The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials.

The in vitro comet assay is a widely applied method for investigating genotoxicity of chemicals including engineered nanomaterials (NMs). A big challenge in hazard assessment of NMs is possible interference between the NMs and reagents or read-out of the test assay, leading to a risk of biased results. Here, we describe both the standard alkaline version of the in vitro comet assay with 12 mini-gels per slide for detection of DNA strand breaks and the enzyme-modified version that allows detection of oxidized DNA bases by applying lesion-specific endonucleases (e.g., formamidopyrimidine DNA glycosylase or endonuclease III). We highlight critical points that need to be taken into consideration when assessing the genotoxicity of NMs, as well as basic methodological considerations, such as the importance of carrying out physicochemical characterization of the NMs and investigating uptake and cytotoxicity. Also, experimental design-including treatment conditions, cell number, cell culture, format and volume of medium on the plate-is crucial and can have an impact on the results, especially when testing NMs. Toxicity of NMs depends upon physicochemical properties that change depending on the environment. To facilitate testing of numerous NMs with distinct modifications, the higher throughput miniaturized version of the comet assay is essential.

How does pycnogenol® influence oxidative damage to DNA and its repair ability in elderly people?

Our purpose in this randomized, double blind, placebo controlled study was to find out the possible effect of a polyphenolic pine bark extract, Pycnogenol® (Pyc) on the level of 8-oxo-7,8-dihydroguanine (8-oxoG) as representative of oxidative damage to DNA and on the DNA repair ability of elderly people. According to our results, three months of Pyc administration had no effect on the level of oxidative damage to DNA or on repair ability, but we found a relationship between the level of 8-oxoG and repair ability of DNA in this group. To conclude, even if the positive effect of Pyc was not confirmed in the case of elderly people it is important to highlight the necessity of further investigations about the mechanisms of Pyc acting on different age groups.

An efficient procedure for genotyping single nucleotide polymorphisms.

Analysis of single nucleotide polymorphisms (SNPs) has been and will be increasingly utilized in various genetic disciplines, particularly in studying genetic determinants of complex diseases. Such studies will be facilitated by rapid, simple, low cost and high throughput methodologies for SNP genotyping. One such method is reported here, named tetra-primer ARMS-PCR, which employs two primer pairs to amplify, respectively, the two different alleles of a SNP in a single PCR reaction. A computer program for designing primers was developed. Tetra-primer ARMS-PCR was combined with microplate array diagonal gel electrophoresis, gaining the advantage of high throughput for gel-based resolution of tetra-primer ARMS-PCR products. The technique was applied to analyse a number of SNPs and the results were completely consistent with those from an independent method, restriction fragment length polymorphism analysis.

Correlations of DNA damage and repair with nuclear and chromosomal damage in HeLa cells caused by methylnitrosamides.

N-Methyl-N-nitrosourethan induces breaks or alkali-labile sites in cellular DNA, many if not all of which are repaired rapidly. Other DNA lesions are repaired by an excision process. Hydroxyurea and 1-beta-D-arabinofuranosylcytosine cause an accumulation of DNA breaks after N-methyl-N-nitrosourethan treatment, probably by inhibiting the DNA-synthetic (but not the nucleolytic) stage of excision repair. Chromosome damage (fragmentation or attenuation of interphase chromosomes and decondensation and radial rearrangement of metaphase chromosomes) is present soon after treatment with N-methyl-N-nitrosourethan and is not reversed during further incubation. It is apparently associated with the longer-lived DNA lesions, probably those which are removed by excision, and is enhanced by incubation with hydroxyurea and 1-beta-D-arabinofuranosylcytosine. N-Methyl-N-nitrosourethan also inhibits cellular protein and the loss of nucleolar structure. N-Methyl-N-nitrosourea is less potent than N-methyl-N-nitrosourethan in causing DNA or chromosome damage and is less cytotoxic.

Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes.

The association between high intake of fruit and vegetables and low incidence of certain cancers is well established. Dietary antioxidants present in these foods are thought to decrease free radical attack on DNA and hence to protect against mutations that cause cancer, but this causal mechanism remains conjectural. We have adopted a molecular epidemiological approach to this question, based on a modified alkaline single-cell gel electrophoresis assay ("comet assay") which specifically detects oxidation of pyrimidines in the DNA of human lymphocytes. In a survey of men 50-59 years of age living in the northeast of Scotland, smokers initially showed significantly more base damage than nonsmokers. Correlations between oxidative base damage and plasma concentrations of various antioxidants were generally negative but not statistically significant. Supplementation of the diet for 20 weeks with vitamin C (100 mg/day), vitamin E (280 mg/day), and beta-carotene (25 mg/day) resulted in a highly significant (P < 0.002) decrease in endogenous oxidative base damage in the lymphocyte DNA of both smokers and nonsmokers. In addition, lymphocytes of antioxidant-supplemented subjects showed an increased resistance to oxidative damage when challenged in vitro with H2O2. These findings strongly support the hypothesis that fruit and vegetables exert a cancer-protective effect via a decrease in oxidative damage to DNA.

Genetic and biochemical characterization of the CHO-UV-1 mutant defective in postreplication recovery of DNA.

The CHO-UV-1 mutant, a Chinese hamster ovary cell with defective postreplication recovery of DNA, is 2- to 4-fold more sensitive than its wild-type counterpart (CHO-77256) to the lethal effects of ethylating agents and UV radiation; it is also hypersensitive (10- to 20-fold) to some DNA-methylating and -cross-linking agents. We studied the CHO-UV-1 mutant further to define its phenotype in terms of DNA damage induction and repair, methyltransferase activity, and effects of caffeine on mutational and lethal responses. Both wild-type and CHO-UV-1 cells incurred similar levels and types of damage when exposed to UV radiation, N-methyl-N'-nitro-N-nitrosoguanidine, or N-methyl-N-nitrosourea. The rate and extent of repair of Micrococcus luteus endonuclease-sensitive sites after UV irradiation or treatment with N-methyl-N'-nitro-N-nitrosoguanidine were also equivalent in these two cell types. Twenty % of the initial endonuclease-sensitive sites induced in either cell line remained at 18 h after UV irradiation; approximately 8% of the sites after N-methyl-N'-nitro-N-nitrosoguanidine exposure were present in both parental and CHO-UV-1 cells after a 17-h repair period. Moreover, the ability of CHO-UV-1 to resynthesize and ligate DNA during excision repair was similar to that of its parent. Neither CHO-UV-1 nor CHO-77256 had appreciable levels of O6-methylguanine-DNA methyltransferase activity which ameliorates the cytotoxicity of alkylating agents. Caffeine, a known inhibitor of postreplication repair, decreased the frequency of mutation induction at the hypoxanthine-guanine phosphoribosyltransferase locus by 40-55% in CHO-77256 but not in CHO-UV-1. These results rule out defective excision repair as a factor in the hypersensitivity of the CHO-UV-1 mutant to DNA-damaging agents. Hence, this cell line appears to derive from a mutation affecting nonexcision repair processes and should be useful in clarifying the mechanism(s) of postreplication recovery of DNA in mammalian cells.

Novel Chinese hamster ultraviolet-sensitive mutants for excision repair form complementation groups 9 and 10.

In this paper we demonstrate that the mutants CHO7PV and CHO4PV isolated by us from the CHO-K1 prol- cell line represent two new complementation groups of UV-sensitive excision repair-defective rodent mutants. We have classified the mutant CHO7PV as representative of Group 9 and CHO4PV as representative of Group 10. Cellular and biochemical characterization of these mutants indicates that they are moderately sensitive to a broad spectrum of mutagens (UV and mono- and bifunctional alkylating agents), partially unable to perform UV-induced DNA repair synthesis, and partially defective in the incision step of the DNA excision repair pathway and in the removal of the two main lesions caused by UV [cyclobutane pyrimidine dimers and (6-4) photo-products]. In terms of UV survival and incision, CHO4PV is apparently more defective than CHO7PV (40% and 50% of wild-type survival, respectively, and 55% and 75% of wild-type incision), whereas when repair DNA synthesis and lesion removal are compared, CHO7PV seems to be more severely affected (30% of wild-type unscheduled DNA synthesis in CHO7PV and 60% in CHO4PV). This suggests a subtlety in the relation between removal of these specific lesions and overall repair capacity and survival.

DNA damage in ultraviolet-irradiated HeLa and CHO-Kl cells examined by alkaline lysis and hydroxyapatite chromatography.

DNA damage and repair activity have been examined in cultured mammalian cells by the technique of alkaline lysis followed by analysis of the single-strand content of DNA by hydroxyapatite chromatography; the rate of unwinding of DNA in alkali is related to the number of single-strand breaks present. When HeLa cells are ultraviolet-irradiated and incubated, on subsequent alkaline lysis the DNA unwinds at a faster rate than the DNA of unirradiated cells. The rate is increased if inhibitors of DNA synthesis (hydroxyurea, deoxyadenosine or 1-beta-D-arabinofuranosyl cytosine) are present during the incubation. Similar effects are seen in CHO-Kl cells synchronised in G1 phase, though mitotic CHO-Kl cells show little effect of post-irradiation incubation or of the presence of inhibitors. It appears that agents known to block replicative DNA synthesis can also inhibit repair DNA synthesis following ultraviolet irradiation, leading to an accumulation of single-strand breaks in DNA produced by repair nuclease activity. This phenomenon is probably responsible for the retarded sedimentation of such DNA on alkaline sucrose gradients.

In vitro repair of oxidative and ultraviolet-induced DNA damage in supercoiled nucleoid DNA by human cell extract.

A human cell extract is capable of carrying out damage-specific incision on supercoiled nucleoid DNA. The DNA breaks are detected by single cell gel electrophoresis. Incubation of the incised DNA with extract and DNA precursors results in resealing of the breaks, with retention of the supercoiled state of the DNA.

Troglitazone inhibits formation of early atherosclerotic lesions in diabetic and nondiabetic low density lipoprotein receptor-deficient mice.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a ligand-activated nuclear receptor expressed in all of the major cell types found in atherosclerotic lesions: monocytes/macrophages, endothelial cells, and smooth muscle cells. In vitro, PPARgamma ligands inhibit cell proliferation and migration, 2 processes critical for vascular lesion formation. In contrast to these putative antiatherogenic activities, PPARgamma has been shown in vitro to upregulate the CD36 scavenger receptor, which could promote foam cell formation. Thus, it is unclear what impact PPARgamma activation will have on the development and progression of atherosclerosis. This issue is important because thiazolidinediones, which are ligands for PPARgamma, have recently been approved for the treatment of type 2 diabetes, a state of accelerated atherosclerosis. We report herein that the PPARgamma ligand, troglitazone, inhibited lesion formation in male low density lipoprotein receptor-deficient mice fed either a high-fat diet, which also induces type 2 diabetes, or a high-fructose diet. Troglitazone decreased the accumulation of macrophages in intimal xanthomas, consistent with our in vitro observation that troglitazone and another thiazolidinedione, rosiglitazone, inhibited monocyte chemoattractant protein-1-directed transendothelial migration of monocytes. Although troglitazone had some beneficial effects on metabolic risk factors (in particular, a reduction of insulin levels in the diabetic model), none of the systemic cardiovascular risk factors was consistently improved in either model. These observations suggest that the inhibition of early atherosclerotic lesion formation by troglitazone may result, at least in part, from direct effects of PPARgamma activation in the artery wall.

A buccal cell model comet assay: development and evaluation for human biomonitoring and nutritional studies.

The comet assay is a widely used biomonitoring tool for DNA damage. The most commonly used cells in human studies are lymphocytes. There is an urgent need to find an alternative target human cell that can be collected from normal subjects with minimal invasion. There are some reports of buccal cells, collected easily from the inside of the mouth, being used in studies of DNA damage and repair, and these were of interest. However, our preliminary studies following the published protocol showed that buccal cells sustained massive damage and disintegrated at the high pH [O. Ostling, K.J. Johanson. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123 (1984) 291-298] used, but that at lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, the aims of this study were to develop a protocol than enabled buccal cell lysis and DNA damage testing in the comet assay, and to use the model to evaluate the potential use of the buccal cell model in human biomonitoring and nutritional study. Specifically, we aimed to investigate intra- and inter-individual differences in buccal cell DNA damage (as strand breaks), the effect of in vitro exposure to both a standard oxidant challenge and antioxidant treatment, as well as in situ exposure to an antioxidant-rich beverage and supplementation-related effects using a carotenoid-rich food. Successful lysis was achieved using 0.25% trypsin for 30 min followed by proteinase K (1mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis (in 0.01 M NaOH, 1mM EDTA, pH 9.1, 18 min at 12 V), a satisfactory comet image was obtained, though inter-individual variation was quite wide. Pre-lysis exposure of cells to a standard oxidant challenge (induced by H2O2) increased DNA strand breaks in a dose related manner, and incubation of cells in Trolox (a water soluble Vitamin E analogue) conferred significant protection (P<0.05) against subsequent oxidant challenge. Exposure of buccal cell in situ (i.e. in the mouth) to antioxidant-rich green tea led to an acute decrease in basal DNA strand breaks. In a controlled human intervention trial, buccal cells from 14 subjects after 28 days' supplementation with a carotenoid-rich berry (Fructus barbarum L.) showed a small but statistically significant (P<0.05) decrease in DNA strand breaks. These data indicate that this buccal cell comet assay is a feasible and potentially useful alternative tool to the usual lymphocyte model in human biomonitoring and nutritional work.

Towards an alternative testing strategy for nanomaterials used in nanomedicine: lessons from NanoTEST.

In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project ( www.nanotest-fp7.eu ) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed.

A reinvestigation of non-disjunction resulting in 47, XXY males of paternal origin.

We have used polymorphisms within the Xp/Yp pseudoautosomal region (PAR 1) to determine the frequency and location of recombination in 80 paternally derived 47, XXY males. Of 64 informative results, there were 10 single cross-overs, one double cross-over and 53 without a cross-over. Therefore 2/3 of 47, XXY males of paternal origin result from meiosis in which the X and Y chromosomes fail to recombine. This failure was not associated with the presence of an increase in recombination in the smaller Xq/Yq pseudoautosomal region (PAR 2) or with the presence of microdeletions within PAR 1.

Molecular epidemiology in cancer research.

The use of molecular biomarkers in epidemiological investigations brings clear advantages of economy, speed and precision. Epidemiology--the study of the factors that control the patterns of incidence of disease--normally requires large numbers of subjects and/or long periods of time, because what is measured (the occurrence of disease) is a rare event. Biomarkers are measurable biological parameters that reflect, in some way, an individual's risk of disease-because they indicate exposure to a causative (or protective) agent, or because they represent an early stage in development of the disease, or because they allow an assessment of individual susceptibility. Biomarkers must be usable on one of the few materials available for biomonitoring of humans, i.e. blood, urine, exfoliated epithelial cells and, with some difficulty, biopsies. The approach of molecular epidemiology has a great potential is several areas of cancer research: investigating the aetiology of the disease; monitoring cancer risk in people exposed to occupational or environmental carcinogens; studying factors that protect from cancer; and assessing intrinsic factors that might predispose to cancer. The biomarkers most commonly employed in cancer epidemiology include: measurements of DNA damage--DNA breaks, altered bases, bulky adducts--in lymphocytes; the surrogate marker of chemical modifications to blood proteins, caused by agents that also damage DNA; the presence of metabolites of DNA-damaging agents (or the products of DNA damage themselves) in urine; chromosome alterations, including translocations, micronuclei and sister chromatid exchange, resulting from DNA damage; mutations in marker genes; DNA repair; and the differential expression of a variety of enzymes, involved in both activation and detoxification of carcinogens, that help to determine individual susceptibility. The molecular approach has been enthusiastically employed in several studies of occupational/environmental exposure to carcinogens. While the estimation of biological markers of exposure has certainly shown the expected effects in terms of DNA damage and adducts, the detection of the biological effects of exposure (e.g. at the level of chromosome alterations) has not been so clear-cut. This is true also when smokers are examined as a group compared with non-smokers. Several markers (especially of chromosome damage and mutation) show a strong correlation with age-indicating either an increasing susceptibility to damage with age, or an accumulation of long-lived changes. DNA repair--a crucial player in the removal of damage before it can cause mutation--may vary between individuals, and may be modulated by intrinsic or extrinsic factors, but limited data are available because of the lack of a reliable assay. Information on other enzymes determining individual susceptibility does exist, and some significant effects of phenotypic or genotypic polymorphisms have emerged, although the interactions between various enzymes make the situation very complex. The important question of whether oxidative DNA damage in normal cells is decreased by dietary antioxidants (vitamin C, carotenoids etc., from fruit and vegetables) has been tackled in antioxidant supplementation experiments. The use of poorly validated assays for base oxidation has not helped us to reach a definitive answer; it seems that, in any case, the level of oxidative damage has been greatly exaggerated. DNA-damaging agents lead to characteristic kinds of base changes (transitions, transversions, deletions). The investigation of the spectrum of mutations in cancer-related genes studied in tumour tissue should lead to a better understanding of the agents ultimately responsible for inducing the tumour. Similarly, studying mutations in a neutral marker gene (not involved in tumorigenesis) can tell us about the origins of the 'background' level of mutations. So far, interpretation of the growing databases is largely speculative. (ABSTRACT

The influence of cell growth, detoxifying enzymes and DNA repair on hydrogen peroxide-mediated DNA damage (measured using the comet assay) in human cells.

Single-cell gel electrophoresis (the comet assay) is a sensitive method for detecting strand breaks at the level of individual cells. Cells embedded in agarose are lysed, electrophoresed, and fluorescently stained. Breaks in the DNA release its supercoiling and allow DNA to extend toward the anode, resembling a comet. We have used the comet assay to investigate the influence of growth state, xenobiotic detoxifying enzymes, and DNA repair processes on the response of cultured human cells to oxidative damage. HepG2 and Caco-2 cells are differentiated liver and colon cell lines, respectively. HeLa and GM1899A cells are relatively unspecialized epithelial and lymphoblastoid cells. Substrate-dependent cells showed a cyclical fluctuation of glutathione (GSH) with respect to growth. Enzyme activities (glutathione reductase, glutathione peroxidase, and catalase) varied considerably between cell types and changed with cell growth state. Hydrogen peroxide induced more DNA damage in actively dividing cells than in confluent cultures. Sensitivity to oxidative injury did not correlate with detoxifying enzyme activity. Rather, differences in susceptibility between cells could be correlated with differences in DNA repair capacity. This study highlights the need to standardize experimental conditions if the comet assay is to be employed in the study of genotoxicity.

DNA damage in diabetes: correlation with a clinical marker.

Levels of DNA damage in groups of 10 patients with insulin-dependent diabetes mellitus and 10 matched controls were compared using the comet assay; DNA strand breaks, oxidized pyrimidines (endonuclease III-sensitive sites) and altered purines (sites sensitive to formamidopyrimidine glycosylase) were measured. Mean values of strand breaks and oxidized pyrimidines were significantly higher in diabetics. Strand breaks correlated with body mass index in the diabetic group. A strong correlation was seen between formamidopyrimidine glycosylase-sensitive sites and serum glucose concentrations. When three patients with normal glucose levels were excluded from the statistical analysis, the mean value of formamidopyrimidine glycosylase-sensitive sites was very significantly elevated compared with normal. DNA damage in lymphocytes is thus a useful marker of oxidative stress, and in particular formamidopyrimidine glycosylase-sensitive sites seem to represent changes specifically related to hyperglycemia.

Anthocyanin-rich extract decreases indices of lipid peroxidation and DNA damage in vitamin E-depleted rats.

Anthocyanins are secondary plant metabolites responsible for the blue, purple, and red color of many plant tissues. The phenolic structure of anthocyanins conveys marked antioxidant activity in model systems via donation of electrons or hydrogen atoms from hydroxyl moieties to free radicals. Dietary intakes of anthocyanins may exceed 200 mg/day, however, little is known about their antioxidant potency in vivo. Consequently, the aim of this study was to establish whether anthocyanins could act as putative antioxidant micronutrients. Rats were maintained on vitamin E-deficient diets for 12 weeks in order to enhance susceptibility to oxidative damage and then repleted with rations containing a highly purified anthocyanin-rich extract at a concentration of 1 g/kg diet. The extract consisted of the 3-glucopyranoside forms of delphinidin, cyanidin, petunidin, peonidin, and malvidin. Consumption of the anthocyanin-repleted diet significantly improved (p <.01) plasma antioxidant capacity and decreased (p <.001) the vitamin E deficiency-enhanced hydroperoxides and 8-Oxo-deoxyguanosine concentrations in liver. These compounds are indices of lipid peroxidation and DNA damage, respectively. Dietary consumption of anthocyanin-rich foods may contribute to overall antioxidant status, particularly in areas of habitually low vitamin E intake.

Analysis of DNA strand breaks, oxidized bases, and glutathione S-transferase P1 in human colon cells from biopsies.

The balance of genetic damage and deactivating enzymes is decisive for cancer risk. To assess these factors in normal human colon cells, we determined background levels of DNA breaks or oxidized bases and of glutathione S-transferases (GSTs) as potential biomarkers of risk and chemoprevention, respectively. Also, genotoxicity by compounds involved in lipid peroxidation was determined to elucidate possible sources of damage. Cells were isolated from sigmoid biopsies of 51 donors and processed with the comet assay to reveal genetic damage. GST proteins were analyzed immunologically. HT29 clone 19A colon tumor cells, resembling primary cells, were treated with 2-trans-hexenal (400 microM) or hydrogen peroxide (75 microM) and processed for damage. Fifteen percent of primary colon cells contained strand breaks; 22% contained additional oxidized bases, with distinct sex differences. Similar damage was found in HT29 clone cells and is induced by both test compounds. GST levels were similar in both cell types. The comet assay is sufficiently sensitive to detect oxidative genetic damage in small amounts of cells from small amounts of biopsies. Lipid peroxidation is a possible risk factor. Together with GST as a potential biomarker of chemoprevention, the technique may serve as a valuable biomarker to assess exposure to risk factors.

Breast cancer diagnosis and prognosis in women following augmentation with silicone gel-filled prostheses.

62 healthy women were studied mammographically before and after augmentation mammoplasty. Postaugmentation mammograms were done using both the implant compression and implant displacement technique. The amount of visualisable tissue was measured in all films before and after augmentation. We concluded: State-of-the-art film-screen mammography is extremely difficult to obtain in most patients augmented with silicone-gel-filled prostheses. On average, there is a decrease in measurable visualised breast tissue after augmentation mammoplasty with silicone-gel-filled prostheses. The area of mammographically measurable tissue is no different whether smooth or textured implants are used. Textured implants are less likely to form an early capsular contracture and are therefore preferred. However, the cancer-causing potential of polyurethane in humans is currently unknown. Anterior breast tissue is generally seen better with displacement mammography; posterior breast tissue with compression mammography. Better films are generally obtained when the implant is in the subpectoral position rather than subglandular. The more severe the capsular contracture, the poorer the mammogram. In addition 42 previously augmented patients developed breast carcinomas an average of 8.4 years after augmentation with silicone-gel-filled implants; 95% had palpable lesions (only 60% of which could be seen on mammography), 90% had infiltrating carcinomas, 45% had metastases to axillary nodes, and 7 patients have recurred, 5 of whom have died. We concluded: Augmented women who develop breast cancer are similar, in terms of tumour size and nodal positivity, to non-augmented breast cancer patients who present with palpable masses. When compared with non-augmented women whose breast cancers are found with screening mammography, augmented patients with breast cancer present with a higher percentage of invasive lesions and involved axillary lymph nodes, resulting in a poorer prognosis. The 40% false negative rate for mammography in this series is unduly high and alarming. Augmentation mammoplasty with silicone-gel-filled implants should be discouraged in women with a high risk of developing breast cancer.

Oxidative damage to DNA: do we have a reliable biomarker?

Oxidized bases in DNA can be measured directly by high-performance liquid chromatography (HPLC). 7,8-Dihydro-8-oxo-guanine (8-OHgua), as the most abundant oxidation product, is often regarded as an indicator of oxidative stress. Estimates of endogenous 8-OHgua levels in human lymphocyte DNA are between 2 and 8 for every 10(5) unaltered bases--a high frequency in view of the potential mutagenicity of this base alteration and of the presence of an effective base excision repair pathway in eukaryotic cells. An alternative approach to the measurement of oxidized bases makes use of repair endonucleases with appropriate lesion specificities--endonuclease III, for oxidized pyrimidines and formamidopyrimidine glycosylase for 8-OHgua. These enzymes introduce breaks at sites of damage in DNA. The comet assay (single cell gel electrophoresis) can then be used to detect the DNA breaks. This modified comet assay, like other enzyme-linked DNA breakage assays, gives a value for endogenous oxidative base damage that is more than 10-fold lower than most estimated from HPLC. It is possible that HPLC-based estimates are artificially high because oxidation of guanine occurs during isolation, storage, or hydrolysis of DNA. Using a revised DNA isolation procedure designed to decrease in vitro oxidation, we have obtained results for 8-OHgua concentrations in human lymphocytes that are closure to the figures obtained by the comet assay. It is still an open question whether 8-OHgua, measured by HPLC or by the comet assay, is a valid marker for oxidative damage.