Folic Acid Supplementation in Postpolypectomy Patients in a Randomized Controlled Trial Increases Tissue Folate Concentrations and Reduces Aberrant DNA Biomarkers in Colonic Tissues Adjacent to the Former Polyp Site.

BACKGROUND: Low folate status is associated with an increased risk of colorectal carcinogenesis. Optimal folate status may be genoprotective by preventing uracil misincorporation into DNA and DNA hypomethylation. Adenomatous polyps have low folate status compared with normal colonic mucosa, and they are surrounded by histologically normal mucosa that also is of low folate status. OBJECTIVE: In a randomized controlled trial conducted at a single Dublin hospital between April 2002 and March 2004, we assessed the effect of folic acid supplementation on tissue folate, uracil misincorporation into DNA, and global DNA hypomethylation in colonocytes isolated from sites of adenomatous polyps and from histologically normal tissue adjacent and 10-15 cm distal to them. METHODS: Twenty patients with adenomatous polyps on initial colonoscopy and polypectomy were randomly assigned to receive either 600 µg folic acid/d [n = 12, 38% men, mean age 64.3 y, and body mass index (BMI, in kg/m(2)) 26.6] or placebo (n = 8, 50% men, mean age 68.4 y, and BMI 27.2) for 6 mo, and then repeat the colonoscopy. Blood and colonocyte tissue folate concentrations were measured with the use of a microbiological assay. Uracil misincorporation and global DNA hypomethylation were measured in colonocytes with the use of modified comet assays. RESULTS: Over time, folic acid supplementation, compared with placebo, increased tissue folate (mean ± SEM) from 15.6 ± 2.62 pg/10(5) cells to 18.1 ± 2.12 pg/10(5) cells (P < 0.001) and decreased the global DNA hypomethylation ratio from 1.7 ± 0.1 to 1.0 ± 0.1 (P < 0.001). The uracil misincorporation ratio decreased by 0.5 ± 0.1 for the site adjacent to the polyp over time (P = 0.05). CONCLUSION: A response to folic acid supplementation, which increased colonocyte folate and improved folate-related DNA biomarkers of cancer risk, was seen in the participants studied. Exploratory analysis points toward the area formerly adjacent to polyps as possibly driving the response. That these areas persist after polypectomy in the absence of folate supplementation is consistent with a potentially carcinogenic field's causing the appearance of the polyp.

Low colonocyte folate is associated with uracil misincorporation and global DNA hypomethylation in human colorectum.

Low folate status is a risk factor for colon carcinogenesis; mechanisms proposed to account for this relationship include uracil misincorporation into DNA and global DNA hypomethylation. We investigated whether such biomarkers are related to folate status in isolated colonocytes from colonoscopy patients. In cases with adenomatous polyps (n = 40) or hyperplastic polyps (n = 16), colonocytes were isolated from biopsies from the polyp, from a site adjacent to the polyp, and from normal mucosa 10-15 cm distal to the polyp. In polyp-free controls (n = 53), biopsies were taken from ascending, transverse, and descending areas of colon. Within adenoma cases, there was a trend (P-trend < 0.001) of decreasing colonocyte folate (pg/105 cells, mean ± CI) from the site distal to the polyp (16.9 ± 2.4), to the site adjacent to the polyp (14.7 ± 2.3), to the polyp (12.8 ± 2.0). Correspondingly, there were increases in uracil misincorporation (P-trend < 0.001) and global DNA hypomethylation (P-trend = 0.012) across the 3 sites. Colonocyte folate concentrations were significantly correlated with RBC folate concentrations, but only in individuals with generally lower (≤484 µg/L) RBC folate status (r = 0.54; P = 0.006; n = 24), and were also significantly lower in normal mucosa of cases with adenomatous polyps than in controls matched for colonic segment. In conclusion, localized folate deficiency in specific areas of colon might create carcinogenic fields and affect the development of colorectal polyps through uracil misincorporation and DNA hypomethylation; alternatively, the polyp itself might deplete folate in the surrounding tissue. Folate supplementation trials aimed at colon cancer prevention should target individuals with suboptimal folate status.

Comet sensitivity in assessing DNA damage and repair in different cell cycle stages.

The comet assay is a sensitive tool for estimation of DNA damage and repair at the cellular level, requiring only a very small number of cells. In comparing the levels of damage or repair in different cell samples, it is possible that small experimental effects could be confounded by different cell cycle states in the samples examined, if sensitivity to DNA damage, and repair capacity, varies with the cell cycle. We assessed this by arresting HeLa cells in various cell cycle stages and then exposing them to ionizing radiation. Unirradiated cells demonstrated significant differences in strand break levels measured by the comet assay (predominantly single-strand breaks) at different cell cycle stages, increasing from G(1) into S and falling again in G(2). Over and above this variation in endogenous strand break levels, a significant difference in susceptibility to breaks induced by 3.5 Gy ionizing radiation was also evident in different cell cycle phases. Levels of induced DNA damage fluctuate throughout the cycle, with cells in G(1) showing slightly lower levels of damage than an asynchronous population. Damage increases as cells progress through S phase before falling again towards the end of S phase and reaching lowest levels in M phase. The results from repair experiments (where cells were allowed to repair for 10 min after exposure to ionizing radiation) also showed differences throughout the cell cycle with G(1)-phase cells apparently being the most efficient at repair and M-phase cells the least efficient. We suggest, therefore, that in experiments where small differences in DNA damage and repair are to be investigated with the comet assay, it may be desirable to arrest cells in a specific stage of the cell cycle or to allow for differential cycle distribution.

Modelling the comet assay.

The single-cell gel electrophoresis technique or comet assay is widely regarded as a quick and reliable method of analysing DNA damage in individual cells. It has a proven track record from the fields of biomonitoring to nutritional studies. The assay operates by subjecting cells that are fixed in agarose to high salt and detergent lysis, thus removing all the cellular content except the DNA. By relaxing the DNA in an alkaline buffer, strands containing breaks are released from supercoiling. Upon electrophoresis, these strands are pulled out into the agarose, forming a tail which, when stained with a fluorescent dye, can be analysed by fluorescence microscopy. The intensity of this tail reflects the amount of DNA damage sustained. Despite being such an established and widely used assay, there are still many aspects of the comet assay which are not fully understood. The present review looks at how the comet assay is being used, and highlights some of its limitations. The protocol itself varies among laboratories, so results from similar studies may vary. Given such discrepancies, it would be attractive to break the assay into components to generate a mathematical model to investigate specific parameters.

In vitro developmental toxicity test detects inhibition of stem cell differentiation by silica nanoparticles.

While research into the potential toxic properties of nanomaterials is now increasing, the area of developmental toxicity has remained relatively uninvestigated. The embryonic stem cell test is an in vitro screening assay used to investigate the embryotoxic potential of chemicals by determining their ability to inhibit differentiation of embryonic stem cells into spontaneously contracting cardiomyocytes. Four well characterized silica nanoparticles of various sizes were used to investigate whether nanomaterials are capable of inhibition of differentiation in the embryonic stem cell test. Nanoparticle size distributions and dispersion characteristics were determined before and during incubation in the stem cell culture medium by means of transmission electron microscopy (TEM) and dynamic light scattering. Mouse embryonic stem cells were exposed to silica nanoparticles at concentrations ranging from 1 to 100 microg/ml. The embryonic stem cell test detected a concentration dependent inhibition of differentiation of stem cells into contracting cardiomyocytes by two silica nanoparticles of primary size 10 (TEM 11) and 30 (TEM 34) nm while two other particles of primary size 80 (TEM 34) and 400 (TEM 248) nm had no effect up to the highest concentration tested. Inhibition of differentiation of stem cells occurred below cytotoxic concentrations, indicating a specific effect of the particles on the differentiation of the embryonic stem cells. The impaired differentiation of stem cells by such widely used particles warrants further investigation into the potential of these nanoparticles to migrate into the uterus, placenta and embryo and their possible effects on embryogenesis.

Systematic random sampling of the comet assay.

The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory 'tail' DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the 'randomness' of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

Use of the comet-FISH assay to demonstrate repair of the TP53 gene region in two human bladder carcinoma cell lines.

The alkaline single-cell gel electrophoresis (comet) assay can be combined with fluorescence in situ hybridization (FISH) methodology to investigate the localization of specific gene domains within an individual cell. The position of the fluorescent hybridization spots in the comet head or tail indicates whether the sequence of interest lies within or in the vicinity of a damaged region of DNA. In this study, we used the comet-FISH assay to examine initial DNA damage and subsequent repair in the TP53 gene region of RT4 and RT112 bladder carcinoma cells after 5 Gy gamma irradiation. In addition to standard comet parameter measurements, the number and location of TP53 hybridization spots within each comet was recorded at each repair time. The results indicate that the rate of repair of the TP53 gene region was fastest during the first 15 min after damage in both cell lines. When compared to overall genomic repair, the repair of the TP53 gene region was observed to be significantly faster during the first 15 min and thereafter followed a rate similar to that for the overall genome. The data indicate that the TP53 domain in RT4 and RT112 cells is repaired rapidly after gamma irradiation. Furthermore, this repair may be preferential compared to the repair of overall genomic DNA, which gives a measure of the average DNA repair response of the whole genome. We suggest that the comet-FISH assay has considerable potential in the study of gene-specific repair after DNA damage.

The effects of atomic force microscopy upon nominated living cells.

This work describes a system for precise re-location of cells within a monolayer after atomic force imaging. As we know little about probe interaction with soft biological surfaces any corroborative evidence is of great importance. For example, it is of paramount importance in living cell force microscopy that interrogated cells can be re-located and imaged by other corroborative technologies. Methodologies expressed here have shown that non-invasive force parameters can be established for specific cell types. Additionally, we show that the same sample can be transferred reliably to an SEM. Results here indicate that further work with live cells should initially establish appropriate prevailing force parameters and that cell damage should be checked for before and after an imaging experiment.

FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair.

Thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1(+) and TK1(-) clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumor suppressor (TP53) and human telomerase reverse transcriptase (hTERT) gene regions, over 1 h after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1(+) compared to TK1(-) cells, a trend also reflected to a lesser degree in genomic DNA repair between the cell-lines. The targeted gene-specific repair in TK(+) cells occurred rapidly, mainly over the first 15 min repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1(+) cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents.

From single cells to tissues: interactions between the matrix and human breast cells in real time.

BACKGROUND: Mammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma--epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis. RESULTS: The formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology. CONCLUSIONS: Localized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs.

Inducing hysteretic spin crossover in solution.

A hysteretic spin transition is induced in a solution assembly of a mononuclear Fe(III) amphiphilic complex which exhibits only gradual spin crossover in the solid state. The hysteretic behavior is dependent on dynamic solution assembly and removal of solvent causes reversion to the original bulk solid magnetic response.

Quantification of nanoparticle uptake by cells using an unbiased sampling method and electron microscopy.

AIMS: By randomly sampling a known fraction of a pellet of cultured cells, we have accurately estimated the mean number of 50 nm gold nanoparticles accumulated within a single cell. Cellular nanoparticle uptake was measured using a combination of stereological sampling techniques and transmission electron microscopy. MATERIALS & METHODS: Nanoparticles were counted individually and their intracellular location was recorded. Quantifying cell and nanoparticle number by analyzing a known fraction of the sample led to precise estimates of intracellular nanoparticle numbers and their spatial locations on an ultrastructural level. We propose a simple and reliable fractionator design and show its applicability and potential using fibroblast cells exposed to 50-nm gold nanoparticles. RESULTS & CONCLUSION: We demonstrate that this approach is suitable for any electron-dense nanomaterial resolvable by electron microscopy and any convex-shaped cells. In addition, the fractionator concept is flexible enough to be used for spatio-temporal or in vivo studies.

Genotoxicity evaluation of amorphous silica nanoparticles of different sizes using the micronucleus and the plasmid lacZ gene mutation assay.

We investigated the potential of four well-characterized amorphous silica nanoparticles to induce chromosomal aberrations and gene mutations using two in vitro genotoxicity assays. Transmission electron microscopy (TEM) was used to verify the manufacturer's nominal size of 10, 30, 80 and 400 nm which showed actual sizes of 11, 34, 34 and 248 nm, respectively. The 80 (34) nm silica nanoparticles induced chromosomal aberrations in the micronucleus assay using 3T3-L1 mouse fibroblasts and the 30 (34) and 80 (34) nm silica nanoparticles induced gene mutations in mouse embryonic fibroblasts carrying the lacZ reporter gene. TEM imaging demonstrated that the majority of nanoparticles were localized in vacuoles and not in the nucleus of 3T3-L1 cells, indicating that the observed DNA damage was most likely a result of indirect mechanisms. Further studies are needed to reveal these mechanisms and to determine the biological relevance of the effects of these particular silica nanoparticles in vivo.

Cryo DualBeam Focused Ion Beam-Scanning Electron Microscopy to Evaluate the Interface Between Cells and Nanopatterned Scaffolds.

With the advance of nanotechnology in biomaterials science and tissue engineering, it is essential that new techniques become available to observe processes that take place at the direct interface between tissue and scaffold materials. Here, Cryo DualBeam focused ion beam-scanning electron microscopy (FIB-SEM) was used as a novel approach to observe the interactions between frozen hydrated cells and nanometric structures in high detail. Through a comparison of images acquired with transmission electron microscopy (TEM), conventional FIB-SEM operated at ambient temperature, and Cryo DualBeam FIB-SEM, the advantages and disadvantages of each technique were evaluated. Ultrastructural details of both (extra)cellular components and cell organelles were best observe with TEM. However, processing artifacts such as shrinkage of cells at the substrate interface were introduced in both TEM and conventional FIB-SEM. In addition, the cellular contrast in conventional FIB-SEM was low; consequently, cells were difficult to distinguish from the adjoining substrate. Cryo DualBeam FIB-SEM did preserve (extra)cellular details like the contour, cell membrane, and mineralized matrix. The three described techniques have proven to be complementary for the evaluation of processes that take place at the interface between tissue and substrate.

Quantification of nanoparticle uptake by cells using microscopical and analytical techniques.

Quantification of nanoparticles in biological systems (i.e., cells, tissues and organs) is becoming a vital part of nanotoxicological and nanomedical fields. Dose is a key parameter when assessing behavior and any potential risk of nanomaterials. Various techniques for nanoparticle quantification in cells and tissues already exist but will need further development in order to make measurements reliable, reproducible and intercomparable between different techniques. Microscopy allows detection and location of nanoparticles in cells and has been used extensively in recent years to characterize nanoparticles and their pathways in living systems. Besides microscopical techniques (light microscopy and electron microscopy mainly), analytical techniques such as mass spectrometry, an established technique in trace element analysis, have been used in nanoparticle research. Other techniques require 'labeled' particles, fluorescently, radioactively or magnetically. However, these techniques lack spatial resolution and subcellular localization is not possible. To date, only electron microscopy offers the resolving power to determine accumulation of nanoparticles in cells due to its ability to image particles individually. So-called super-resolution light microscopy techniques are emerging to provide sufficient resolution on the light microscopy level to image or 'see' particles as individual particles. Nevertheless, all microscopy techniques require statistically sound sampling strategies in order to provide quantitative results. Stereology is a well-known sampling technique in various areas and, in combination with electron microscopy, proves highly successful with regard to quantification of nanoparticle uptake by cells.

Fate and effects of CeO2 nanoparticles in aquatic ecotoxicity tests.

Cerium dioxide nanoparticles (CeO2 NPs) are increasingly being used as a catalyst in the automotive industry. Consequently, increasing amounts of CeO2 NPs are expected to enter the environment where their fate in and potential impacts are unknown. In this paper we describe the fate and effects of CeO2 NPs of three different sizes (14, 20, and 29 nm) in aquatic toxicity tests. In each standard test medium (pH 7.4) the CeO2 nanoparticles aggregated (mean aggregate size approximately 400 nm). Four test organisms covering three different trophic levels were investigated, i.e., the unicellular green alga Pseudokirchneriella subcapitata, two crustaceans: Daphnia magna and Thamnocephalus platyurus, and embryos of Danio rerio. No acute toxicity was observed for the two crustaceans and D. rerio embryos, up to test concentrations of 1000, 5000, and 200 mg/L, respectively. In contrast, significant chronic toxicity to P. subcapitata with 10% effect concentrations (EC10s) between 2.6 and 5.4 mg/L was observed. Food shortage resulted in chronic toxicity to D. magna, for wich EC10s of > or = 8.8 and < or = 20.0 mg/L were established. Chronic toxicity was found to increase with decreasing nominal particle diameter and the difference in toxicity could be explained by the difference in surface area. Using the data set, PNEC(aquatic)S > or = 0.052 and < or = 0.108 mg/L were derived. Further experiments were performed to explain the observed toxicity to the most sensitive organism, i.e., P. subcapitata. Toxicity could not be related to a direct effect of dissolved Ce or CeO2 NP uptake or adsorption, nor to an indirect effect of nutrient depletion (by sorption to NPs) or physical light restriction (through shading by the NPs). However, observed clustering of NPs around algal cells may locally cause a direct or indirect effect.

Reproducible comet assay of amorphous silica nanoparticles detects no genotoxicity.

Genotoxicity of commercial colloidal and laboratory-synthesized silica nanoparticles was tested using the single cell gel electrophoresis or Comet assay. By using a carefully developed protocol and careful characterization of the nanoparticle dispersions, Comet assays were performed on 3T3-L1 fibroblasts with 3, 6, and 24 h incubations and 4 or 40 microg/ml of silica nanoparticles. No significant genotoxicity was observed for the nanoparticles tested under the conditions described, and results were independently validated in two separate laboratories, showing that in vitro toxicity testing can be quantitatively reproducible.

Global DNA and p53 region-specific hypomethylation in human colonic cells is induced by folate depletion and reversed by folate supplementation.

There is increasing evidence to suggest that reduced folate status may be a causative factor in carcinogenesis, particularly colorectal carcinogenesis. Folate is essential for the synthesis of S-adenosylmethionine, the methyl donor required for all methylation reactions in the cell, including the methylation of DNA. Global DNA hypomethylation appears to be an early, and consistent, molecular event in carcinogenesis. We have examined the effects of folate depletion on human-derived cultured colon carcinoma cells using 2 novel modifications to the Comet (single cell gel electrophoresis) assay to detect global DNA hypomethylation and gene region-specific DNA hypomethylation. Colon cells cultured in folate-free medium for 14 d showed a significant increase in global DNA hypomethylation compared with cells grown in medium containing 3 micromol/L folic acid. This was also true at a gene level, with folate-deprived cells showing significantly more DNA hypomethylation in the region of the p53 gene. In both cases, the effects of folate depletion were completely reversed by the reintroduction of folic acid to the cells. These results confirm that decreased folate levels are capable of inducing DNA hypomethylation in colon cells and particularly in the region of the p53 gene, suggesting that a more optimal folate status in vivo may normalize any DNA hypomethylation, offering potential protective effects against carcinogenesis. This study also introduces 2 novel functional biomarkers of DNA hypomethylation and demonstrates their suitability to detect folate depletion-induced molecular changes.