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.

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.

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.

Effects of unconjugated bilirubin on chromosomal damage in individuals with Gilbert's syndrome measured with the micronucleus cytome assay.

Circulating unconjugated bilirubin (UCB) has been reported to protect against lung and colorectal cancer. The present study aimed to explore, for the first time, whether mildly elevated circulating UCB, as found in Gilbert`s syndrome (GS), is associated with changes of DNA damage. A random 76 individuals, matched for age and gender, were recruited from the general population and allocated into the GS group (UCB ≥ 17.1 µM; n = 38) or control group (UCB <17.1 µM; n = 38). Chromosomal and cytological changes were determined in lymphocytes and buccal cells using the cytokinesis-block micronucleus cytome assay (CBMN) and buccal micronucleus cytome assay (BMcyt). No significant differences were found between GS subjects and the control group in the CBMN and BMcyt determined endpoints. Subsequently, when age dependency of effects were analysed, lower formation of buccal micronucleated cells (by 73.3%) and buccal nuclear buds (by 70.9%) in the GS subgroup ≥ 30 years were found, compared to the GS subgroup <30 years. These findings suggest DNA protection in epithelial tissue of older individuals with GS.

Investigations of genotoxic activity of antimicrobial/antiviral agent FS-1 in human lymphocytes and tumor cell.

A very promising antiviral and antimicrobial agent FS-1 was studied for its ability to induce DNA damage and micronuclei in human tumor cell lines HeLa and Caco-2 at concentrations of 200, 500 and 1000 microg/ml without exogenous metabolic activation. The compound was additionally tested for DNA damaging ability in human lymphocytes at concentrations of 200, 400 and 800 microg/ml. Neither DNA damage nor micronucleus formation was observed after treatment of all types of cells with FS-1. Based on these results, FS-1 can be further studied for its safety to humans for potential application in clinical medicine as an antimicrobial/antiviral drug.

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.

Micronuclei and other nuclear anomalies levels in exfoliated buccal cells and DNA damage in leukocytes of patients with polycystic ovary syndrome.

PURPOSE: To evaluate the genetic instability in somatic cells of patients with polycystic ovary syndrome (PCOS) by means of study of micronuclei (MN) level in exfoliated buccal cells and DNA damage in leukocytes. METHODS: The levels of MN in exfoliated buccal cells and DNA damage in leukocytes of 17 PCOS patients and 17 healthy women were studied. Except MN, other nuclear anomalies connected both with genotoxicity and cytotoxicity were evaluated. DNA damage was evaluated by means of the comet or singlecell gel electrophoresis assay in leukocytes. RESULTS: The results of our study showed significantly increased frequencies of MN but not of other nuclear anomalies in exfoliated buccal cells of PCOS patients. DNA in leukocytes was also found significantly damaged compared with healthy females. CONCLUSION: Genetic instability can have very serious consequences for PCOS patients because of established correlations of increased levels of MN and chromosomal aberration with cancer incidence. Hence, more scrupulous investigations in this area are certainly warranted.

The influence of three newly synthesized pyrimidine-containing compounds on micronucleus induction and tumor growth.

PURPOSE: To study the micronucleus (MN)-inducing (both in vivo and in vitro) and antitumor activity in vivo of 3 newly synthesized compounds (DGS-658, DGB-664A and DGS-666), and the influence of these compounds on MN-inducing and antitumor activity of cyclophosphamide (CP). MATERIALS AND METHODS: The compounds were tested for their toxicity and MN-inducing activities in HeLa tumor cell line and Swiss mice. Antitumor activity was studied on mouse Ehrlich ascites carcinoma (EAC) by means of evaluation of tumor (ascites) volume and mean lifespan (MLS). To study the influence of the compounds on MN-inducing effect of CP (30 mg/kg), one hour after i.p. injection, mice were treated with the compounds at doses equal to (1/2) of LD(50) (lethal dose). To study the effect of possible enhancement of antitumor activity, the compounds were injected one hour after CP (at doses equal to maximum tolerated dose / MTD), for 6 consecutive days. One day after the last injection half of the mice with EAC were sacrificed and antitumor activity was assessed by means of ascites volume inhibition. Also the frequency of MN and the number of viable cells (by means of trypan blue exclusion) was evaluated in ascites. The rest of the mice were kept until death and then the MLS was calculated. RESULTS: Only DGS-666 induced significant increase in the number of MN and prolonged the MLS of mice with EAC. Combined action of DGS-658, DGS-664A, DGS-666 and CP showed a significant increase in the number of EAC cells with MN by 17.5%, 23.1% and 50.2%, respectively, compared with CP action (p <0.001). Antitumor effect of combined action of the compounds with CP (based on the ascites volume) was increased compared with CP effect by 17.7% (p >0.05; DGS-658 and DGS-664A) and 28.2% (p <0.001; DGS- 666). Combined action of CP and the DGS-658, DGS-664A, DGS-666 prolonged significantly the MLS of mice compared with CP action by 51.2%, 56.0% and 110.4%, respectively (p <0.001). CONCLUSION: These newly synthesized compounds, practically inactive in MN induction and possessing no or slight antitumor activity, increased significantly the mutagenic and antitumor activity of CP, one of the most frequently used chemotherapeutic agents in clinical oncology. The compounds are practically non-toxic making them very attractive for further studies.

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.

The micronucleus assay in exfoliated human cells: a mini-review of papers from the CIS.

The critical analysis of the data concerning micronucleus assay in exfoliated epithelial cells presented by the investigators from the CIS is carried out. Twenty two articles are evaluated, and shortcomings of some of them are discussed. Presented results are compared whenever possible with literature data. The aim of the mini-review is a criticism of shortcomings of the papersforfurther improvement of the presentation of the data on micronucleus assay which will give the possibility to compare the results with the data presented by foreign investigators.

Possible role of the micronucleus assay in diagnostics and secondary prevention of cervix cancer: a minireview.

Recent literature data are presented concerning micronuclei (MN) frequency in exfoliated cells of cervix cancer patients. These data strongly support a positive correlation between the MN level and malignization (changes from premalignant stage to cancer). It is suggested that the evaluation of frequency of MN in exfoliated cervical cells may be an additional criterion for establishing cervical cancer risk and the study of MN in cervix smears will increase the sensitivity and specificity of cytology which could impact in diagnostics and secondary prevention of cervical cancer.

Relationship between the chemical structures and biological activities (toxicity, mutagenic and antitumor) in newly synthesized derivatives of pyrazolo pyrimidines.

The relationship between chemical structure, micronucleus-inducing and antitumor activities was studied in four newly synthesized pyrazolo pyrymidine compounds (DGB-216, DGB-227, DGB-228 and DGB-331). In bone marrow erythrocytes of mice no one of compounds was active. Only DGB-216 has slight antitumor activity and increases the mean life span of mice with Ehrlich ascite carcinoma by 11%, while others were practically non-active. Changes in the chemical structures of the compounds lead to substantial changes in the acute toxicity only. The search of antitumor compounds among the derivatives of -6-etoxycarbonyl-pyrazolo[1,5a]-pyrymidine and -2-methyl-pyrazolo[1,5a]-pyrymidine is useless, as it has been shown in the present investigation. But the search of compounds with antitumor properties among derivatives of pyrazolo pyrymidines is a perspective idea because recently some very active antitumor compounds based on mentioned strusture were synthesized in Italy and the USA.

Micronucleus-inducing and antitumor activity of three newly synthesized bridged nitrogen atom-containing pyrimidines.

PURPOSE: To study micronucleus (MN)-inducing and antitumor activity of 3 newly synthesized compounds having condensed nitrogen-containing heterocyclic structures with a bridged nitrogen atom (code numbers - DGB-216, DGS-618 and DGS-623). MATERIALS AND METHODS: The compounds were tested for MN-inducing activity in SH-SY5Y and HeLa tumor cell lines at doses close to IC50 (assessed by means of trypan blue dye exclusion technique ) and 1/2 of IC50 after 24 h incubation without recovery time. In parallel, apoptotic cells were also registered after 24 h incubation of cells with the compounds, staining with Hoechst 33258 and investigation under fluorescent microscope. The compounds were also studied in albino mice bone marrow cells at doses of 1/2, 1/5 and 1/10 of LD50 injected intraperitoneally (i.p.) twice at 0 and 24 h and preparing bone marrow smears 24 h after the last injection. The antitumor activity of the compounds was studied on mouse Ehrlich ascites carcinoma assay by measuring the mean survival time. RESULTS: Only DGS-618 showed cytotoxity at concentrations close to 75.0-80.0 microg/ml; the others were not cytoxic at concentration about 250.0 microg/ml. No one substance induced significant number of cells with MN and apoptosis compared with the negative control. Only DGS-618 was slightly mutagenic in MN-assay at dose of 1/2 of LD50. In contrast, this compound was absolutely inactive in Ehrlich tumor assay. Only DGS-623 was active and induced significant increase in the mean lifespan of mice by 31.0-24.0% in 2 experiments. CONCLUSION: The compound DGS-618 which does not induce MN both in vivo and in vitro and shows antitumor activity in vivo is worth testing in other tumor models. Recent publications show that the search of antitumor agents among pyrazolyl-pyrimidine-containing compounds could be successful because some of them synthesized in the USA and Japan possess expressed antitumor activity.

Chromosomal aberrations level in peripheral blood lymphocytes of women with polycystic ovary syndrome.

PURPOSE: Polycystic ovary syndrome (PCOS), characterized by polycystic ovaries, hyperandrogenism, chronic anovulation and hirsutism, is a common endocrine disease in females worldwide. Many investigations have shown oxidative stress in such patients and the relationship between genetic instability and oxidative stress is well known. The aim of the present study was to investigate the background chromosomal aberrations (CAs) level in lymphocytes of females with PCOS. PATIENTS AND METHODS: Fifteen females, diagnosed with PCOS (hirsutism score >6; significantly increased level of testosterone in blood; increased ovarian volume) and 15 healthy women of similar physical parameters (controls) were included in this investigation. The frequency of CAs in cultures lymphocytes was used as a biomarker of cytogenetic damage. RESULTS: The frequencies of all types of CAs were significantly higher in patients with PCOS, and the mitotic index was significantly lower. CONCLUSION: Females with PCOS have increased CAs level in lymphocytes which is a sign of genetic instability.

Methods for the detection of antioxidants which prevent age related diseases: a critical review with particular emphasis on human intervention studies.

It is well documented that reactive oxygen species (ROS) are involved in the aetiology of age related diseases. Over the last decades, strong efforts have been made to identify antioxidants in human foods and numerous promising compounds have been detected which are used for the production of supplements and functional foods. The present paper describes the advantages and limitations of methods which are currently used for the identification of antioxidants. Numerous in vitro methods are available which are easy to perform and largely used in screening trials. However, the results of such tests are only partly relevant for humans as certain active compounds (e.g. those with large molecular configuration) are only poorly absorbed in the gastrointestinal tract and/or may undergo metabolic degradation. Therefore experimental models are required which provide information if protective effects take place in humans under realistic conditions. Over the last years, several methods have been developed which are increasingly used in human intervention trials. The most widely used techniques are chemical determinations of oxidised guanosine in peripheral blood cells or urine and single cell gel electrophoresis (comet) assays with lymphocytes which are based on the measurement of DNA migration in an electric field. By using of DNA-restriction enzymes (formamidopyrimidine DNA glycosylase and endonuclease III) it is possible to monitor the endogenous formation of oxidised purines and pyrimidines; recently also protocols have been developed which enable to monitor alterations in the repair of oxidised DNA. Alternatively, also the frequency of micronucleated cells can be monitored with the cytokinesis block method in peripheral human blood cells before and after intervention with putative antioxidants. To obtain information on alterations of the sensitivity towards oxidative damage, the cells can be treated ex vivo with ROS (H(2)O(2) exposure, radiation). The evaluation of currently available human studies shows that in approximately half of them protective effects of dietary factors towards oxidative DNA-damage were observed. Earlier studies focused predominantly on the effects of vitamins (A, C, E) and carotenoids, more recently also the effects of fruit juices (from grapes, kiwi) and beverages (soy milk, tea, coffee), vegetables (tomato products, berries, Brussels sprouts) and other components of the human diet (coenzyme Q(10), polyunsaturated fatty acids) were investigated. On the basis of the results of these studies it was possible to identify dietary compounds which are highly active (e.g. gallic acid). At present, strong efforts are made to elucidate whether the different parameters of oxidative DNA-damage correlates with life span, cancer and other age related diseases. The new techniques are highly useful tools which provide valuable information if dietary components cause antioxidant effects in humans and can be used to identify individual protective compounds and also to develop nutritional strategies to reduce the adverse health effects of ROS.