Veronika Ehrlich

Use of conventional and -omics based methods for health claims of dietary antioxidants: a critical overview.

This article describes the principles and limitations of methods used to investigate reactive oxygen species (ROS) protective properties of dietary constituents and is aimed at providing a better understanding of the requirements for science based health claims of antioxidant (AO) effects of foods. A number of currently used biochemical measurements aimed of determining the total antioxidant capacity and oxidised lipids and proteins are carried out under unphysiological conditions and are prone to artefact formation. Probably the most reliable approaches are measurements of isoprostanes as a parameter of lipid peroxidation and determination of oxidative DNA damage. Also the design of the experimental models has a strong impact on the reliability of AO studies: the common strategy is the identification of AO by in vitro screening with cell lines. This approach is based on the assumption that protection towards ROS is due to scavenging, but recent findings indicate that activation of transcription factors which regulate genes involved in antioxidant defence plays a key role in the mode of action of AO. These processes are not adequately represented in cell lines. Another shortcoming of in vitro experiments is that AO are metabolised in vivo and that most cell lines are lacking enzymes which catalyse these reactions. Compounds with large molecular configurations (chlorophylls, anthocyans and polyphenolics) are potent AO in vitro, but weak or no effects were observed in animal/human studies with realistic doses as they are poorly absorbed. The development of -omics approaches will improve the scientific basis for health claims. The evaluation of results from microarray and proteomics studies shows that it is not possible to establish a general signature of alterations of transcription and protein patterns by AO. However, it was shown that alterations of gene expression and protein levels caused by experimentally induced oxidative stress and ROS related diseases can be normalised by dietary AO.

Structurally related mycotoxins ochratoxin A, ochratoxin B, and citrinin differ in their genotoxic activities and in their mode of action in human-derived liver (HepG2) cells: Implications for risk assessment

Abstract: To elucidate the effects of three structurally related mycotoxins, namely, ochratoxin A (OTA), ochratoxin B (OTB), and citrinin (CIT), on human health, we investigated their acute toxic, mitogenic, and genotoxic effects in the human-derived liver cell line (HepG2). These compounds are found in moldy foods in endemic areas of nephropathy, which is associated with urinary tract cancers. In agreement with previous experiments, we found that OTA causes a dose-dependent induction of micronuclei (MN) and DNA migration in the single-cell gel electrophoresis (SCGE) assay, which was statistically significant at concentrations of ≥5 μg/ml. In contrast, OTB was devoid of genotoxic activity under identical conditions, but the compound caused pronounced inhibition of cell division even at doses lower than OTA (10 μg/ml). CIT caused an effect similar to that of OTA in MN assays (significant at dose levels of ≥2.5 μg/ml) but was negative in the SCGE test. All compounds failed to induce mutations in Salmonella/microsome assays in strains TA 98 and TA 100 after addition of HepG2-derived enzyme homogenate (S9-mix). By use of DNA-centromeric probes we found that induction of MN by OTA involves chromosome breaking effects (55-60% of the MN were centromere negative), whereas CIT-induced MN were predominantly centromere positive (78-82%). Our findings indicate that OTB is devoid of genotoxic activity in human-derived cells and therefore probably not a genotoxic carcinogen in humans. In contrast, CIT was an equally potent inducer of MN in HepG2 cells as OTA, but this effect is caused by a different mechanism, namely, aneuploidy. Furthermore, our data suggest that combined exposure to structurally related mycotoxins that cause DNA damage via completely different mechanisms may significantly increase the cancer risk of humans consuming moldy foods.

Inhalative exposure to vanadium pentoxide causes DNA damage in workers: results of a multiple end point study.

Background Inhalative exposure to vanadium pentoxide (V2O5) causes lung cancer in rodents. Objective The aim of the study was to investigate the impact of V2O5 on DNA stability in workers from a V2O5 factory. Methods We determined DNA strand breaks in leukocytes of 52 workers and controls using the alkaline comet assay. We also investigated different parameters of chromosomal instability in lymphocytes of 23 workers and 24 controls using the cytokinesis-block micronucleus (MN) cytome method. Results Seven of eight biomarkers were increased in blood cells of the workers, and vanadium plasma concentrations in plasma were 7-fold higher than in the controls (0.31 μg/L). We observed no difference in DNA migration under standard conditions, but we found increased tail lengths due to formation of oxidized purines (7%) and pyrimidines (30%) with lesion-specific enzymes (formamidopyrimidine glycosylase and endonuclease III) in the workers. Bleomycin-induced DNA migration was higher in the exposed group (25%), whereas the repair of bleomycin-induced lesions was reduced. Workers had a 2.5-fold higher MN frequency, and nucleoplasmic bridges (NPBs) and nuclear buds (Nbuds) were increased 7-fold and 3-fold, respectively. Also, apoptosis and necrosis rates were higher, but only the latter parameter reached statistical significance. Conclusions V2O5 causes oxidation of DNA bases, affects DNA repair, and induces formation of MNs, NPBs, and Nbuds in blood cells, suggesting that the workers are at increased risk for cancer and other diseases that are related to DNA instability.

Impact of paper filtered coffee on oxidative DNA-damage: results of a clinical trial.

Coffee is among the most frequently consumed beverages worldwide and epidemiological studies indicate that its consumption is inversely related to the incidence of diseases in which reactive oxygen species (ROS) are involved (liver cirrhosis, certain forms of cancer and neurodegenerative disorders). It has been postulated that antioxidant properties of coffee may account for this phenomenon. To find out if consumption of paper filtered coffee which is the most widely consumed form in Central Europe and the US protects humans against oxidative DNA-damage, a controlled intervention trial with a cross-over design was conducted in which the participants (n=38) consumed 800ml coffee or water daily over 5 days. DNA-damage was measured in peripheral lymphocytes in single cell gel electrophoresis assays. The extent of DNA-migration attributable to formation of oxidised purines (formamidopyrimidine glycosylase sensitive sites) was decreased after coffee intake by 12.3% (p=0.006). Biochemical parameters of the redox status (malondialdehyde, 3-nitrotyrosine and the total antioxidant levels in plasma, glutathione concentrations in blood, intracellular ROS levels and the activities of superoxide dismutase and glutathione peroxidase in lymphocytes) were not markedly altered at the end of the trial, also the urinary 8-isoprostaglandine F2α concentrations were not affected. Overall, the results indicate that coffee consumption prevents endogenous formation of oxidative DNA-damage in human, this observation may be causally related to beneficial health effects of coffee seen in earlier studies.

No Acute and Persistent DNA Damage after an Ironman Triathlon

During acute and strenuous exercise, the enhanced formation of reactive oxygen species can induce damage to lipids, proteins, and nucleic acids. The aim of this study was to investigate the effect of an Ironman triathlon (3.8 km swim, 180 km cycle, 42 km run), as a prototype of ultra-endurance exercise, on DNA stability. As biomarkers of genomic instability, the number of micronuclei, nucleoplasmic bridges, and nuclear buds were measured within the cytokinesis-block micronucleus cytome assay in once-divided peripheral lymphocytes of 20 male triathletes. Blood samples were taken 2 days before, within 20 min after the race, and 5 and 19 days post-race. Overall, the number of micronuclei decreased (P < 0.05) after the race, remained at a low level until 5 days post-race, and declined further to 19 days post-race (P < 0.01). The frequency of nucleoplasmic bridges and nuclear buds did not change immediately after the triathlon. The number of nucleoplasmic bridge declined from 2 days pre-race to 19 days post-exercise (P < 0.05). The frequency of nuclear buds increased after the triathlon, peaking 5 days post-race (P < 0.01) and decreased to basic levels 19 days after the race (P < 0.01). The results suggest that an Ironman triathlon does not cause long-lasting DNA damage in well-trained athletes. (Cancer Epidemiol Biomarkers Prev 2008;17(8):1913–9)

The endonuclease Ankle1 requires its LEM and GIY-YIG motifs for DNA cleavage in vivo

The LEM domain (for lamina-associated polypeptide, emerin, MAN1 domain) defines a group of nuclear proteins that bind chromatin through interaction of the LEM motif with the conserved DNA crosslinking protein, barrier-to-autointegration factor (BAF). Here, we describe a LEM protein annotated in databases as ‘Ankyrin repeat and LEM domain-containing protein 1’ (Ankle1). We show that Ankle1 is conserved in metazoans and contains a unique C-terminal GIY-YIG motif that confers endonuclease activity in vitro and in vivo. In mammals, Ankle1 is predominantly expressed in hematopoietic tissues. Although most characterized LEM proteins are components of the inner nuclear membrane, ectopic Ankle1 shuttles between cytoplasm and nucleus. Ankle1 enriched in the nucleoplasm induces DNA cleavage and DNA damage response. This activity requires both the catalytic C-terminal GIY-YIG domain and the LEM motif, which binds chromatin via BAF. Hence, Ankle1 is an unusual LEM protein with a GIY-YIG-type endonuclease activity in higher eukaryotes.

Use of four new human-derived liver-cell lines for the detection of genotoxic compounds in the single-cell gel electrophoresis (SCGE) assay.

One of the main problems of in vitro genotoxicity assays is that the lack of adequate representation of drug-metabolising enzymes in indicator cell lines that are currently used in routine testing may lead to false results. In the present study, we investigated the ability of four new human-derived livercell lines to detect the DNA-damaging effects of representatives of different classes of genotoxic carcinogens that require metabolic activation, namely the nitrosamine N-nitrosodimethylamine (NDMA), the heterocyclic aromatic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), the polycyclic aromatic hydrocarbon benzo(a)pyrene (B(a)P) and the mycotoxin aflatoxin B1 (AFB1). Hydrogen peroxide (H2O2) was used in all experimental series as a positive control and parallel experiments were carried out with human HepG2 cells, which have been used in earlier studies. DNA damage was monitored in single-cell gel electrophoresis (SCGE) assays. Furthermore, RT-PCR experiments were carried out to study the expression of genes encoding for a panel of different phase-I and phase-II enzymes, which are involved in the activation/detoxification of genotoxic carcinogens. With one of the newly isolated hepatocellular lines, HCC1.2, positive results were obtained with all model compounds, two other new lines (HCC2 and HCC3), HepG2 and the virally immortalized line NKNT-3 were less sensitive and/or failed to detect some of the genotoxins. PCR analyses showed that all cell lines express genes coding for a variety of xenobiotic drug-metabolising enzymes. The highest levels were found in general in HCC1.2, while in NKNT-3 cells some genes were not transcribed. Overall, our results indicate that the line HCC1.2 may be useful for the development of improved in vitro genotoxicity test systems.

Use of human-derived cell lines for the investigation of dietary factors which affect DNA stability

One of the key problems in in vitro genotoxicity testing concerns the adequate representation of xenobiotic drug metabolising enzymes in the indicator cells. Conventional cell lines (V-79, CHO) which are currently used lack most phase I as well as phase II enzymes, which catalyse the activation/detoxification of chemical carcinogens. As shown earlier by our working group, the human cell line HepG2 possesses several phase I/phase II enzymes in an inducible form, but one of the disadvantages of HepG2 cells is their instability and the lack of specific enzymes (e.g. CYP2E1, NAT2, CYP1A2) which are important in the activation of dietary carcinogens (nitrosamines, heterocyclic aromatic amines). Therefore we studies the metabolic capacity of four new isolated human-derived cell lines (HCC1.2, HCC2, HCC3 und NKNT-3) by use of RT-PCR and investigated the sensibility of these lines towards induction of DNA damage by selected representatives of different classes of genotoxic dietary carcinogens which require activation (NDMA, PhIP, Trp-P-1, AFB1, B(a)P). Two of theses cell lines were found to possess a variety of enzymes monitored, including those which are lacking in HepG2, and positive results were obtained in the single cell gel electrophoresis assay with all of the model compounds in the line HCC1.2. Taken together, our results indicate that these cell lines may be highly useful tools for the detection of dietary mutagens and antimutagens. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Testing for Food Safety Using Competent Human Liver Cells

Publisher Summary The risk assessment process, defined as the determination of the probability of harm resulting from exposure to a food component, has to be based on sound scientific data and carried out to internationally agreed standards in a transparent manner. This is essential to ensure consumer confidence in the safety of the food supply, particularly when foods are traded on an international basis. The traditional risk assessment process applied to food additives relies on toxicology testing in vitro and in vivo assays. In vitro assays with metabolically incompetent cells require the addition of exogenous enzyme homogenates to catalyze the activation of genotoxic food derived carcinogens. This process is generally dependent on cytochrome P450 enzymatic activities present in rat liver. In vivo assays in animals use intake levels many times higher than is likely in humans. Employing safety factors then carries out extrapolation of the data to determine the safe level for humans.

Investigation of genotoxic effects of inhalative occupational exposure to vanadium: results of a multiple endpoint study

Animal experiments showed that inhalative exposure to vanadate causes lung cancer. To assess DNA damage in exposed workers (EW, n = 52) of a metal factory, we monitored DNA migration and formation of micronuclei in blood cells of EW, who are exposed to vanadium pentoxide via inhalation and in a matched control group (CG, n = 54). Median level of vanadium serum concentrations of EW (2.23 μg/l) was found to be 7-fold increased compared to CG (0.31 μg/l). In the standard single cell gel electrophoresis (SCGE) assay with leucocytes, no differences were detected between EW and CG, but increased levels of oxidised DNA bases (detected with FPG and ENDO III) were found (p < 0.05). Pretreatment of leukocytes with bleomycin resulted in a higher extent of DNA migration (27%, p < 0.001) and reduced capacity of DNA repair (by 42%, p < 0.001) in EW. Furthermore, the cytochalasin blocked micronucleus (CBMN) assay was carried out with peripheral lymphocytes (n = 24 per group). EW showed a 2.5-fold higher MN frequency as CG and a 7fold increase of nucleoplasmic bridges (which reflect formation of dicentric chromosomes) was seen. Nuclear buds (attributable to gene amplification events) were 3fold higher in EW (p < 0.001 for all events). Apoptosis rates did not differ significantly between the two groups, whereas necrosis rates doubled in EW (p < 0.001). Taken together, our results show increased genetic damage in individuals which inhale vanadium dust, possibly indicating increased risk for cancer.