Genotoxicity of water samples from Dianchi lake detected by the Vicia faba micronucleus test.

Dianchi Lake covers about a 300 km2 area. Kunming city on the edge of the lake is surrounded by industrial establishments. Farm land surrounds the remaining areas of the lake. The lake water is polluted by the Kunming city municipal sewage from 3 million inhabitants, the industrial effluent and farm runoff. Water samples were collected from 12 sites along the shore of the lake during the dry (May) and rainy (August) seasons for genotoxicity testing with the Vicia micronucleus assay during the year 1995. Genotoxicity in terms of micronuclei (MCN) frequencies in the root tip cells of Vicia showed a consistent elevated frequency of MCN over the control sample at the 0.05-0.01 levels of significance in both seasons. The MCN frequencies of sites A (Daguanhe), B (Gaoqiao) and C (Xiyuan) were 3.5-4 times as high as the control values (5.25/1000 cells) in the dry season. In the rainy season, the MCN frequencies of water samples from most of the 12 sites were relatively lower than those of the dry season except sites J (Haikou), and K (Kunyang). The average MCN frequency of the dry season samples was 14.97 per 1000 cells and that of the rainy season samples was 12.24 per 1000 cells while the average control value was around 5.00/1000 cells.     

Mutagenicity of instant coffee and its interaction with dimethylnitrosamine in the micronucleus test

The mutagenicity of instant coffee and its interaction with dimethylnitrosamine (DMN) were examined in mice using the micronucleus test. Although neither a single nor multiple administration of instant coffee by gavage induced a significant rise in micronucleated cells over the dose range tested (100-2500 mg/kg), there was a tendency for the number of micronucleated cells to increase in a dose-related fashion. When coffee was administered with DMN, the difference in the frequency of micronucleated cells was small in comparison to a single treatment with DMN alone, thus indicating a lack of synergism between coffee and DMN.     

Radiosensitivity detected by the micronucleus test is not generally increased in sporadic prostate cancer patients

The micronucleus test (MNT) has shown increased micronuclei (MN) frequencies in BRCA associated and sporadic breast cancer patients, Ataxia telangiectasia and Nijmegen Breakage Syndrome patients, demonstrating a common cellular phenotype of increased radiosensitivity. Some genes, causative of these diseases, have also recently been associated with prostate cancer. In order to investigate if prostate cancer exhibits the cellular phenotype of increased radiosensitivity, we performed MNT analysis on 22 sporadic prostate cancer patients and 43 male controls. We determined the baseline MN frequency, in order to see in vivo chromosomal damage without radiation, and induced (after irradiation with 2 Gy) frequency of MN, both in binucleated cells (BNC) obtained from cultured peripheral blood lymphocytes. An automated image analysis system was used to score the MN employing two different classifiers (Classifier A and B) for detection of BNC. The mean baseline frequencies were 48/43 MN/1000 BNC (A/B) for the controls and 42/50 (A/B) for prostate cancer patients. The induced MN frequencies amounted to 107/111 MN/1000 BNC (A/B) for controls and 111/114 MN/1000 BNC (A/B) for prostate cancer patients. The obtained MN frequencies did not result in a statistically significant difference between unselected cases and controls. However, restricting the analysis to young patients (50-60 years, N = 7) and age-matched controls (N = 7) revealed marginally significant higher MN frequencies in patients. We conclude that increased radiosensitivity is not a property of prostate cancer patients in general.     

Genotoxic effects of butadiene in mouse lung cells detected by an ex vivo micronucleus test

Lung fibroblasts from BD-exposed mice have been analysed for the occurrence of micronuclei. Primary cultures set up 24h after the end of exposure were treated with cytochalasin B and micronuclei scored in binucleate cells. A three-fold statistically significant increase of micronucleated cells was detected after exposure to 500ppm, the lowest tested concentration. A linear dose effect relationship was observed between 500 and 1300ppm. Immunofluorescent staining of kinetochore proteins was applied to distinguish between acentric micronuclei produced by chromosome breaks and micronuclei containing a centromeric region, most likely induced by chromosome loss. A statistically significant increase of both types of MN in 1300ppm-exposed females and a significant increase in centromeric MN in 500ppm-exposed males were detected. These data demonstrate that an intermediate of BD metabolism with a potential for clastogenic and aneugenic effects is active in lung cells after inhalation exposure. These effects can play a role in the initiation and promotion of BD-induced lung tumours.     

The micronucleus assay in Anodonta cygnea for the detection of drinking water mutagenicity

A micronucleus test in gill cells of the freshwater mussel Anodonta cygnea has been proposed for the detection of drinking water genotoxicity. Animals were exposed for 28 days to a drinking water sample and collected every week. Highly significant increases in spontaneous MN frequency were observed at each sampling, especially after 13 days of exposure. As positive control 2 doses of mytomicin C (MMC) were used (10(-8) and 10(-7) M). A second experiment was performed at a municipal waterworks in order to assess the role of water treatment processes in the production of mutagenic compounds. The most prevalent genotoxic effects were detected after chlorination (mean: 10.47% +/- 3.05, p less than 0.001).     

Evaluation of the genotoxicity of treated urban sludge in the Tradescantia micronucleus assay

The sludge produced in sewage treatment plants can contain toxic substances. Among these, the genotoxic substances are of great concern. The present paper aimed at evaluating the genotoxicity of treated sludge samples collected at four different sewage treatment plants (STP) located in the State of S?o Paulo, Brazil using the Trad-MN assay. Another objective of the study was to compare the responses of the Clone #4430 with the Tradescantia pallida. Sludge samples mixed with reference soil in concentrations of 10, 25 and 50% (v/v) were tested in experiments with 3 months exposure in the field. Negative and positive controls (arsenic trioxide) were also tested with both plants. In Clone #4430 two sludge samples induced genotoxicity while in T. pallida three were positive, although no clear dose-response were observed for both plants. Results with the negative and positive controls suggest that T. pallida presented similar results when compared to the Clone #4430. The protocol using plants chronically exposed to sludge mixed with soil seems to be a promising tool to assess the genotoxicity of sludge although time consuming.     

Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test

As part of a larger literature study on transgenic animals in mutagenicity testing, test results from the transgenic mutagenicity assays (lacI model; commercially available as the Big Blue(R) mouse, and the lacZ model; commercially available as the Mutatrade markMouse), were compared with the results on the same substances in the more traditional mouse bone marrow micronucleus test. 39 substances were found which had been tested in the micronucleus assay and in the above transgenic mouse systems. Although, the transgenic animal mutation assay is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation, the results for the majority of substances were in agreement. Both test systems, the transgenic mouse assay and the mouse bone marrow micronucleus test, have advantages and they complement each other. However, the transgenic animal assay has some distinct advantages over the micronucleus test: it is not restricted to one target organ and detects systemic as well as local mutagenic effects.     

Risk assessment of adenoviruses detected in treated drinking water and recreational water

AIMS: Human adenoviruses (HAds) have previously been detected in sewage and polluted river and dam water, as well as treated drinking water. The 51 serotypes of HAds cause a wide range of infections with clinical manifestations associated with the gastrointestinal, respiratory and urinary tracts, and the eyes. Water may play a meaningful role in the transmission of many of these HAd serotypes, specifically the enteric HAds which are transmitted via the faecal-oral route. The presence of these viruses in water used for drinking and recreational purposes is considered to constitute a potential health risk. In this study, the risk of infection by the group of HAds previously detected over a period of 1 year in selected drinking water supplies, as well as river and dam water used for recreational purposes, was assessed. METHODS AND RESULTS: Adenoviruses were previously detected in nine of 204 (4.41%) samples of two drinking water supplies (A and B) treated and disinfected according to international specifications, in four of 51 (7.8%) samples of river water and nine of 51 (17.7%) samples of dam water. Application of these previously published results in an exponential risk assessment model indicated an annual risk of infection of 1.01 x 10(-1) and 1.7 x 10(-1) for drinking water supplies A and B, respectively, assuming a daily consumption of 2 l day(-1). The daily risk of infection constituted by HAds in the river water was calculated as 1.71 x 10(-4), and in the dam water as 3.12 x 10(-5), assuming a consumption of 30 ml of water per day. CONCLUSIONS: The risk of infection exceeded the tolerable risk of one infection per 10 000 consumers per year proposed for drinking water. However, the results for river and dam water used for recreational purposes were within the tolerable risk of one infection per 1000 bathers per day proposed for environmental waters used for recreational purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed that the risk of HAd infection calculated for the drinking water supplies and the recreational water may overestimate the actual risk of infection, as conservative values were assumed for some of the variables. For a more accurate assessment of the potential risk of infection research should at least include a thorough investigation of the water consumption of individuals in South Africa, and the efficiency of recovery of the glass wool adsorption-elution method.     

Ten model mutagens evaluated by the micronucleus test

The following ten mutagenic compounds were subjected to the micronucleus bone marrow test (MNT) in the mouse: cyclophosphamide (CTX), thiotepa (TT), vincristin (VCR), colcemid (COLC), adriamycin (AM), bleomycin (BM), cytosin arabinoside (ARA C), 6-mercaptopurine (6-MP), methotrexate (MTX) and 5-fluorouracil (5-FU). Dose-effect curves were established for all compounds. With the exception of CTX, COLC and AM, the drugs also were subjected to chromosome analyses on Chinese hamster fibroblasts in vitro.The MNT revealed loss of chromatin due to chromosome breakage and rearrangements by CTX, TT and AM, to breakage by ARA C, 6-MP, MTX and 5-FU, as well as loss of entire chromosomes caused by impairment of the spindle by VCR and COLC. With the exception of BM, the effects were demonstrable in the therapeutic dose range.The MNT, provided it is carried out by the methodology of the authors, not only reveals chromatin loss but permits important conclusions in regard to the proliferative state of the bone marrow and the specific time of action of the mutagens in the cell cycle. If arrest of the cell cycle occurs, as in the case of anti-metabolites MTX and 5-FU particularly, the routine scheme of investigation needs to be modified since micronucleated cells appear only after release of the metabolic block, i.e. after a delay of 24 h.The negative bone marrow results obtained with BM emphasize the importance of combining in vivo and in vitro tests.     

Mutagenicity studies on paracetamol in human volunteers. II. Unscheduled DNA synthesis and micronucleus test

The possible genotoxic effect of paracetamol (PC) was studied in a group of 11 healthy volunteers. PC was administered in the form of tablets 3 x 1000 mg in the course of 8 h. Blood samples and buccal mucosa cells were taken 0, 24, 72 and 168 h after the first administration of the drug. Each blood sample was used for the termination of the unscheduled DNA synthesis (UDS) in peripheral lymphocytes and ascorbemia in plasma. Buccal mucosa cells were analysed for micronuclei. After PC administration the level of UDS induced by MNNG was decreased to T/C = 4.11 +/- 0.56 after 24 h vs. T/C = 5.02 +/- 0.47 (p less than 0.01) at 0 h. The frequency of micronucleated cells in the buccal mucosa was increased after 72 h to 0.38 +/- 0.07% vs. 0.19 +/- 0.06% (p less than 0.01) before PC administration. If PC was administered simultaneously with ascorbic acid (AA), also in a dose of 3 X 1000 mg, a decreased level of UDS was observed after 24, 72 and 168 h and the increased number of micronuclei was qualitatively the same as the PC alone: 0.38 +/- 0.09% after 72 h vs. 0.20 +/- 0.05% at 0 h AA did not decrease the genotoxic effect of PC, but prolonged the influence of PC on UDS.     

Genotoxicity of 2,4- and 2,6-dinitrotoluene as measured by the Tradescantia micronucleus (Trad-MCN) bioassay

The phytogenotoxicity of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) was assessed using the Tradescantia micronucleus (Trad-MCN) bioassay. Tradescantia cuttings bearing young inflorescences were exposed for 6h to 2,4- or 2,6-DNT amended water solutions up to their respective solubilities. The nominal concentrations were 0, 1.9, 3.8, 7.5, 15, 30, 60, 100, 150, 200mg/l of 2,4-DNT, and 0, 7.5, 15, 30, 60, 90, 120, 180mg/l of 2,6-DNT. Each treatment was repeated three or four times. Chemical concentrations in test solutions were analyzed prior to and after the exposure. Cadmium chloride (0-20mM) was used as the positive control. Micronuclei (MCN) were scored in the tetrad-stage pollen mother cells. The MCN frequency (%), i.e. the number of micronuclei scored in 100 tetrads, was the measurement endpoint. Results indicated that both 2,4-DNT and 2,6-DNT were genotoxic with the minimum effective dose (MED) of 30 and 135mg/l, respectively. Longer exposure (30h) without recovery time at 150mg/l of 2,4-DNT and 180mg/l of 2,6-DNT did not induce significantly higher MCN frequencies.     

Mutagenicity of ground water (in the bore-holes) in Ararat Valley (Armenia) detected by Trad-SHM bioassay

The genotoxicity of ground water from four bore-holes of different depths (40-120m) in the Ararat valley (Armenia) used both for drinking and irrigation was investigated. The frequency of recessive somatic mutations was determined using the Tradescantia-stamen-hair-mutation (Trad-SHM) test. The Tradescantia clone 02 was used. The pink mutation events (PMEs) were increased by 3.18-6.81-fold in comparison with the control depending both on the depth of subterranean water location and the increase of Na(+) ion concentration in these water samples. The peak frequency was found in water from the 40-45m depth. The deeper the bore-holes, the lower the mutagenicity of water and the concentration of Na(+) ions. Different types of mutant sector arrangements and their frequencies changed depending on the subterranean water depth.     

Ten model mutagens evaluated by the micronucleus test.

The following ten mutagenic compounds were subjected to the micronucleus bone marrow test (MNT) in the mouse: cyclophosphamide (CTX), thiotepa (TT), vincristin (VCR), colcemid (COLC), adriamycin (AM), bleomycin (BM), cytosin arabinoside (ARA C), 6-mercaptopurine (6-MP), methotrexate (MTX) and 5-fluorouracil (5-FU). Dose-effect curves were established for all compounds. With the exception of CTX, COLC and AM, the drugs also were subjected to chromosome analyses on Chinese hamster fibroblasts in vitro. The MNT revealed loss of chromatin due to chromosome breakage and rearrangements by CTX, TT and AM, to breakage by ARA C, 6-MP, MTX and 5-FU, as well as loss of entire chromosomes caused by impairment of the spindle by VCR and COLC. With the exception of BM, the effects were demonstrable in the therapeutic dose range. The MNT, provided it is carried out by the methodology of the authors, not only reveals chromatin loss but permits important conclusions in regard to the proliferative state of the bone marrow and the specific time of action of the mutagens in the cell cycle. If arrest of the cell cycle occurs, as in the case of anti-metabolites MTX and 5-FU particularly, the routine scheme of investigation needs to be modified since micronucleated cells appear only after release of the metabolic block, i.e. after a delay of 24 h. The negative bone marrow results obtained with BM emphasize the importance of combining in vivo and in vitro tests.     

Aneuploidy-inducing chemicals in yeast evaluated by the micronucleus test

Chinese hamsters were exposed to acetone, methyl ethyl ketone, ethyl acetate and 2-methoxy ethyl acetate, known to be strong inducers of aneuploidy in the yeast Saccharomyces cerevisiae. All solvents yielded negative results in the micronucleus test, whereas the vinca alkaloid vindesine--used as a positive control substance--proved to act as a spindle poison in mammals in vivo.     

Statistical analysis of the micronucleus test

Various statistical procedures are discussed to compare the incidence of micronucleated polychromatic erythrocytes between various treatment groups and a negative control. The effect of toxicity on the choice of statistical procedure is discussed. The procedure which is recommended is based on likelihood ratio tests comparing each dose group separately against the negative control. The significance level is controlled to allow for the multiple comparisons.     

Genetic toxicological testing of some dyes by the micronucleus test

Three widely used dyes, acridine orange, blue VRS and fast green FCF were administered to male mice in order to study the induction of gross chromosomal anomalies using the micronucleus test. All 3 compounds were shown to be clastogenic.     

Strain difference in the micronucleus test

The Collaborative Study Group for the Micronucleus Test, a task group of the Environmental Mutagen Society of Japan, has earlier addressed the question of sex difference as a source of variation in the micronucleus test. Strain difference, another issue in test protocols requiring urgent clarification, was selected as the subject of the second study. Male mice of strains Slc: ddY (ddY), CRJ: CD-1(ICR) (CD-1), Slc: BDF₁ (BDF₁), and ms: Hal (ms) were treated with 6 different chemicals chosen from various classes of micronucleus inducers: colchicine, 7,12-dimethylbenz[a]anthracene, ethyl methanesulfonate, N-ethyl-N-nitrosourea, 6-mercaptopurine, and potassium chromate. All 4 strains gave positive results with all 6 chemicals, although ms tended to show the highest responses. ddY and CD-1 were low responders, while BDF₁ was intermediate between ms and the other two. Although ms seemed superior to the other strains, its high responses became manifest mostly at high dose levels. ms was not always the most sensitive strain; it responded moderately to ethyl methanesulfonate. Also the background level of micronucleated polychromatic erythrocytes was the highest in ms, but this did not explain the apparent high sensitivity of this strain. Despite the strain differences, it can be concluded that any of the other strains used seems to suffice as a tester for the micronucleus test.