-on’ fluorescent genosensor for the determination of DNA damage induced by CNS acting drugs

The genotoxic and carcinogenic adverse effects of various drugs should be considered for assessing drug benefit/risk ratio. On that account, the scope of this study is to examine the kinetics of DNA damage triggered by three CNS acting drugs; carbamazepine, quetiapine and desvenlafaxine. Two precise, simple and green approaches were proposed for probing drug induced DNA impairment; MALDI-TOF MS and terbium (Tb3+) fluorescent genosensor. The results revealed that all the studied drugs induced DNA damage manifested by the MALDI-TOF MS analysis as a significant disappearance of the DNA molecular ion peak with the appearance of other peaks at smaller m/z indicating the formation of DNA strand breaks. Moreover, significant enhancement of Tb3+ fluorescence occurred, proportional to the amount of DNA damage, upon incubation of each drug with dsDNA. Furthermore, the DNA damage mechanism is examined. The proposed Tb3+ fluorescent genosensor showed superior selectivity and sensitivity and is significantly simpler and less expensive than other methods reported for the detection of DNA damage. Moreover, the DNA damaging potency of these drugs was studied using calf thymus DNA in order to clarify the potential safety hazards associated with the studied drugs on natural DNA.

Culture medium-dependent isoproterenol stability and its impact on DNA strand breaks formation and repair

In vitro mechanistic research is mostly performed without taking into consideration the potential influence of cell culture media and/or their supplements and therefore, interactions between compounds of interest and medium ingredients may be overlooked. Isoproterenol (isoprenaline) is a synthetic catecholamine used as sympathomimetic drug that stimulates β-adrenergic receptors and is widely used in biomedical research. Clinical studies have shown that isoproterenol is rapidly metabolized in the human body with a plasma half-life of about 2–5 min. However, despite its use in many in vitro and ex vivo studies, the stability of isoproterenol in cell culture media has not been characterized. Our results show a decrease of isoproterenol concentration in RPMI medium but high stability of the compound in TexMACS medium. The isoproterenol oxidation product isoprenochrome forms during treatment in both media. However, isoprenochrome formation is significantly lower in TexMACS medium. The effective level of isoproterenol and the formation of oxidation products might explain the discrepancies observed in isoproterenol-induced genotoxicity and cytotoxicity.

Mechanistic studies on the adverse effects of manganese overexposure in differentiated LUHMES cells

Manganese (Mn) is an essential trace element, but overexposure is associated with toxicity and neurological dysfunction. Accumulation of Mn can be observed in dopamine-rich regions of the brain in vivo and Mn-induced oxidative stress has been discussed extensively. Nevertheless, Mn-induced DNA damage, adverse effects of DNA repair, and possible resulting consequences for the neurite network are not yet characterized. For this, LUHMES cells were used, as they differentiate into dopaminergic-like neurons and form extensive neurite networks. Experiments were conducted to analyze Mn bioavailability and cytotoxicity of MnCl2, indicating a dose-dependent uptake and substantial cytotoxic effects. DNA damage, analyzed by means of 8-oxo-7,8-dihydro-2′-guanine (8oxodG) and single DNA strand break formation, showed significant dose- and time-dependent increase of DNA damage upon 48 h Mn exposure. Furthermore, the DNA damage response was increased which was assessed by analytical quantification of poly(ADP-ribosyl)ation (PARylation). Gene expression of the respective DNA repair genes was not significantly affected. Degradation of the neuronal network is significantly altered by 48 h Mn exposure. Altogether, this study contributes to the characterization of Mn-induced neurotoxicity, by analyzing the adverse effects of Mn on genome integrity in dopaminergic-like neurons and respective outcomes.

Okamoto model for necrosis and its expansions, CD38-cyclic ADP-ribose signal system for intracellular Ca2+ mobilization and Reg (Regenerating gene protein)-Reg receptor system for cell regeneration.

In pancreatic islet cell culture models and animal models, we studied the molecular mechanisms involved in the development of insulin-dependent diabetes. The diabetogenic agents, alloxan and streptozotocin, caused DNA strand breaks, which in turn activated poly(ADP-ribose) polymerase/synthetase (PARP) to deplete NAD+, thereby inhibiting islet β-cell functions such as proinsulin synthesis and ultimately leading to β-cell necrosis. Radical scavengers protected against the formation of DNA strand breaks and inhibition of proinsulin synthesis. Inhibitors of PARP prevented the NAD+ depletion, inhibition of proinsulin synthesis and β-cell death. These findings led to the proposed unifying concept for β-cell damage and its prevention (the Okamoto model). The model met one proof with PARP knockout animals and was further extended by the discovery of cyclic ADP-ribose as the second messenger for Ca2+ mobilization in glucose-induced insulin secretion and by the identification of Reg (Regenerating gene) for β-cell regeneration. Physiological and pathological events found in pancreatic β-cells have been observed in other cells and tissues.

Natural Merosesquiterpenes Activate the DNA Damage Response via DNA Strand Break Formation and Trigger Apoptotic Cell Death in p53-Wild-type and Mutant Colorectal Cancer.

Simple SummaryBowel cancer is a serious disease, which affects many people worldwide. Unfortunately, the disease is often diagnosed in an advanced stage, which impairs the chance of survival. Furthermore, resistance to therapy occurs frequently. Thus, novel therapeutic approaches are required to improve cancer therapy. Here, we studied whether merosesquiterpenes might be useful for cancer treatment. These compounds occur in marine sponges and were isolated by our group. We were able to identify three compounds with potent cytotoxic activity in different cell lines established from human large bowel cancer. Our experiments provided evidence that the compounds cause DNA damage and trigger cell death, so-called mitochondrial apoptosis, which was attested in cancer cells with expression of wild-type and mutated p53 tumor suppressor. Finally, we show that merosesquiterpenes also kill intestinal tumor organoids, an ex vivo model of large bowel cancer.Colorectal cancer (CRC) is a frequently occurring malignant disease with still low survival rates, highlighting the need for novel therapeutics. Merosesquiterpenes are secondary metabolites from marine sponges, which might be useful as antitumor agents. To address this issue, we made use of a compound library comprising 11 isolated merosesquiterpenes. The most cytotoxic compounds were smenospongine > ilimaquinone ≈ dactylospontriol, as shown in different human CRC cell lines. Alkaline Comet assays and γH2AX immunofluorescence microscopy demonstrated DNA strand break formation in CRC cells. Western blot analysis revealed an activation of the DNA damage response with CHK1 phosphorylation, stabilization of p53 and p21, which occurred both in CRC cells with p53 knockout and in p53-mutated CRC cells. This resulted in cell cycle arrest followed by a strong increase in the subG1 population, indicative of apoptosis, and typical morphological alterations. In consistency, cell death measurements showed apoptosis following exposure to merosesquiterpenes. Gene expression studies and analysis of caspase cleavage revealed mitochondrial apoptosis via BAX, BIM, and caspase-9 as the main cell death pathway. Interestingly, the compounds were equally effective in p53-wild-type and p53-mutant CRC cells. Finally, the cytotoxic activity of the merosesquiterpenes was corroborated in intestinal tumor organoids, emphasizing their potential for CRC chemotherapy.

Novel inexpensive ‘turn-on’ fluorescent biosensor for the sensitive detection of DNA damage induced by epirubicin

For several decades, DNA has been well-known as the cellular target for various cytotoxic anticancer drugs. An active research area between chemistry, medicine and molecular biology is studying mechanism of interaction of drug molecules with DNA. In this study, we introduce a novel inexpensive and simple fluorescent biosensor to directly quantitate the DNA damage induced by epirubicin, an anticancer agent, and investigate the underlying molecular mechanisms of damage. The proposed fluorescent biosensor is established using terbium chloride. In damaged DNA, there are ssDNA regions facing the damaged nucleobases. In the damaged DNA, the terbium biosensor coordinates directly with the unpaired nucleobases accompanied by enhancement of its fluorescence, generating a measureable signal proportionate to the amount of damage. Moreover, dithiothreitol is used in this study to asses any possible DNA damage mechanisms that may occur other than the DNA oxidative damage that is commonly induced by various anthracyclines. In this work, the kinetics of the damage induced in DNA by epirubicin in absence and presence of dithiothreitol were compared using the proposed Tb3+ fluorescence biosensor. The results were confirmed using MALDI-TOF mass spectrometric analysis. It revealed that in absence of dithiothreitol, epirubicin induces oxidative DNA damage due to the interaction with reactive oxygen species causing strand breaks where the mass spectra show a significant reduction of the molecular-ion-peak of DNA, and appearance of peaks of smaller m/z indicating the DNA strand-breaks formation. While, in presence of dithiothreitol, epirubicin covalently binds to DNA to form DNA adducts with m/z values larger than the molecular-ion-peak of DNA target. Furthermore, the potency of epirubicin was examined on calf-thymus natural DNA samples with the proposed fluorescence biosensor. Whilst the interaction mechanism of epirubicin with DNA in presence of dithiothreitol shows slower DNA damage kinetics, but it has more potent damaging effect on DNA than in the case when no dithiothreitol is added. This introduces a novel alternative anticancer therapy that requires further investigation. Moreover, the low cost of the proposed fluorescent biosensor allows it to be an automatic simple process for the investigation of any DNA-drug interactions without the need to couple it with any other analytical method.

Repair of O6-carboxymethylguanine adducts by O6-methylguanine-DNA methyltransferase in human colon epithelial cells.

The protein O6-methylguanine-DNA methyltransferase (MGMT) is able to repair the mutagenic O6-methylguanine (O6-MeG) adduct back to guanine. In this context, it may protect against colorectal cancer formation associated with N-nitroso compounds. Such compounds may be endogenously formed by nitrosylation of amino acids, which can give rise to mutagenic O6-MeG and O6-carboxymethylguanine (O6-CMG) adducts. It is well established that O6-MeG is repaired by MGMT. However, up to now, whether O6-CMG is repaired by this enzyme remains unresolved. Therefore, the aim of the present study was to analyze the fate of both types of O6-guanine adducts in the presence and absence of MGMT activity. To this end, MGMT activity was efficiently blocked by its chemical inhibitor O6-benzylguanine in human colon epithelial cells (HCECs). Exposure of cells to azaserine (AZA) caused significantly higher levels of both O6-MeG and O6-CMG adducts in MGMT-inhibited cells, with O6-CMG as the more abundant DNA lesion. Interestingly, MGMT inhibition did not result in higher levels of AZA-induced DNA strand breaks in spite of elevated DNA adduct levels. In contrast, MGMT inhibition significantly increased DNA strand break formation after exposure to temozolomide (TMZ), a drug that exclusively generates O6-MeG adducts. In line with this finding, the viability of the cells was moderately reduced by TMZ upon MGMT inhibition, whereas no clear effect was observed in cells treated with AZA. In conclusion, our study clearly shows that O6-CMG is repaired by MGMT in HCEC, thereby suggesting that MGMT might play an important role as a tumor suppressor in diet-mediated colorectal cancer.

Protective effect of leaf extract of Abutilon indicum on DNA damage and peripheral blood lymphocytes in combating the oxidative stress

The current research explores in vitro antioxidant characteristics, radiation-induced DNA damage protection and quenching effects of the oxidative stress by the ethanolic leaf extract of Abutilon indicum (EEAI) on human peripheral blood lymphocytes (PBLs). PBLs were incubated with various concentrations of EEAI accompanied by pre- and post-treatment with hydrogen peroxide. Cell viability was investigated by MTT assay. In addition, quenching of free radicals were measured in vitro using DPPH, superoxide anion, hydrogen peroxide, reducing power and nitric oxide radical scavenging assays. These activities were compared with ascorbic acid as standard antioxidants. Furthermore, inhibition of UV radiation-induced strand break formation in plasmid pBR322 DNA and anti-Fenton reactions in calf thymus DNA was evaluated. Cytotoxic effects of hydrogen peroxide on PBLs were significantly reduced with EEAI pre-treatment compared to post-treatment in a dose-dependent manner comparable with similar cytoprotective effects of ascorbic acid (p > 0.05). EEAI has shown strong antioxidant effects in the scavenging of DPPH, superoxide anion, hydrogen peroxide, and nitric oxide. EEAI also has a strong protective effect of UV-induced plasmid pBR322 DNA cleavage and Fenton-induced DNA damage. Overall, the results revealed that Abutilon indicum has a cytoprotective, potent antioxidant and DNA protective effect that provide pharmacological credence to justify its overall biological activity. Furthermore, future studies to identifying bioactive molecules and its molecular mechanisms responsible for promising therapeutic applications in the rescue of disease-induced cellular oxidative damage.

Sensitive spectrofluorimetric and mass spectroscopic methods for the determination of nucleic acid damage induced by photosensitized anti-inflammatory drugs: Comparative study

Anti-inflammatory drugs are reported to induce changes in nucleic-acids upon UV-irradiation. Such changes have the potential to cause apoptosis, carcinogenesis, and mutagenesis. In this work, the kinetics of the damage induced in DNA by some anti-inflammatory drugs were compared after UV-irradiation. Five commonly used anti-inflammatory drugs; diclofenac, ketoprofen, leflunomide, piroxicam and tolmetin, were studied. Simple, sensitive and eco-friendly methods for the analysis of DNA-damage were proposed including absorption spectroscopy, MALDI-TOF mass spectrometry and fluorescence using TbCl3. Results show that all drugs induced DNA-damage after UV-irradiation. Absorption spectroscopy results demonstrated hyperchromic shift in the absorption band characteristic to DNA, indicating distortion of the double-strand. Mass spectra showed a significant decrease of the molecular-ion-peak of DNA, together with peaks of smaller m/z that indicated the formation of DNA strand-breaks. TbCl3 fluorescence was observed to increase with incubation time of each drug with DNA, indicating the presence of more single-stranded regions in DNA due to damage. TbCl3 fluorescence was used to obtain the kinetics of the induced damage. Results show that DNA-damage occurred via photoinduced oxidative mechanism. Also, the potency of the studied drugs was examined on calf-thymus real DNA samples using TbCl3 fluorescence with ketoprofen and leflunomide being the most photogenotoxic anti-inflammatory drugs.

Aluminium Induced DNA-damage and Oxidative Stress in Cultures of the Marine Sponge Hymeniacidon perlevis

Aluminium is the most abundant element in the earth crust, and has no known biological function. However, it is an established neurotoxicant in its trivalent oxidation state, with exposure resulting in neurodegenerative diseases like Parkinson’s disease and presenile dementia. Although, the potential genotoxic and carcinogenic effects of aluminium are established in mammalian and other model system, there is however very limited information on aluminium genotoxicity in aquatic invertebrates. Mechanism of aluminium toxicity is also largely unclear. With a concentration range between 0.001– 0.05mg/L in near neutral pH water, and up to 0.5-1mg/L in an acidic water , aluminium poses a potential threat to the marine ecosystem, however it is poorly studied. This study, therefore presents for the first time, aluminium-induced DNA damage using the comet assay and reactive oxygen Species (ROS) formation using 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCF-DA) assay as biomarkers of genotoxicity and oxidative stress in the inter-tidal marine sponge Hymeniacidon perlevis, respectively. H. perlevis is widely distributed in the British Isles, Mediterranean and the Arctic sea and has been reported as a model for environmental biomonitoring in aquatic ecosystem and as a suitable alternative to bivalves. In this study, cryopreserved single sponge cells of H. perlevis were cultured as viable aggregates and were thereafter treated with 0.1, 0.2, 0.3 and 0.4mg/L aluminium chloride (AlCl3) for 12 hours. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Our results showed that non-cytotoxic concentrations of AlCl3 caused a statistically significant concentration-dependent increase in the level of DNA-strand break and reactive oxygen species formation single sponge cells of H. perlevis. There was also a statistically significant positive linear correlation between aluminium-induced DNA strand break and ROS formation suggesting the involvement of ROS in the causative mechanism of the aluminium induced DNA-strand breaks observed.

DNA-damaging activities of twenty-four structurally diverse unsubstituted and substituted cyclic compounds in embryo-fetal chicken livers

DNA-damaging activities of twenty-four structurally diverse unsubstituted and substituted cyclic compounds were assessed in embryo-fetal chicken livers. Formation of DNA adducts and strand breaks were measured using the nucleotide 32P-postlabelling (NPL) and comet assays, respectively. Unsubstituted monocyclic benzene, polycyclic fused ring compound naphthalene, covalently connected polycyclic ring compound biphenyl, and heterocyclic ring compound fluorene did not produce DNA damage. Amino-substituted monocyclic compounds, aniline and p-phenylenediamine, as well as polycyclic 1-naphthylamine were also negative. In contrast, carcinogenic monocyclic methyl-substituted anilines: o-toluidine, 2,6-xylidine, 3,4-dimethylaniline, 4-chloro-o-toluidine; 2 methoxy-substituted methylaniline: p-cresidine; 2,4 and 2,6 diamino- or dinitro- substituted toluenes all produced DNA damage. Genotoxic polycyclic amino-substituted 2-naphthylamine, 4-aminobiphenyl, benzidine, methyl-substituted 3,2′-dimethyl-4-aminobiphenyl and 4-dimethylaminoazobenzene as well as amino- and nitro- fluorenes substituted at the 1 or 2 positions also were positive in at least one of the assays. Overall, the DNA damaging activity of cyclic compounds in embryo-fetal chicken livers reflected the type and position of the substitution on the aromatic ring. Additionally, substituted polycyclic compounds exhibited higher DNA-damaging potency compared to monocyclic chemicals. These results are congruent with in vivo findings in other species, establishing chicken eggs as a reliable system for structure-activity assessment of members of groups of related chemicals.

Propolis Extracts Inhibit UV-Induced Photodamage in Human Experimental In Vitro Skin Models.

The aim of this study was to assess the antioxidant, photoprotective, and antiaging effects of Greek propolis. Propolis was subjected to n-heptane or methanol extraction. Total phenolic/flavonoid content and antioxidant potential were determined in the extracts. Promising extracts were evaluated for their cytoprotective properties using human immortalized keratinocyte (HaCaT) or reconstituted human skin tissue following exposure to UVB. Assessment of cytotoxicity, DNA damage, oxidative status, and gene/protein expression levels of various matrix metalloproteinases (MMPs) were performed. The propolis methanolic fractions exhibited higher total phenolic and flavonoid contents and significant in vitro antioxidant activity. Incubation of HaCaT cells with certain methanolic extracts significantly decreased the formation of DNA strand breaks following exposure to UVB and attenuated UVB-induced decrease in cell viability. The extracts had no remarkable effect on the total antioxidant status, but significantly lowered total protein carbonyl content used as a marker for protein oxidation in HaCaT cells. MMP-1, -3, -7, and -9, monitored as endpoints of antiaging efficacy, were significantly reduced by propolis following UVB exposure in a model of reconstituted skin tissue. In conclusion, propolis protects against the oxidative and photodamaging effects of UVB and could be further explored as a promising agent for developing natural antiaging strategies.

Analysis of DNA Damage Biomarkers in Human Leukocytes by PAHs Exposure

Aims: To study the potential genotoxicity of two polynuclear aromatic hydrocarbons (PAHs), exposed cultured human leukocytes in vitro using two types of biomarkers genotoxicity: DNA strand breaks (DNA-SB) and adduct formation (DNA-PAHs).
 Study Design: Human leukocytes were exposed to toxic cultures with different concentrations of anthracene (ANT), phenanthrene (PHE) and benzo(a)pyrene (B(a)P) for 24 hours. Four toxic test groups, PAHs, control group, analytic blank group and standard fluorescence group were considered. 
 Place and Duration of Study: Laboratorio de Citopatología Ambiental, of Departamento de Morfología at Escuela Nacional de Ciencias Biológicas, IPN; between february 2016 and july 2018.
 Methodology: Human leukocytes cells were isolated and cell viability was previously verified by Trypan dye exclusion test. Firstly, the lethal concentrations with neutral red (NR50) assay for each one PAHs was obtained. Then sublethal concentrations range of these toxics for both biomarkers were used. In case of DNA fragmentation, a fluorochrome was used to mark DNA strandbreaks and isolation with alkaline solution finally determined with fluorescence spectroscopy. To test the formation of DNA-PAHs adducts, first of all they were isolated with a solvent system with polarity gradient and finally determined with fluorescence spectroscopy.
 Results: PAHs with 3 aromatic rings showed lethal cytotoxicity, lower in case of the B(a)P with 5 aromatic rings. These results are in contrast with previously reported observations, in which non-adduct with DNA and DNA strandbreaks formation were detected with Anthracene and Phenanthrene while DNA adduction formation and DNA strandbreaks were produced in case of B(a)P.
 Conclusion: Biomarkers may be used as suitable discriminants of genotoxic agents as well as of environmental pollutants with genotoxic potential and for application in studies of environmental risk assessment and in hazardous waste evaluation. The application of both genotoxic biomarkers, DNA strand breaks and production of adducts DNA-PAHs, as genotoxicity assays are quickly and accurate techniques for determining the carcinogenic potential of environmental samples.

Genotoxic effects of neurotoxin ß-N-methylamino-l-alanine in human peripheral blood cells

The non-proteinogenic amino acid ß-N-methylamino-l-alanine (BMAA) is associated with the development of neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC) and amyotrophic lateral sclerosis. BMAA is known to induce neurotoxic effects leading to neurodegeneration via multiple mechanisms including misfolded protein accumulation, glutamate induced excitotoxicity, calcium dyshomeostasis, endoplasmic reticulum stress and oxidative stress. In the present study, for the first time, genotoxic activity of BMAA (2.5, 5, 10 and 20 μg/mL) was studied in human peripheral blood cells (HPBCs) using the comet and cytokinesis-block micronucleus cytome assays. In addition, the influence of BMAA on the oxidative stress was assessed. At non-cytotoxic concentrations BMAA did not induce formation of DNA strand breaks in HPBCs after 4 and 24 h exposure; however, it significantly increased the number of micronuclei after 24 and 48 h at 20 μg/mL and nucleoplasmic bridges after 48 h at 20 μg/mL. The frequency of nuclear buds was slightly though non-significantly increased after 48 h. Altogether, this indicates that in HPBCs BMAA is clastogenic and induces complex genomic alterations including structural chromosomal rearrangements and gene amplification. No influence on oxidative stress markers was noticed. These findings provide new evidence that environmental neurotoxin BMAA, in addition to targeting common pathways involved in neurodegeneration, can also induce genomic instability in non-target HPBCs suggesting that it might be involved in cancer development. Therefore, these data are important in advancing our current knowledge and opening new questions in the understanding of the mechanisms of BMAA toxicity, particularly in the context of genotoxicity.

Does exposure to reduced pH and diclofenac induce oxidative stress in marine bivalves? A comparative study with the mussel Mytilus galloprovincialis and the clam Ruditapes philippinarum

CO2-driven acidification and emerging contaminants, such as pharmaceuticals, pose new threats for the maintenance of natural populations of marine organisms by interfering with their normal biochemical pathways and defences. The combined effects of seawater acidification, as predicted in climate change scenarios, and an emerging contaminant (the non-steroidal anti-inflammatory drug, NSAID, diclofenac) on oxidative stress-related parameters were investigated in the Mediterranean mussel Mytilus galloprovincialis and the Manila clam Ruditapes philippinarum. A flow-through system was used to carry out a three-week exposure experiment with the bivalves. First, the animals were exposed to only three pH values for 7 days. The pH was manipulated by dissolving CO2 in the seawater to obtain two reduced pH treatments (pH −0.4 units and pH −0.7 units), which were compared with seawater at the natural pH level (8.1). Thereafter, the bivalves were concomitantly exposed to the three experimental pH values and environmentally relevant concentrations of diclofenac (0.00, 0.05 and 0.50 μg/L) for an additional 14 days. The activities of superoxide dismutase, catalase and cyclooxygenase, and lipid peroxidation and DNA strand-break formation were measured in both the gills and digestive gland after 7, 14 and 21 days of exposure to each experimental condition. The results show that the biochemical parameters measured in both the mussels and clams were more influenced by the reduced pH than by the contaminant or the pH*contaminant interaction, although the biomarker variation patterns differed depending on the species and tissues analysed. Generally, due to increases in its antioxidant defence, M. galloprovincialis was more resistant than R. philippinarum to both diclofenac exposure and reduced pH. Conversely, reduced pH induced a significant decrease in COX activity in both the gills and digestive gland of clams, possibly resulting in the increased DNA damage observed in the digestive gland tissue.

Camptothecin Efficacy to Poison Top1 Is Altered by Bisphenol A in Mouse Embryonic Fibroblasts.

Bisphenol A (BPA) is used heavily in the production of polycarbonate plastics, thermal receipt paper, and epoxies. Ubiquitous exposure to BPA has been linked to obesity, diabetes, and breast and reproductive system cancers. Resistance to chemotherapeutic agents has also been shown in cancer cell models. Here, we investigated BPA's ability to confer resistance to camptothecin (CPT) in mouse embryonic fibroblasts (MEFs). MEFs are sensitive to CPT; however, co-exposure of BPA with CPT improved cell survival. Co-exposure significantly reduced Top1-DNA adducts, decreasing chromosomal aberrations and DNA strand break formation. This decrease occurs despite BPA treatment increasing the protein levels of Top1. By examining chromatin structure after BPA exposure, we determined that widespread compaction and loss of nuclear volume occurs. Therefore, BPA reduced CPT activity by reducing the accessibility of DNA to Top1, inhibiting DNA adduct formation, the generation of toxic DNA strand breaks, and improving cell survival.

Dose-Rate Effects in Breaking DNA Strands by Short Pulses of Extreme Ultraviolet Radiation.

In this study, we examined dose-rate effects on strand break formation in plasmid DNA induced by pulsed extreme ultraviolet (XUV) radiation. Dose delivered to the target molecule was controlled by attenuating the incident photon flux using aluminum filters as well as by changing the DNA/buffer-salt ratio in the irradiated sample. Irradiated samples were examined using agarose gel electrophoresis. Yields of single- and double-strand breaks (SSBs and DSBs) were determined as a function of the incident photon fluence. In addition, electrophoresis also revealed DNA cross-linking. Damaged DNA was inspected by means of atomic force microscopy (AFM). Both SSB and DSB yields decreased with dose rate increase. Quantum yields of SSBs at the highest photon fluence were comparable to yields of DSBs found after synchrotron irradiation. The average SSB/DSB ratio decreased only slightly at elevated dose rates. In conclusion, complex and/or clustered damages other than cross-links do not appear to be induced under the radiation conditions applied in this study.

Capsicodendrin from Cinnamosma fragrans Exhibits Antiproliferative and Cytotoxic Activity in Human Leukemia Cells: Modulation by Glutathione.

Capsicodendrin (CPCD, 1), an epimeric mixture of a dimeric drimane-type sesquiterpene, is one of the major compounds present in the three endemic species of Madagascan traditional chemopreventive plants: Cinnamosma species ( C. fragrans, C. macrocarpa, and C. madagascariensis). Despite the popular use of Cinnamosma in Madagascan traditional medicine and the reported antiproliferative properties of CPCD, elucidation of its mechanism(s) of action is still to be accomplished. In the present study, CPCD at low micromolar concentrations was cytotoxic and induced apoptosis in human myeloid leukemia cells in a time- and concentration-dependent manner. The activity of CPCD in HL-60 and K562 cells was modulated by glutathione (GSH), since depletion of this intracellular thiol-based antioxidant with buthionine sulfoximine resulted in significantly ( p < 0.05) greater potency in antiproliferation assays. GSH depletion also significantly potentiated the cytotoxic activity in CPCD-treated human HL-60 cells. Single-cell gel electrophoresis (Comet) assays revealed that GSH depletion in HL-60 cells enhanced the formation of DNA strand breaks in the presence of CPCD. Although CPCD does not contain an obvious Michael acceptor in its structure, 1H NMR analyses indicated that cinnamodial (2), a monomer of CPCD, was formed within a few hours when dissolved in DMSO- d6 and interacts with GSH to form a covalent bond via Michael addition at the C-7 carbon. Together the results strongly suggest that 2 is responsible for the DNA-damaging, pro-apoptotic, and cytotoxic effects of CPCD and that depletion of GSH enhances overall activity by diminishing covalent interaction between GSH and this 2-alkenal decomposition product of CPCD.

Genotoxic and mutagenic potential of camphorquinone in L5178/TK+/− mouse lymphoma cells

ObjectivesCamphorquinone (CQ) is the most important photoinitiator used in dental composite resins. Sparse data indicate a mutagenic potential of CQ. Therefore, it was aim of this study to evaluate the cytotoxicity, genotoxicity, and mutagenicity of CQ in L5178Y TK+/− mouse lymphoma cells. MethodsL5178Y/TK+/− cells were exposed to different concentrations of non-irradiated CQ (0.25–2.5mM). Cytotoxicity was evaluated by propidium iodide assay, determination of suspension growth rate, relative total growth and the mitotic index. Intracellular levels of reactive oxygen/nitrogen species (ROS/RNS) were quantified by 2′,7′-dichlorofluoresceine diacetate (DCFH-DA). Early induction of DNA strand breaks and oxidative DNA base lesions was assessed using the 8-hydroxyguanine DNA-glycosylase 1 (hOGG1)-modified alkaline comet assay, whereas mutagenicity of CQ was determined in the mouse lymphoma TK assay (MLA), according to OECD Guideline No. 490. ResultsCQ (0.5–2.5mM) induced concentration- and time-dependent inhibition of cell growth associated with increased ROS/RNS production, amounting to 2342%±1108% of controls after 90min at 2.5mM. Additionally, CQ concentration-dependently caused direct DNA-damage, i.e. formation of DNA strand breaks and 8-hydroxy-2′-deoxyguanosine. Whereas the MLA indicated lack of mutagenicity of CQ after a 4h of treatment, CQ concentration-dependently increased total mutant frequency (MF) after 24h (about 2-fold at 2.5mM). But, based on the global evaluation factor concept, increase in MF did not reach biologically relevance. SignificanceCQ induced concentration-dependent, cytotoxic and genotoxic effects in L5178Y/TK+/− cells, most likely due to oxidative stress, but without mediating obvious biological relevant mutagenicity.

Size-dependent genotoxicity of silver, gold and platinum nanoparticles studied using the mini-gel comet assay and micronucleus scoring with flow cytometry.

Metallic nanoparticles (NPs) are promising nanomaterials used in different technological solutions as well as in consumer products. Silver (Ag), gold (Au) and platinum (Pt) represent three metallic NPs with current or suggested use in different applications. Pt is also used as vehicle exhaust catalyst leading to a possible exposure via inhalation. Despite their use, there is limited data on their genotoxic potential and possible size-dependent effects, particularly for Pt NPs. The aim of this study was to explore size-dependent genotoxicity of these NPs (5 and 50 nm) following exposure of human bronchial epithelial cells. We characterised the NPs and assessed the viability (Alamar blue assay), formation of DNA strand breaks (mini-gel comet assay) and induction of micronucleus (MN) analysed using flow cytometry (in vitro microflow kit). The results confirmed the primary size (5 and 50 nm) but showed agglomeration of all NPs in the serum free medium used. Slight reduced cell viability (tested up to 50 µg/ml) was observed following exposure to the Ag NPs of both particle sizes as well as to the smallest (5 nm) Au NPs. Similarly, at non-cytotoxic concentrations, both 5 and 50 nm-sized Ag NPs, as well as 5 nm-sized Au NPs, increased DNA strand breaks whereas for Pt NPs only the 50 nm size caused a slight increase in DNA damage. No clear induction of MN was observed in any of the doses tested (up to 20 µg/ml). Taken together, by using the comet assay our study shows DNA strand breaks induced by Ag NPs, without any obvious differences in size, whereas effects from Au and Pt NPs were size-dependent in the sense that the 5 nm-sized Au NPs and 50 nm-sized Pt NPs particles were active. No clear induction of MN was observed for the NPs.