Inhibition of survivin expression after using oxaliplatin and vinflunine to induce cytogenetic damage in vitro in lymphocytes from colon cancer patients and healthy individuals.

Chemotherapy drugs usually inflict a lethal dose to tumour cells with the consequence that these cells are being killed by cell death. However, each round of chemotherapy also causes damage to normal somatic cells. The DNA cross-linking agent oxaliplatin (OXP), which causes DNA double-strand breaks, and vinflunine (VFN), which disrupts the mitotic spindle, are two of these chemotherapy drugs which were evaluated in vitro using peripheral lymphocytes from colorectal cancer patients and healthy individuals to determine any differential response. Endpoints examined included micronucleus (MN) induction using the cytokinesis-blocked micronucleus (CBMN) assay and pancentromeric fluorescence in situ hybridisation. Also, survivin expression was monitored since it regulates the mitotic spindle checkpoint and inhibits apoptosis. OXP produced cytogenetic damage (micronuclei in binucleated cells) via its clastogenic but also previously unknown aneugenic action, possibly through interfering with topoisomerase II, whilst VFN produced micronuclei in mononucleated cells because of incomplete karyokinesis. Survivin expression was found to be significantly reduced in a concentration-dependent manner by not only OXP but surprisingly also VFN. This resulted in large numbers of multinucleated cells found with the CBMN assay. As survivin is upregulated in cancers, eliminating apoptosis inhibition might provide a more targeted chemotherapy approach; particularly, when considering VFN, which only affects cycling cells by inhibiting their mitotic spindle, and alongside possibly other pro-apoptotic compounds. Hence, these newly found properties of VFN -the inhibition of survivin expression-might demonstrate a promising chemotherapeutic approach as VFN induces less DNA damage in normal somatic cells compared to other chemotherapeutic compounds.

Analysis of human invasive cytotrophoblasts using multicolor fluorescence in situ hybridization.

Multicolor fluorescence in situ hybridization, or FISH, is a widely used method to assess fixed tissues or isolated cells for numerical and structural chromosome aberrations. Unlike other screening procedures which provide average chromosome numbers for heterogeneous samples, FISH is a sensitive cell-by-cell method to analyze the distribution of abnormal cells in complex tissues. Here, we applied FISH to characterize chromosomal composition of a rare, but very important class of human cells that stabilize the fetal-maternal interface connecting the placenta to the uterine wall during early pregnancy, called invasive cytotrophoblasts (iCTBs). Combining differently-labeled, chromosome-specific DNA probes, we were able to unambiguously determine the number of up to six different autosomes and gonosomes in individual cell nuclei from iCTBs selected on the basis of their invasive behavior. In this manuscript, we describe a method for generation of iCTBs from placental villi, and provide the complete workflow of our FISH experiments including a detailed description of reagents and a trouble-shooting guide. We also include an in-depth discussion of the various types and sources of DNA probes which have evolved considerably in the last two decades. Thus, this communication represents both a complete guide as well as a valuable resource, intended to allow an average laboratory to reproduce the experiments and minimize the amount of specialized, and often costly, equipment.

Cigarette smoke-induced transgenerational alterations in genome stability in cord blood of human F1 offspring.

The relevance of preconceptional and prenatal toxicant exposures for genomic stability in offspring is difficult to analyze in human populations, because gestational exposures usually cannot be separated from preconceptional exposures. To analyze the roles of exposures during gestation and conception on genomic stability in the offspring, stability was assessed via the Comet assay and highly sensitive, semiautomated confocal laser scans of γH2AX foci in cord, maternal, and paternal blood as well as spermatozoa from 39 families in Crete, Greece, and the United Kingdom. With use of multivariate linear regression analysis with backward selection, preconceptional paternal smoking (% tail DNA: P>0.032; γH2AX foci: P>0.018) and gestational maternal (% tail DNA: P>0.033) smoking were found to statistically significantly predict DNA damage in the cord blood of F1 offspring. Maternal passive smoke exposure was not identified as a predictor of DNA damage in cord blood, indicating that the effect of paternal smoking may be transmitted via the spermatozoal genome. Taken together, these studies reveal a role for cigarette smoke in the induction of DNA alterations in human F1 offspring via exposures of the fetus in utero or the paternal germline. Moreover, the identification of transgenerational DNA alterations in the unexposed F1 offspring of smoking-exposed fathers supports the claim that cigarette smoke is a human germ cell mutagen.

Analysis of human invasive cytotrophoblasts demonstrates mosaic aneuploidy.

A total of 24 chromosome-specific fluorescence in situ hybridization probes for interphase nucleus analysis were developed to determine the chromosomal content of individual human invasive cytotrophoblasts derived from in vitro cultured assays. At least 75% of invasive cytotrophoblasts were hyperdiploid and the total number of chromosomes ranged from 47 to 61. The results also demonstrated that these hyperdiploid invasive cytotrophoblasts showed significant heterogeneity. The most copy number gains were observed for chromosomes 13, 14, 15, 19, 21, and 22 with average copy number greater than 2.3. A parallel study using primary invasive cytotrophoblasts also showed a similar trend of copy number changes. Conclusively, 24-chromosome analysis of human non-proliferating cytotrophoblasts (interphase nuclei) was achieved. Hyperdiploidy and chromosomal heterogeneity without endoduplication in invasive cytotrophoblasts may suggest a selective advantage for invasion and short lifespan during normal placental development.

In vitro sensitivities to UVA of lymphocytes from patients with colon and melanoma cancers and precancerous states in the micronucleus and the Comet assays.

To use lymphocytes as surrogate cells to investigate their in vitro sensitivities to ultraviolet (UV) treatment in different cancers and precancerous states by comparison with lymphocytes from healthy control individuals was the main aim of this research. UV light induces precise cellular and genomic mutations. In this study, the effect of ultraviolet A (UVA) (320-400 nm) was used as a generic mutagen to evaluate in vitro different sensitivities from lymphocytes of patients with suspected melanoma (SM), malignant melanoma (MM), polyposis coli (PC) and colorectal cancer (CRC). DNA damage was evaluated by two different methods: the micronucleus (MN) assay and the Comet assay. The baseline frequency of MNs was significantly increased in lymphocytes from all patients (SM, MM, PC and CRC) when compared to healthy individuals. After UV irradiation, MN frequencies were significantly increased in lymphocytes of all groups, both patients and healthy individuals. However, the MN frequency in all patient groups was significantly higher than in the healthy individual group. Similar results for the induction of genomic DNA damage were obtained for the Comet assay. Also for the Comet assay, UVA-induced DNA damage for all four patient groups was significantly increased when compared to healthy individuals (SM, MM, PC and CRC groups: P < 0.001). Conclusively, peripheral lymphocytes from patients with cancers MM and CRC or precancerous states SM and PC are more sensitive to a generic mutagen such as UVA than lymphocytes from healthy individuals. This feature may be used as an essential biomarker to screen and diagnose precancerous states and cancers in early stages.

Bioinformatic Tools Identify Chromosome-Specific DNA Probes and Facilitate Risk Assessment by Detecting Aneusomies in Extra-embryonic Tissues.

Despite their non-diseased nature, healthy human tissues may show a surprisingly large fraction of aneusomic or aneuploid cells. We have shown previously that hybridization of three to six non-isotopically labeled, chromosome-specific DNA probes reveals different proportions of aneuploid cells in individual compartments of the human placenta and the uterine wall. Using fluorescence in situ hybridization, we found that human invasive cytotrophoblasts isolated from anchoring villi or the uterine wall had gained individual chromosomes. Chromosome losses in placental or uterine tissues, on the other hand, were detected infrequently. A more thorough numerical analysis of all possible aneusomies occurring in these tissues and the investigation of their spatial as well as temporal distribution would further our understanding of the underlying biology, but it is hampered by the high cost of and limited access to DNA probes. Furthermore, multiplexing assays are difficult to set up with commercially available probes due to limited choices of probe labels. Many laboratories therefore attempt to develop their own DNA probe sets, often duplicating cloning and screening efforts underway elsewhere. In this review, we discuss the conventional approaches to the preparation of chromosome-specific DNA probes followed by a description of our approach using state-of-the-art bioinformatics and molecular biology tools for probe identification and manufacture. Novel probes that target gonosomes as well as two autosomes are presented as examples of rapid and inexpensive preparation of highly specific DNA probes for applications in placenta research and perinatal diagnostics.

Ex vivo/in vitro effects of aspirin and ibuprofen, bulk and nano forms, in peripheral lymphocytes of prostate cancer patients and healthy individuals.

Inhibiting inflammatory processes or eliminating inflammation represents a logical role in the suppression and treatment strategy of cancer. Several studies have shown that anti-inflammatory drugs (NSAIDs) act as anticancer agents while reducing metastases and mortality rate. NSAIDs are seriously limited by their side effects and toxicity, which can become cumulative with their long-term administration for chemoprevention. In the current ex vivo / in vitro study, the genotoxicity mechanisms of NSAIDS in bulk and nanoparticle forms allowed a strategy to prevent and minimise the damage in human lymphocytes. When compared to their bulk forms, acetylsalicylic acid (Aspirin) nano and ibuprofen nano (IBU N), both NSAIDs in 500 µg/mL concentration significantly decreased DNA damage measured by alkaline comet assay. Micronuclei (MNi) frequency also decreased after ASP N (500 µg/mL), ASP B (500 µg/mL) and IBU N (200 µg/mL) in prostate cancer patients and healthy individuals, however, the ibuprofen bulk (200 µg/mL) showed a significant increase in MNi formation in lymphocytes from healthy and prostate cancer patients when compared to the respective untreated lymphocytes. These findings suggest that a reduction in particle size had an impact on the reactivity of the drug, further emphasising the potential of nanoparticles to improve the current treatment options.

DNA probe pooling for rapid delineation of chromosomal breakpoints.

Structural chromosome aberrations are hallmarks of many human genetic diseases. The precise mapping of translocation breakpoints in tumors is important for identification of genes with altered levels of expression, prediction of tumor progression, therapy response, or length of disease-free survival, as well as the preparation of probes for detection of tumor cells in peripheral blood. Similarly, in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for carriers of balanced, reciprocal translocations benefit from accurate breakpoint maps in the preparation of patient-specific DNA probes followed by a selection of normal or balanced oocytes or embryos. We expedited the process of breakpoint mapping and preparation of case-specific probes by utilizing physically mapped bacterial artificial chromosome clones. Historically, breakpoint mapping is based on the definition of the smallest interval between proximal and distal probes. Thus, many of the DNA probes prepared for multiclone and multicolor mapping experiments do not generate additional information. Our pooling protocol, described here with examples from thyroid cancer research and PGD, accelerates the delineation of translocation breakpoints without sacrificing resolution. The turnaround time from clone selection to mapping results using tumor or IVF patient samples can be as short as 3 to 4 days.

Flavonoids inhibit the genotoxicity of hydrogen peroxide (H(2)O(2)) and of the food mutagen 2-amino-3-methylimadazo[4,5-f]-quinoline (IQ) in lymphocytes from patients with inflammatory bowel disease (IBD).

Inflammatory bowel disease (IBD) including Crohn's disease (CD) and ulcerative colitis (UC) is a chronic inflammatory gastrointestinal autoimmune condition with an inappropriate immune response. We investigated DNA damage induced in vitro in lymphocytes from IBD patients caused by oxidative stress through H(2)O(2) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and whether the plant flavonoids, quercetin and epicatechin, found in fruits, tea and soybeans could effectively reduce such stress. Lymphocytes from IBD patients and healthy volunteers were treated with 50 microg/ml H(2)O(2) or IQ in the presence of quercetin (0-250 microg/ml) or epicatechin (0-100 microg/ml). Flavonoid supplementation (250 microM quercetin or 100 microM epicatechin) caused an overall significant decrease of induced DNA damage resulting in a 48.6% (P < 0.001) reduction of H(2)O(2)-induced and a 43% (P < 0.001) reduction of IQ-induced DNA damage within the patient groups; for the control groups, reductions in DNA damage were 35.2 and 57.1%, respectively (both, P < 0.001). There was less induced DNA damage within lymphocytes from UC patients compared to CD patients for both series of experiments (H(2)O(2) and quercetin, IQ and epicatechin). In conclusion, flavonoids dramatically reduced oxidative stress in vitro in lymphocytes from IBD patients and healthy individuals. Thus, flavonoids could be very effective in the treatment of oxidative stress and encouraged in the diet of IBD patients.

Antioxidants and the Comet assay.

It is widely accepted that antioxidants, either endogenous or from the diet, play a key role in preserving health. They are able to quench radical species generated in situations of oxidative stress, either triggered by pathologies or xenobiotics, and they protect the integrity of DNA from genotoxicants. Nevertheless, there are still many compounds with unclear or unidentified prooxidant/antioxidant activities. This is of concern since there is an increase in the number of compounds synthesized or extracted from vegetables to which humans might be exposed. Despite the well-established protective effects of fruit and vegetables, the antioxidant(s) responsible have not all been clearly identified. There might also be alternative mechanisms contributing to the protective effects for which a comprehensive description is lacking. In the last two decades, the Comet assay has been extensively used for the investigation of the effects of antioxidants and many reports can be found in the literature. The Comet assay, a relatively fast, simple, and sensitive technique for the analysis of DNA damage in all cell types, has been applied for the screening of chemicals, biomonitoring and intervention studies. In the present review, several of the most well-known antioxidants are considered. These include: catalase, superoxide dismutase, glutathione peroxidase, selenium, iron chelators, melatonin, melanin, vitamins (A, B, C and E), carotenes, flavonoids, isoflavones, tea polyphenols, wine polyphenols and synthetic antioxidants. Investigations showing beneficial as well as non-beneficial properties of the antioxidants selected, either at the in vitro, ex vivo or in vivo level are discussed.

Use of spermatozoal mRNA profiles to study gene-environment interactions in human germ cells.

Paternal exposure to genotoxic compounds is thought to contribute to diseases in their offspring. Therefore, it is of importance to develop biomarkers of male germ cell exposure to genotoxins. Unfortunately, the testis cannot be reached for routine biomonitoring, but mRNA-profiles in spermatozoa may reflect the processes that have occurred in the testis after exposures to genotoxins, since spermatozoa are largely transcriptionally inactive. Therefore, mRNA profiles from sperm in ejaculates of cigarette smokers (N=4) were compared with non-smokers (N= 4). Smoking behaviour was verified by assessing cotinine levels in seminal plasma. High expression of the germ cell specific gene protamine 2 (PRM2) was observed in spermatozoal mRNA isolates by Q-PCR, which was absent in reference mRNA isolates obtained from a pool of other organs. Gene-expression analysis was subsequently performed using microarray technology and a total of 781 genes were found to be differentially expressed in spermatozoa of smokers compared to non-smokers (fold change >40%; p < 0.05). To further limit the number of false positive results, genes were additionally selected on basis of the correlation between their expression levels with cotinine concentrations in seminal plasma (r > 0.80 as arbitrary cut-off value, p < 0.05), and a total of 200 transcripts remained, of which the germ cell specific transcription factor SALF was the highest up-regulated gene (5.4-fold) and the zinc finger encoding gene TRIM26 most down regulated (7.4-fold). Although no altered pathways could be identified for the differentially expressed genes, an enrichment was observed for NF-kappaB regulated genes (46% vs. 27%, p = 0.004) playing a central role in stress response. Accordingly, subsequent analysis of transcription factor networks suggests that apoptosis was inhibited in smokers. These data show the feasibility of using gene-expression profiles in mature sperm to elucidate gene-environment interactions in male testis.

Chaga mushroom extract inhibits oxidative DNA damage in lymphocytes of patients with inflammatory bowel disease.

Inflammatory Bowel Disease (IBD) is partly caused by oxidative stress from free radicals and reduced antioxidant levels. Using hydrogen peroxide to induce oxidative stress in vitro in peripheral lymphocytes we investigated the induction of DNA damage supplemented with ethanolic extract of Chaga mushroom as a protective antioxidant. Lymphocytes were obtained from 20 IBD patients and 20 healthy volunteers. For treatment, a constant H_{2}O_{2 } dose (50 microg/ml) was used with variable doses of Chaga extract (10-500 microg/ml). DNA damage was evaluated in 50 cells per individual and dose using the Comet assay (making 1000 observations per experimental point ensuring appropriate statistical power). Chaga supplementation resulted in a 54.9% (p < 0.001) reduction of H_{2}O_{2 } induced DNA damage within the patient group and 34.9% (p < 0.001) within the control group. Lymphocytes from Crohn's disease (CD) patients had a greater basic DNA damage than Ulcerative Colitis (UC) patients (p < 0.001). Conclusively, Chaga extract reduces oxidative stress in lymphocytes from IBD patients and also healthy individuals when challenged in vitro. Thus, Chaga extract could be a possible and valuable supplement to inhibit oxidative stress in general.

An evaluation of DNA damage in human lymphocytes and sperm exposed to methyl methanesulfonate involving the regulation pathways associated with apoptosis.

Exposure to DNA-damaging agents produces a range of stress-related responses. These change the expression of genes leading to mutations that cause cell cycle arrest, induction of apoptosis and cancer. We have examined the contribution of haploid and diploid DNA damage and genes involved in the regulation of the apoptotic process associated with exposure, The Comet assay was used to detect DNA damage and quantitative RT-PCR analysis (qPCR) to detect gene expression changes in lymphocytes and sperm in response to methyl methanesulfonate. In the Comet assay, cells were administered 0-1.2 mM of MMS at 37 °C for 30 min for lymphocytes and 32 °C for 60 min for sperm to obtain optimal survival for both cell types. In the Comet assay a significant increase in Olive tail moment (OTM) and % tail DNA indicated DNA damage at increasing concentrations compared to the control group. In the qPCR study, cells were treated for 4 h, and RNA was isolated at the end of the treatment. qPCR analysis of genes associated with DNA stress responses showed that TP53 and CDKN1A are upregulated, while BCL2 is downregulated compared with the control. Thus, MMS caused DNA damage in lymphocytes at increasing concentrations, but appeared not to have the same effect in sperm at the low concentrations. These results indicate that exposure to MMS increased DNA damage and triggered the apoptotic response by activating TP53, CDKN1A and BCL2. These findings of the processing of DNA damage in human lymphocytes and sperm should be taken into account when genotoxic alterations in both cell types are produced when monitoring human exposure.

Chromosome-specific DNA repeat probes.

In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with alpha-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

Time-course of radiation-induced chromosomal aberrations in tumor patients after radiotherapy.

PURPOSE: Radiation-induced chromosome aberrations are routinely used in biologic dosimetry to monitor radiation exposure. Translocations are considered stable aberrations with time after exposure. This study was performed to determine the temporal persistence of radiation-induced translocations during a 36-month period in therapeutically irradiated testicular seminoma patients who underwent partial body exposure (>10% of bone marrow). METHODS AND MATERIALS: Chromosome analyses were carried out in peripheral lymphocytes of 11 patients with testicular seminoma (n = 9), germinoma (n = 1), or follicular non-Hodgkin's lymphoma (n = 1). All patients received radiotherapy with photons from a linear accelerator; in 1 case, additional electron beams were used. Doses ranged from 26 Gy (seminoma) to 45 Gy (non-Hodgkin's lymphoma). None of the patients received chemotherapy. From each patient, blood samples were taken during the 36 months after irradiation at defined points. Chromosomal aberrations were scored after fluorescence in situ hybridization painting of chromosomes 1, 4, and 12 in combination with a pancentromeric probe. RESULTS: For 9 patients (7 with testicular seminoma, 1 with germinoma, and 1 with non-Hodgkin's lymphoma), a significant temporal decline of translocations, with a mean decline rate of 4.4% +/- 0.4% monthly, could be detected. Two testicular seminoma patients showed no temporal decline of aberration frequencies. CONCLUSION: Most partial body irradiated patients (9 of 11) showed a significant temporal decline of translocation frequencies during a 36-month period. Thus, reciprocal translocations after partial body irradiation cannot be regarded as stable over time. The temporal decline of aberration frequencies has to be taken into account for retrospective dose estimations.

Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay).

Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose-response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.

Male-mediated developmental toxicity.

Male-mediated developmental toxicity has been of concern for many years. The public became aware of male-mediated developmental toxicity in the early 1990s when it was reported that men working at Sellafield might be causing leukemia in their children. Human and animal studies have contributed to our current understanding of male-mediated effects. Animal studies in the 1980s and 1990s suggested that genetic damage after radiation and chemical exposure might be transmitted to offspring. With the increasing understanding that there is histone retention and modification, protamine incorporation into the chromatin and DNA methylation in mature sperm and that spermatozoal RNA transcripts can play important roles in the epigenetic state of sperm, heritable studies began to be viewed differently. Recent reports using molecular approaches have demonstrated that DNA damage can be transmitted to babies from smoking fathers, and expanded simple tandem repeats minisatellite mutations were found in the germline of fathers who were exposed to radiation from the Chernobyl nuclear power plant disaster. In epidemiological studies, it is possible to clarify whether damage is transmitted to the sons after exposure of the fathers. Paternally transmitted damage to the offspring is now recognized as a complex issue with genetic as well as epigenetic components.

Comparative genomic hybridization (CGH) in genotoxicology.

In the past two decades comparative genomic hybridization (CGH) and array CGH have become crucial and indispensable tools in clinical diagnostics. Initially developed for the genome-wide screening of chromosomal imbalances in tumor cells, CGH as well as array CGH have also been employed in genotoxicology and most recently in toxicogenomics. The latter methodology allows a multi-endpoint analysis of how genes and proteins react to toxic agents revealing molecular mechanisms of toxicology. This chapter provides a background on the use of CGH and array CGH in the context of genotoxicology as well as a protocol for conventional CGH to understand the basic principles of CGH. Array CGH is still cost intensive and requires suitable analytical algorithms but might become the dominating assay in the future when more companies provide a large variety of different commercial DNA arrays/chips leading to lower costs for array CGH equipment as well as consumables such as DNA chips. As the amount of data generated with microarrays exponentially grows, the demand for powerful adaptive algorithms for analysis, competent databases, as well as a sound regulatory framework will also increase. Nevertheless, chromosomal and array CGH are being demonstrated to be effective tools for investigating copy number changes/variations in the whole genome, DNA expression patterns, as well as loss of heterozygosity after a genotoxic impact. This will lead to new insights into affected genes and the underlying structures of regulatory and signaling pathways in genotoxicology and could conclusively identify yet unknown harmful toxicants.

The protective effect of the flavonoids on food-mutagen-induced DNA damage in peripheral blood lymphocytes from colon cancer patients.

The food mutagens IQ (2-amino-3-methylimidazo[4,5-f]quinoline) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) are heterocyclic amines (HCA), generated when heating proteinaceous food. This study investigates the protective potential of the flavonoids quercetin (Q) and rutin (R) against oxidative stress induced in vitro by IQ and PhIP in lymphocytes from healthy individuals and untreated, newly diagnosed colon cancer patients using the Comet assay. In the presence of up to 500µM Q and R, the DNA damage resulting from a high dose of PhIP (75µM) or IQ (150µM) was significantly reduced (P<0.001) to levels comparable to six times lower IQ or 7.5 times lower PhIP doses. Lymphocytes from colon cancer patients had greater baseline DNA damage than those from healthy individuals (P<0.01) and this higher level of damage was also observed throughout in vitro treatment. Except for the >50years of age group and male gender, confounding factors such as smoking, drinking and/or dietary habits were not found to be significant. In conclusion, flavonoids reduced oxidative stress caused by food mutagens in vitro in lymphocytes of healthy individuals and colon cancer patients. Thus, dietary supplementation with flavonoid-rich vegetables and fruits may prove very effective in protecting against oxidative stress.

Micronutrients intake is associated with improved sperm DNA quality in older men.

OBJECTIVE: To investigate whether lifestyle factors such as increased dietary intake of micronutrients reduce the risks of sperm DNA damage, and whether older men benefit more than younger men. DESIGN: Cross-sectional study design with equalized assignments into age groups. SETTING: National laboratory and university. PATIENT(S): Nonclinical group of 22-80-year-old nonsmoking men (n = 80) who reported no fertility problems. MAIN OUTCOME MEASURE(S): Sperm DNA damage measured by alkaline and neutral DNA electrophoresis (i.e., sperm Comet assay). RESULT(S): Sociodemographics, occupational exposures, medical and reproductive histories, and lifestyle habits were determined by questionnaire. The average daily dietary and supplement intake of micronutrients (vitamin C, vitamin E, b-carotene, zinc, and folate) was determined using the 100-item Modified Block Food Frequency Questionnaire (FFQ). Men with the highest intake of vitamin C had approximately 16% less sperm DNA damage (alkaline sperm Comet) than men with the lowest intake, with similar findings for vitamin E, folate, and zinc (but not ß-carotene). Older men (>44 years) with the highest vitamin C intake had approximately 20% less sperm DNA damage compared with older men with the lowest intake, with similar findings for vitamin E and zinc. The older men with the highest intake of these micronutrients showed levels of sperm damage that were similar to those of the younger men. However, younger men (<44 years) did not benefit from higher intakes of the micronutrients surveyed. CONCLUSION(S): Men with higher dietary and supplement intake of certain micronutrients may produce sperm with less DNA damage, especially among older men. This raises the broader question of how lifestyle factors, including higher intakes of antioxidants and micronutrients, might protect somatic as well as germ cells against age-associated genomic damage.