Standards for Quantitative Measurement of DNA Damage in Mammalian Cells.

As the potential applications of DNA diagnostics continue to expand, there is a need for improved methods and standards for DNA analysis. This report describes several methods that could be considered for the production of reference materials for the quantitative measurement of DNA damage in mammalian cells. With the focus on DNA strand breaks, potentially useful methods for assessing DNA damage in mammalian cells are reviewed.

Development of a Reference Method and Materials for Quantitative Measurement of UV-Induced DNA Damage in Mammalian Cells: Comparison of Comet Assay and Cell Viability.

Application of DNA damage diagnostic tests is rapidly growing, in particular for ovarian, prostate, and skin cancers; environmental monitoring; chronic and degenerative diseases; and male infertility. Such tests suffer from significant variability among different laboratories due the lack of standardization, experimental validation, and differences in data interpretation. Reference methods and materials for quantitative measurement of UVA-induced DNA damage in mammalian cells are frequently needed.

Molecular networking-driven isolation of 8'-Glycosylated biscoumarins from Cruciata articulata.

A molecular networking-guided phytochemical investigation of Cruciata articulata led to the isolation of five unreported biscoumarins, four of which were characterized by a shared 6-methoxy-7,8'-dihydroxy-3,7'-biscoumarin aglycone. These were isolated alongside two known coumarin glycosides, daphnetin-8-O-β-D-glucoside and 6'-acetoxy-daphnetin-8-O-β-D-glucoside. Their structures were elucidated by extensive 1D and 2D NMR experiments, in combination with chemical transformation and MS/MS fragmentation analysis.

Cruciasides C-G, monoterpenoid glycosides from Cruciata articulata.

Cruciata articulata (L.) Ehrend. is a herbaceous species distributed in parts of Western Asia and the Mediterranean region.

Feruloyl sucrose derivatives from the root of Xerophyllum tenax.

A phytochemical investigation of the roots of Xerophyllum tenax led to the isolation of three undescribed feruloyl sucrose derivatives along with two known feruloyl sucrose derivatives, heloniosides A and B. This is the first report of their occurrence in the genus Xerophyllum and the family Melanthiaceae. The structures of these compounds were elucidated on the basis of chemical and spectroscopic analysis including 1D and 2D NMR and analysis of MS-MS fragmentation.

Genotoxic Effects of Etoposide, Bleomycin, and Ethyl Methanesulfonate on Cultured CHO Cells: Analysis by GC-MS/MS and Comet Assay.

To evaluate methods for analysis of genotoxic effects on mammalian cell lines, we tested the effect of three common genotoxic agents on Chinese hamster ovary (CHO) cells by single-cell gel electrophoresis (comet assay) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Suspension-grown CHO cells were separately incubated with etoposide, bleomycin, and ethyl methanesulfonate and analyzed by an alkaline comet assay and GC-MS/MS. Although DNA strand breaks were detected by the comet assay after treatment with all three agents, GC-MS/MS could only detect DNA nucleobase lesions oxidatively induced by bleomycin.

Cellular Reference Materials for DNA Damage Using Electrochemical Oxidation.

Reference materials are needed to quantify the level of DNA damage in cells, to assess sources of measurement variability and to compare results from different laboratories. The comet assay (single cell gel electrophoresis) is a widely used method to determine DNA damage in the form of strand breaks. Here we examine the use of electrochemical oxidation to produce DNA damage in cultured mammalian cells and quantify its percentage using the comet assay.

Potential anti-neuroblastoma agents from Juniperus oblonga.

Neuroblastoma (NB) is a neuroendocrine tumor derived from neural crest cells. Approximately 90% of cases occur in children less than 5 years old. The amplification of MYCN correlates with high-risk neuroblastoma and patients with MYCN amplified showed poorer prognosis than those without MYCN amplification.

Unusual Flavones from Primula macrocalyx as Inhibitors of OAT1 and OAT3 and as Antifungal Agents against Candida rugosa.

A bioactivity guided program exploring the interaction of phytochemicals in the entire plant Primula macrocalyx with the organic anion transporters (OAT1 and OAT3) and microorganisms led to the elucidation of ten known flavones (1-4, 6-10, 12) and two previously undescribed flavones (5, 11). The structures of the compounds were determined by extensive analysis of spectroscopic data, as well as by comparison with data from previous reports. Two known flavones (9, 12) are reported for the first time from the family Primulaceae.

Labdane and Abietane Diterpenoids from and Their Cytotoxic Activity.

A phytochemical investigation of the whole plant of led to the isolationof one previously undescribed labdane diterpenoid, (4,5,9,10)-13-des-ethyl-13-oxolabda-8(17),11-dien-19-oic acid (), together with nine known diterpenoids (-, -), two lignans (, ),and a coumarin (). The structures of all the compounds were elucidated on the basis ofspectrometric data, primarily one-dimensional (1D)- and two-dimensional (2D)-NMR and massspectrometry. Electronic circular dichroism (ECD) calculations determined the absoluteconfiguration of .

Biflavonoids from Juniperus oblonga inhibit organic anion transporter 3.

Organic anion transporters (OATs in humans, Oats in rodents) play an important role in the distribution and excretion of numerous endogenous metabolic products and exogenous organic anions, including a host of widely prescribed drugs. Their ligand recognition is also important for drug therapy and development. In this study, the n-butanol and dichloromethane soluble fractions of Juniperus oblonga were found to inhibit OAT3 in vitro and three biflavonoids were found to be responsible for this activity.

Isoflavones from Camphorosma lessingii Inhibit the Organic Anion Transporters OAT1 and OAT3.

Phytochemical investigation of has resulted in the isolation of four previously unreported isoflavones (1: -4: ) and eight known compounds (5: -12: ). Nine of these compounds (1: -6, 8: -10: ) are reported for the first time from members of the family Amaranthaceae. The structures of all isolated compounds were determined by spectroscopic methods, primarily one-dimensional and two-dimensional nuclear magnetic resonance and mass spectrometry.

Controlled potential electro-oxidation of genomic DNA.

Exposure of mammalian cells to oxidative stress can result in DNA damage that adversely affects many cell processes. Lack of dependable DNA damage reference materials and standardized measurement methods, despite many case-control studies hampers the wider recognition of the link between oxidatively degraded DNA and disease risk. We used bulk electrolysis in an electrochemical system and gas chromatographic mass spectrometric analysis (GC/MS/MS) to control and measure, respectively, the effect of electrochemically produced reactive oxygen species on calf thymus DNA (ct-DNA).

Quantifying engineered nanomaterial toxicity: comparison of common cytotoxicity and gene expression measurements.

Background: When evaluating the toxicity of engineered nanomaterials (ENMS) it is important to use multiple bioassays based on different mechanisms of action. In this regard we evaluated the use of gene expression and common cytotoxicity measurements using as test materials, two selected nanoparticles with known differences in toxicity, 5 nm mercaptoundecanoic acid (MUA)-capped InP and CdSe quantum dots (QDs). We tested the effects of these QDs at concentrations ranging from 0.

The Comet Assay: Automated Imaging Methods for Improved Analysis and Reproducibility.

Sources of variability in the comet assay include variations in the protocol used to process the cells, the microscope imaging system and the software used in the computerized analysis of the images. Here we focus on the effect of variations in the microscope imaging system and software analysis using fixed preparations of cells and a single cell processing protocol. To determine the effect of the microscope imaging and analysis on the measured percentage of damaged DNA (% DNA in tail), we used preparations of mammalian cells treated with etoposide or electrochemically induced DNA damage conditions and varied the settings of the automated microscope, camera, and commercial image analysis software.

Electrochemical Potential Gradient as a Quantitative in Vitro Test Platform for Cellular Oxidative Stress.

Oxidative stress in a biological system is often defined as a redox imbalance within cells or groups of cells within an organism. Reductive-oxidative (redox) imbalances in cellular systems have been implicated in several diseases, such as cancer. To better understand the redox environment within cellular systems, it is important to be able to characterize the relationship between the intensity of the oxidative environment, characterized by redox potential, and the biomolecular consequences of oxidative damage.

Scrophularia orientalis extract induces calcium signaling and apoptosis in neuroblastoma cells.

Effective neuroblastoma (NB) treatments are still limited despite treatment options available today. Therefore, this study attempted to identify novel plant extracts that have anticancer effects. Cytotoxicity and increased intracellular calcium levels were determined using the Sulforhodamine B (SRB) assay and Fluo4-AM (acetoxymethyl) staining and fluorescence microscopy in NB cells in order to screen a library of plant extracts.

Functionalization-dependent induction of cellular survival pathways by CdSe quantum dots in primary normal human bronchial epithelial cells.

Quantum dots (QDs) are semiconductor nanocrystals exhibiting unique optical properties that can be exploited for many practical applications ranging from photovoltaics to biomedical imaging and drug delivery. A significant number of studies have alluded to the cytotoxic potential of these materials, implicating Cd-leaching as the causal factor. Here, we investigated the role of heavy metals in biological responses and the potential of CdSe-induced genotoxicity.

Manganese-induced oxidative DNA damage in neuronal SH-SY5Y cells: attenuation of thymine base lesions by glutathione and N-acetylcysteine.

Manganese (Mn) is an essential trace element required for normal function and development. However, exposure to this metal at elevated levels may cause manganism, a progressive neurodegenerative disorder with neurological symptoms similar to idiopathic Parkinson's disease (IPD). Elevated body burdens of Mn from exposure to parental nutrition, vapors in mines and smelters and welding fumes have been associated with neurological health concerns.

Copper oxide nanoparticle mediated DNA damage in terrestrial plant models.

Engineered nanoparticles, due to their unique electrical, mechanical, and catalytic properties, are presently found in many commercial products and will be intentionally or inadvertently released at increasing concentrations into the natural environment. Metal- and metal oxide-based nanomaterials have been shown to act as mediators of DNA damage in mammalian cells, organisms, and even in bacteria, but the molecular mechanisms through which this occurs are poorly understood. For the first time, we report that copper oxide nanoparticles induce DNA damage in agricultural and grassland plants.

NIST gold nanoparticle reference materials do not induce oxidative DNA damage.

One primary challenge in nanotoxicology studies is the lack of well-characterised nanoparticle reference materials which could be used as positive or negative nanoparticle controls. The National Institute of Standards and Technology (NIST) has developed three gold nanoparticle (AuNP) reference materials (10, 30 and 60 nm). The genotoxicity of these nanoparticles was tested using HepG2 cells and calf-thymus DNA.

Standards for immunohistochemical imaging: a protein reference device for biomarker quantitation.

We are developing a reference device to be used in the validation of immunohistochemical imaging of biomarkers by microscopy. The prototype device consists of p53 protein immobilized at various concentrations on a glass slide. The device is designed as a reference control to be used with assays that incorporate commercially available anti-p53 antibodies.

Improved methods and standards for telomerase detection: quantitative histopathology using antibody staining.

Evaluation of telomerase as an early detection biomarker for cancer has been hindered by a lack of reliable methods and standards for in situ histochemical measurement. Improved histochemical methods for measuring telomerase could expedite the acceptance of telomerase as a biomarker for use in diagnostic and clinical applications. The lack of a crystal structure for telomerase coupled with high variability in the antibodies available for immunohistochemical analysis has led to confusion in the literature regarding the binding specificity of these antibodies.

High-throughput DNA diagnostic measurements using capillary electrophoresis: p53, fragile X and telomerase.

Capillary electrophoresis (CE) has become recognized as a powerful tool for the characterization of DNA. It has numerous advantages over slab-gel electrophoresis in that it is fast, highly reproducible and easy to automate. It is well known for its contribution to success in sequencing the human genome, but it is equally important in a wide range of forensic and pharmaceutical applications.

Standards for validation of cancer biomarkers.

As large scale genomics and proteomics efforts identify an increasingly complex list of biomarkers to identify human disease, populations predictive for that disease, and drug or other therapy responses for treatment, attention is needed in the research and development arena to bring initial discoveries to clinical utility. This article reviews the process of biomarker test verification and analytical validation, utilizing measurement standardization. Two such measurement programs are described in this manuscript: the identification of mutations in human mitochondrial DNA, and the measurement of telomerase activity in cancer.