Intake of a resveratrol-containing dietary supplement has no impact on DNA stability in healthy subjects.

It has been postulated that the beneficial health effects of dietary supplements and of red wines which contain resveratrol (RES) are due to the anti-oxidative properties of this phenolic compound, but evidence for protection against reactive oxygen species is mainly based on results of in vitro experiments and high-dose animal experiments. Aim of this study was to find out if intake of a RES-containing supplement protects healthy humans against oxidative DNA-damage and alters their redox status. Therefore, an intervention trial was conducted in which the participants (n=12) consumed a RES-containing supplement over a period of five days. At the start, after one day and after five days of consumption, and after a washout period DNA stability was measured in single cell gel electrophoresis (SCGE) assays with peripheral blood lymphocytes. These tests were conducted (a) under standard conditions, which reflect single- and double-strand DNA breaks, (b) after treatment of the cells with hydrogen peroxide, which enables detection of alterations of the ROS sensitivity, and (c) by use of formamidopyrimidine DNA-glycosylase (FPG), which provides information on formation of oxidatively damaged bases (pyrimidines). Furthermore, the biochemical parameters TAC (total antioxidant capacity) and oxLDL (oxidized low-density lipoprotein), which reflect the redox status, and C-reactive protein (CRP), a marker of inflammation, were monitored. The intake of the supplement had no significant impact on the DNA stability parameters and on the different biomarkers of the redox status. Our results indicate that intake of 6mg RES per day via the supplement does not cause DNA-protective or antioxidant effects. This amount is equivalent to or lower than that reached after intake of many (ca. 50%) of the RES-containing preparations which are currently on the market in Middle Europe, and is contained in 0.3-2L red wine.

Search for the optimal genotoxicity assay for routine testing of chemicals: Sensitivity and specificity of conventional and new test systems.

Many conventional in vitro tests that are currently widely used for routine screening of chemicals have a sensitivity/specificity in the range between 60 % and 80 % for the detection of carcinogens. Most procedures were developed 30-40 years ago. In the last decades several assays became available which are based on the use of metabolically competent cell lines, improvement of the cultivation conditions and development of new endpoints. Validation studies indicate that some of these models may be more reliable for the detection of genotoxicants (i.e. many of them have sensitivity and specificity values between 80 % and 95 %). Therefore, they could replace conventional tests in the future. The bone marrow micronucleus (MN) assay with rodents is at present the most widely used in vivo test. The majority of studies indicate that it detects only 5-6 out of 10 carcinogens while experiments with transgenic rodents and comet assays seem to have a higher predictive value and detect genotoxic carcinogens that are negative in MN experiments. Alternatives to rodent experiments could be MN experiments with hen eggs or their replacement by combinations of new in vitro tests. Examples for promising candidates are ToxTracker, TGx-DDI, multiplex flow cytometry, γH2AX experiments, measurement of p53 activation and MN experiments with metabolically competent human derived liver cells. However, the realization of multicentric collaborative validation studies is mandatory to identify the most reliable tests.

Recommendations and quality criteria for micronucleus studies with humans.

Micronucleus (MN) analyses in peripheral blood lymphocytes and exfoliated cells from different organs (mouth, nose, bladder and cervix) are at present the most widely used approaches to detect damage of genetic material in humans. MN are extranuclear DNA-containing bodies, which can be identified microscopically. They reflect structural and numerical chromosomal aberrations and are formed as a consequence of exposure to occupational, environmental and lifestyle genotoxins. They are also induced as a consequence of inadequate intake of certain trace elements and vitamins. High MN rates are associated with increased risk of cancer and a range of non-cancer diseases in humans. Furthermore, evidence is accumulating that measurements of MN could be a useful tool for the diagnosis and prognosis of different forms of cancer and other diseases (inflammation, infections, metabolic disorders) and for the assessment of the therapeutic success of medical treatments. Recent reviews of the current state of knowledge suggest that many clinical studies have methodological shortcomings. This could lead to controversial findings and limits their usefulness in defining the impact of exposure concentrations of hazardous chemicals, for the judgment of remediation strategies, for the diagnosis of diseases and for the identification of protective or harmful dietary constituents. This article describes important quality criteria for human MN studies and contains recommendations for acceptable study designs. Important parameters that need more attention include sufficiently large group sizes, adequate duration of intervention studies, the exclusion of confounding factors which may affect the results (sex, age, body mass index, nutrition, etc.), the evaluation of appropriate cell numbers per sample according to established scoring criteria as well as the use of proper stains and adequate statistical analyses.

Micronucleus assays with Tradescantia pollen tetrads: an update.

Micronucleus (MN) assays with early pollen tetrad cells of Tradescantia (Trad-MN assays) are at present the most widely used bioassays with plants for the detection of genotoxins in the environment. So far, ∼ 160 chemicals have been tested and ∼ 100 articles that concern complex environmental mixtures were published. This article summarises the results of Trad-MN studies, which have been carried out during the last 15 years with individual compounds and investigations concerning the pollution of environmental compartments (soil, water and air). The evaluation shows that the effects of certain genotoxins such as heavy metals, radionuclides, pesticides and air pollutants can be easily detected with this test. Comparisons with results obtained in MN studies with mitotic (root tip) cells indicate that meiotic tetrad cells are in general more sensitive. Important issues for future research concern the evaluation of the suitability of wildlife Tradescantia species that are sometimes used instead of specific clones (such as #4430 for which standardised protocols have been developed) as well as the assessment of the predictive value of Trad-MN results in regard to the prediction of cancer hazards in humans and adverse effects at the ecosystem level. The fact that the genotoxic effects of certain compound such as metals, which can be detected with plant bioassays, in particular with the Trad-MN assay but not in other commonly used bioassays (e.g. in bacterial tests) makes them an essential element in the batteries for environmental monitoring.

Impact of infections, preneoplasia and cancer on micronucleus formation in urothelial and cervical cells: A systematic review.

Approximately 165,000 and 311,000 individuals die annually from urothelial (UC) and cervical (CC) cancer. The therapeutic success of these cancers depends strongly on their early detection and could be improved by use of additional diagnostic tools. We evaluated the current knowledge of the use of micronucleus (MN) assays (which detect structural and numerical chromosomal aberrations) with urine- (UDC) and cervix-derived (CDC) cells for the identification of humans with increased risks and for the diagnosis of UC and CC. Several findings indicate that MN rates in UDC are higher in individuals with inflammation and schistosomiasis that are associated with increased prevalence of UC; furthermore, higher MN rates were also found in CDC in women with HPV, Candidiasis and Trichomonas infections which increase the risks for CC. Only few studies were published on MN rates in UDS in patients with UC, two concern the detection of recurrent bladder tumors. Strong correlations were found in individuals with abnormal CC cells that are scored in Pap tests and histopathological abnormalities. In total, 16 studies were published which concerned these topics. MN rates increased in the order: inflammation < ASC-US/ASC-H < LSIL < HSIL < CC. It is evident that MNi numbers increase with the risk to develop CC and with the degree of malignant transformation. Overall, the evaluation of the literature indicates that MNi are useful additional biomarkers for the prognosis and detection of CC and possibly also for UC. In regard to the diagnosis/surveillance of UC, further investigations are needed to draw firm conclusions, but the currently available data are promising. In general, further standardization of the assays is needed (i.e. definition of optimal cell numbers and of suitable stains as well as elucidation of the usefulness of parameters reflecting cytotoxicity and mitotic activity) before MN trials can be implemented in routine screening.

Benzalkonium chloride (BAC) and dimethyldioctadecyl-ammonium bromide (DDAB), two common quaternary ammonium compounds, cause genotoxic effects in mammalian and plant cells at environmentally relevant concentrations.

Quaternary ammonium compounds (QACs) are cationic surfactants that are widely used as disinfectants. In the present study, we tested two important representatives, namely, benzalkonium chloride (BAC) and dimethyldioctadecyl-ammonium bromide (DDAB) in four genotoxicity tests, namely, in the Salmonella/microsome assay with strains TA 98, TA 100 and TA 102, in the single-cell gel electrophoresis (SCGE) assay with primary rat hepatocytes and in micronucleus (MN) assays with peripheral human lymphocytes and with root tip cells of Vicia faba. In the bacterial experiments, consistently negative results were obtained in the dose range between 0.001 and 110 microg per plate in the presence and absence of metabolic activation while significant induction of DNA migration was detected in the liver cells. With BAC, a moderate but significant effect was found with an exposure concentration of 1.0 mg/l while DDAB caused damage at lower doses (0.3 mg/l). The effects were not altered when the nuclei were treated with formamidopyridine glycosylase, indicating that they are not due to formation of oxidized purines. The MN assays with blood cells were carried out under identical conditions to the SCGE experiments and a significant increase was seen at the highest dose levels (BAC: 1.0 and 3.0 mg/l; DDAB: 1 mg/l). Both compounds also caused significant induction of MN as well as inhibition of cell division in plant cells, the lowest effective levels were 1.0 and 10 mg/l for DDAB and BAC, respectively. Our findings show that both chemicals induce moderate but significant genotoxic effects in eukaryotic cells at concentrations which are found in wastewaters and indicate that their release into the environment may cause genetic damage in exposed organisms. Furthermore, the direct contact of humans to QAC-containing detergents and pharmaceuticals that contain substantially higher concentrations than those which were required to cause effects in eukaryotic cells in the present study should be studied further in regard to potential DNA-damaging effects in man.