Siegfried Knasmüller

Single cell gel electrophoresis assay: a new technique for human biomonitoring studies.

Human biomonitoring using the single cell gel electrophoresis (SCGE) or comet assay is a novel approach for the assessment of genetic damage in exposed populations. This assay enables the detection of various forms of DNA damage in individual cells with ease and speed and is, therefore, well suited to the analysis of a large group in a population. Here, application of SCGE assay in the identification of dietary protective factors, in clinical studies and in monitoring the risk of DNA damage resulting from occupational, environmental or lifestyle exposures is reviewed. Also, the comparative sensitivity of SCGE assay and conventional cytogenetic tests to detect genetic damage is discussed. Finally, strengths and shortcomings of the SCGE assay are addressed.

Use of metabolically competent human hepatoma cells for the detection of mutagens and antimutagens.

The human hepatoma line (Hep G2) has retained the activities of various phase I and phase II enzymes which play a crucial role in the activation/detoxification of genotoxic procarcinogens and reflect the metabolism of such compounds in vivo better than experimental models with metabolically incompetent cells and exogenous activation mixtures. In the last years, methodologies have been developed which enable the detection of genotoxic effects in Hep G2 cells. Appropriate endpoints are the induction of 6-TGr mutants, of micronuclei and of comets (single cell gel electrophoresis assay). It has been demonstrated that various classes of environmental carcinogens such as nitrosamines, aflatoxins, aromatic and heterocyclic amines and polycyclic aromatic hydrocarbons can be detected in genotoxicity assays with Hep G2 cells. Furthermore, it has been shown that these assays can distinguish between structurally related carcinogens and non-carcinogens, and positive results have been obtained with rodent carcinogens (such as safrole and hexamethylphosphoramide) which give false negative results in conventional in vitro assays with rat liver homogenates. Hep G2 cells have also been used in antimutagenicity studies and can identify mechanisms not detected in conventional in vitro systems such as induction of detoxifying enzymes, inactivation of endogenously formed DNA-reactive metabolites and intracellular inhibition of activating enzymes.

Detection of genotoxic effects of heavy metal contaminated soils with plant bioassays

Aim of the present study was the development of a bioassay which enables the detection of genotoxic effects of heavy metal contaminated soils. In the first part of the present study, the data base on metal effects in plant bioassays was extended. Four metal salts, namely Cr(VI)O3, Cr(III)Cl3, Ni(II)Cl2 and Sb(III)Cl3 were tested comparatively in MN tests with pollen tetrad cells of Tradescantia clone #4430 and in meristematic root tip cells of Vicia faba. With Cr6+ and Ni2+, clear-cut dose-effects were observed in a range between 0.75 and 10.0 mM, whereas this was not the case with Cr3+ (range tested 1.25-10 mM) and Sb3+ (range 0.30-5.25 mM). In Vicia, negative results were obtained with the four metal salts under all conditions of test. To compare the mutagenic potencies of the metals, the increases of the regression curves (k-values) were calculated, they indicate the number of MN induced per mM in 100 tetrad cells. The corresponding values for Cr6+ and Ni2+ are 0.87 and 1.05, respectively. It appears that the Tradescantia system is in particular sensitive towards those metal species which cause DNA damage in animals and man such as Cr6+, Cd2+, Ni2+, and Zn2+, whereas no clear positive results were obtained with less harmful metal ions such as Cu2+, Cr3+ or Sb3+. In the second part of the study, the mutagenic effects of four metal contaminated soils and two types of standardized leachates (pH 4.0 and pH 7.0) of these soils were tested in Tradescantia and in Vicia. In addition, chemical analyses were carried out to determine the metal concentrations in the soils and in the extracts. Two of the samples contained highly elevated levels of a number of metals (Zn, Pb, Cu, Cd, Sb, As), one soil came from the Central Austrian Alps and contained high As levels only. Direct exposure of the Tradescantia plants in the soils resulted in a drastic increase of the MN frequencies over the background. The lowest effect was seen with the Slovakian soil which contained in particular Sb and As (4.5-fold increase over the background), with the other soils, the induced frequencies were 11-15-fold over the control values. On the contrary, negative results were obtained upon exposure of Tradescantia cuttings in the leachates and upon implantation of germinated Vicia beans in the soils. The results of the present study indicate that Trad-MN assays with direct exposure of intact plants is an appropriate method which enables to detect genotoxic effects of metal contaminated soils in situ. This simple and fast biomonitoring assays might be a valuable supplement to analytical analyses of contaminated soils.

Genotoxic effects of heavy metals: Comparative investigation with plant bioassays

The potential use of micronucleus assays in plants for the detection of genotoxic effects of heavy‐metal ions was investigated. Three different plant systems were comparatively investigated in micronucleus with Tradescantia pollen mother cells (Trad MCN) and micronucleus tests with meristematic root tip cells of Allium cepa and Vicia faba (Allium/MCN).

Effects of cruciferous vegetables and their constituents on drug metabolizing enzymes involved in the bioactivation of DNA-reactive dietary carcinogens

Epidemiological studies give evidence that cruciferous vegetables (CF) protect humans against cancer, and also results from animal experiments show that they reduce chemically induced tumor formation. These properties have been attributed to alterations in the metabolism of carcinogens by breakdown products of glucosinolates, which are constituents of CF. The present article gives an overview on the present state of knowledge on the impact of CF and their constituents on enzymes that are involved in the metabolism of DNA-reactive carcinogens. The development of in vitro models with metabolically competent cell lines led to the detection of potent enzyme inducers contained in CF such as sulforaphane. Recently, we showed that Brassica juices induce glutathione-S-transferases (GST) and cytochrome P-450 1A2 in human hepatoma cells (HepG2) and protect against the genotoxic effects of B(a)P and other carcinogens. Earlier in vivo experiments with rodents indicated that indoles and isothiocyanates, two major groups of glucosinolate breakdown products, attenuate the effects of polycyclic aromatic hydrocarbons (PAHs) and nitrosamines via induction of GST and inhibition of cytochrome-P450 isoenzymes, respectively. Our own investigations showed that CF are also protective towards heterocyclic amines (HAs): Brussels sprouts- and garden cress juices attenuated IQ-induced DNA-damage and preneoplastic lesions in colon and liver of rats. These effects were paralleled by induction of uridine-di-phospho-glucuronosyl transferase (UDPGT) which is very probably the mechanism of protection against HAs by cruciferous vegetables. There is also evidence that consumption of CF might protect humans against cancer. In matched control intervention studies with these vegetables, it was shown that they induce GST-activities in humans but overall, results were inconclusive. Recently, we carried out crossover intervention studies and found pronounced GST-induction upon consumption of Brussels sprouts and red cabbage, whereas no effects were seen with white cabbage and broccoli. Furthermore, we found that the isoenzyme induced was GST-pi which plays an important role in protection against breast, bladder, colon and testicular cancer. No induction of the GST-alpha isoform could be detected. Urinary mutagenicity experiments gave further evidence that CF affect drug metabolism in humans. Consumption of red cabbage led to changes in the pattern of meat-derived urinary mutagenicity. Overall, CF are among the most promising chemopreventive dietary constituents and further elucidation of their protective mechanisms and the identification of active constituents may contribute to the development of highly protective Brassica varieties.

Use of the micronucleus assay with exfoliated epithelial cells as a biomarker for monitoring individuals at elevated risk of genetic damage and in chemoprevention trials.

This review summarises the current database on the micronucleus (MN) assay with exfoliated cells (MEC assay) and evaluates the predictive value of this model for the detection of human cancer risks. The MEC test is a cost effective, non-invasive method, in which the formation of MN in exfoliated cells from different organs, such as oral and nasal cavity, bladder, cervix, and oesophagus is used as an endpoint to detect endogenous, lifestyle, occupational and environmental exposures to genotoxins as well as chemoprotection of various compounds in intervention studies. The results suggest that the MN assay might be a useful approach to identify antimutagens which are protective in humans. Based on the comparison of the data from MN experiments with results from epidemiological cancer studies, we conclude that the MEC assay is a useful biomarker for the detection of human cancer risk in organs to which the MEC test can be applied. However, the current data base is not sufficient to draw a firm conclusion on the specificity of this approach.

Evaluation of the single cell gel electrophoresis assay with human hepatoma (Hep G2) cells

Human Hep G2 cells have retained the activities of phase I and phase II enzymes which are involved in the metabolism of environmental genotoxins. The present study describes the results of single cell gel electrophoresis (SCGE) assays with a panel of different model compounds with these cells. With genotoxic carcinogens such as aflatoxin B(1) (AFB(1)), benzo(a)pyrene (B(a)P), nitrosodimethylamine (NDMA) and cyclophosphamide (CP), statistically significant dose dependent induction of DNA migration was measured. With the two heterocyclic amines, 2-amino-3-methyl-3H-imidazo[4, 5-f]quinoline (IQ) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), and also with rodent carcinogens such as safrole, hexamethylphosphoramide (HMPA) and the pyrrolizidine alkaloid isatidine, which give negative results in other in vitro genotoxicity tests, positive results were obtained in Hep G2/SCGE assays. Nitrosomethylurea (NMU) was the only directly acting compound tested in the study and was by far (ca. 10(3)-fold) more active than the corresponding nitrosamine. The exposure concentrations required to cause significant effects varied over a broad range. The most pronounced effect was seen with AFB(1) (0.008 microM) followed by HMPA (15 microM), B(a)P (25 microM), NMU (100 microM), isatidin (500 microM), CP (900 microM), IQ (1200 microM), safrol (4000 microM), and NDMA (90x10(3) microM). Numbers in parenthesis give the lowest concentrations, which caused a significant increase of DNA migration. With two compounds, namely, the non-carcinogen pyrene and the synthetic hormone tamoxifen (TF), negative results were obtained under all test conditions. These findings are in agreement with the results of recent investigations which indicated that human hepatocytes are unable to convert TF to DNA reactive metabolites, whereas it is activated by rat liver cells and causes DNA adducts in these cells. Comparisons of the present results with data from earlier experiments indicate that the Hep G2/SCGE assay enables the detection of genotoxins including compounds which give misleading results in other in vitro genotoxicity tests and appears to be an alternative to tests with primary liver cells from laboratory animals.

Impact of bacteria in dairy products and of the intestinal microflora on the genotoxic and carcinogenic effects of heterocyclic aromatic amines.

This article gives a short overview on the present state of knowledge of the effects of the intestinal microflora on the health hazards of heterocyclic aromatic amines (HAs). Results of single cell gel electrophoresis assays with conventional, germ free and human flora associated rats indicate that the presence of intestinal microorganisms strongly enhances the induction of DNA-damage in colon and liver cells by IQ. Furthermore, it was found that supplementation of the feed with Lactobacilli attenuates the induction of colon cancer by this same amine. These recent findings suggest that the intestinal microflora and lactic acid bacilli in dairy products strongly affect the health risks of HAs. Nevertheless, most previous experiments with HAs focused on the involvement of mammalian enzymes in the biotransformation of these compounds and only a few articles are available which concern interactions of bacteria with HAs. Some of these studies suggested that the formation of directly mutagenic hydroxy-metabolites of the amines by fecal bacteria might be an important activation pathway but it turned out that the hydroxy-derivative of IQ is not genotoxic in mammalian cells and does not cause colon cancer in laboratory rodents. There is some evidence that hydrolysis of HA-metabolites by bacterial ss-glucuronidase might play a role in the activation of HAs but experimental data are scarce and no firm conclusions can be drawn at present. The most important detoxification mechanism appears to be the direct binding of the HAs to the cell walls of certain bacterial strains contained in fermented foods. It was shown that these effects do also take place under physiologically relevant conditions. Overall, it seems that intestinal bacteria play a key role in the activation and detoxification of HAs which has been an area of research long ignored. The elucidation of these mechanisms may enable the development of biomarkers for colon cancer risk and nutritional strategies of protection.

Enhancement of Glutathione and γ-Glutamylcysteine Synthetase, the Rate Limiting Enzyme of Glutathione Synthesis, by Chemoprotective Plant-Derived Food and Beverage Components in the Human Hepatoma Cell Line HepG2

Glutathione (GSH) is an important antioxidant and cofactor of detoxifying metabolism. Therefore, elevation of GSH as achieved by inducing γ-glutamylcysteine synthetase (GCS), the limiting enzyme of GSH synthesis, may contribute to chemoprevention against cancer. In previous animal studies, increases in GCS were mainly found in liver and other organs that are not easily accessible in humans. Thus, employment and evaluation of alternative systems such as human-derived cell lines are encouraged. In the present experiment, we used the hepatoma cell line HepG2 to investigate the response of GCS and GSH to five plant-derived chemoprotectants contained in regularly consumed foodstuffs and beverages (kahweol/cafestol [K/C] [15.5-62.0 μM], α-angelicalactone [100-400 μM], benzyl isothiocyanate [1.7-5.0 μM], diallyl sulfide [175-700 μM], and quercetin [10-50 μM]). All treatments led to dose-dependent increases in both GCS activity and GSH concentration. Time course studies with K/C indicated that the enhancement of GCS preceded that of GSH, suggesting a causal relationship. K/C did not enhance γ-glutamyl transpeptidase, a further enzyme that assists GSH-related chemoprotection. Although GCS induction has been suggested to require an initial short-lived GSH depletion, we did not find any decrease in GSH after 3 h of incubation with K/C. In summary, HepG2 cells were shown to be a useful model to investigate the capacity of potential chemoprotectants to enhance GCS and GSH. To our knowledge, the present study is also the first to show increases in GCS by K/C and a-angelicalactone in vitro and by diallyl sulfide and quercetin in any system.

Genotoxic effects of crude juices from Brassica vegetables and juices and extracts from phytopharmaceutical preparations and spices of cruciferous plants origin in bacterial and mammalian cells

Crude juices of eight Brassica vegetables as well as juices and extracts of spices and phytopharmaceutical preparations from cruciferous vegetables were tested for induction of point mutations in Salmonella TA98 and TA100, repairable DNA damage in E.coli K-12 cells and clastogenic effects in mammalian cells. In bacterial assays, all juices caused genotoxic effects in the absence of metabolic activation, the ranking order being: Brussels sprouts > white cabbage > cauliflower > green cabbage > kohlrabi > broccoli > turnip > black radish. In experiments with mammalian cells, six juices induced structural chromosome aberrations. Brussels sprouts, white and green cabbage caused the strongest effects (800 microliters of juice induced a 5-fold increase over the background). In sister chromatid exchange assays, positive results were measured as well, but the effects were less pronounced. With all juices the genotoxic effects seen in mammalian cells were paralleled by a pronounced decrease in cell viability. Column fractionation experiments showed that 70-80% of the total genotoxic activity of the juices is found in the fraction which contains isothiocyanates and other breakdown products of glucosinolates, whereas phenolics and flavonoids contributed to a lesser extent to the overall effects. On the basis of these findings, and considering the negative results obtained with non-cruciferous vegetables (tomato, carrot and green pepper), it seems likely that the genotoxic effects of the juices are due to specific constituents of cruciferous plants such as glucosinolates and/or their breakdown products, in particular, isothiocyanates, which we found previously to be potent genotoxins in bacterial and mammalian cells. Finally, spices (mustards and horse radish paste) and phytopharmaceutical preparations were tested in bacterial assays. Mustards and horse radish caused very weak effects while most of the pharmaceutical preparations gave negative results, except cabbage tablets, which caused a strong and dose dependent induction of his revertants in Salmonella TA100. The present findings clearly indicate that cruciferous vegetables contain DNA damaging constituents. These observations are in contrast to earlier findings, which emphasized the antimutagenic effects of vegetable juices and also raise the question whether greatly increased consumption of Brassica vegetables or their concentrated constituents as a means for cancer prevention is indeed recommendable.