Genotoxicity of Heterocyclic PAHs in the Micronucleus Assay with the Fish Liver Cell Line RTL-W1

Heterocyclic aromatic hydrocarbons are, together with their un-substituted analogues, widely distributed throughout all environmental compartments. While fate and effects of homocyclic PAHs are well-understood, there are still data gaps concerning the ecotoxicology of heterocyclic PAHs: Only few publications are available investigating these substances using in vitro bioassays. Here, we present a study focusing on the identification and quantification of clastogenic and aneugenic effects in the micronucleus assay with the fish liver cell line RTL-W1 that was originally derived from rainbow trout (Oncorhynchus mykiss). Real concentrations of the test items after incubation without cells were determined to assess chemical losses due to, e.g., sorption or volatilization, by means of gas chromatography-mass spectrometry. We were able to show genotoxic effects for six compounds that have not been reported in vertebrate systems before. Out of the tested substances, 2,3-dimethylbenzofuran, benzothiophene, quinoline and 6-methylquinoline did not cause substantial induction of micronuclei in the cell line. Acridine caused the highest absolute induction. Carbazole, acridine and dibenzothiophene were the most potent substances compared with 4-nitroquinoline oxide, a well characterized genotoxicant with high potency used as standard. Dibenzofuran was positive in our investigation and tested negative before in a mammalian system. Chemical losses during incubation ranged from 29.3% (acridine) to 91.7% (benzofuran) and may be a confounding factor in studies without chemical analyses, leading to an underestimation of the real potency. The relative potency of the investigated substances was high compared with their un-substituted PAH analogues, only the latter being typically monitored as priority or indicator pollutants. Hetero-PAHs are widely distributed in the environment and even more mobile, e.g. in ground water, than homocyclic PAHs due to the higher water solubility. We conclude that this substance class poses a high risk to water quality and should be included in international monitoring programs.     

Aneugenic Effects of Epirubicin in Somatic and Germinal Cells of Male Mice

The ability of the antineoplastic agent epirubicin to induce aneuploidy and meiotic delay in the somatic and germinal cells of male mice was investigated by fluorescence in situ hybridization assay using labeled DNA probes and BrdU-incorporation assay. Mitomycin C and colchicine were used as positive controls for clastogen and aneugen, respectively, and these compounds produced the expected responses. The fluorescence in situ hybridization assay with a centromeric DNA probe for erythrocyte micronuclei showed that epirubicin is not only clastogenic but also aneugenic in somatic cells in vivo. By using the BrdU-incorporation assay, it could be shown that the meiotic delay caused by epirubicin in germ cells was approximately 48 h. Disomic and diploid sperm were shown in epididymal sperm hybridized with DNA probes specific for chromosomes 8, X and Y after epirubicin treatment. The observation that XX- and YY-sperm significantly prevailed over XY-sperm indicates missegregation during the second meiotic division. The results also suggest that earlier prophase stages contribute less to epirubicin-induced aneuploidy. Both the clastogenic and aneugenic potential of epirubicin can give rise to the development of secondary tumors and abnormal reproductive outcomes in cured cancer patients and medical personnel exposed to epirubicin.     

Association of the ACE I/D gene polymorphism with DNA damage in hypertensive men

The aim of the study was to evaluate the association between the angiotensin-converting enzyme ACE I/D (rs 4340) polymorphism and DNA damage in patients with essential hypertension (EH). The I/D polymorphism of ACE was determined by polymerase chain reaction in 170 male hypertensive patients and 64 normotensive blood donors. We used flow cytometry to determine the levels of cell death, micronuclei and accumulation of peripheral blood leukocytes in G1/G0, S, G2/M phases of the cell cycle. Additionally, the whole blood samples were incubated in vitro at 4°C for 24 h to investigate the genotype effects on the susceptibility of cells to DNA damage. We found lower frequency of cells in DNA synthesis S phase and higher levels of micronuclei in the hypertensive compared to normotensive group (p < 0.05); increased formation of micronuclei was seen due to elevated micronuclei frequencies in patients with the ACE II genotype (p < 0.05), but not in ID or DD genotype carriers. Incubation of whole blood samples of normotensive individuals lead to the most active cell death (p < 0.05) and micronuclei formation (p > 0.05) in the II genotype carriers too. However, hypertensive patients displayed different cellular response to incubation-induced DNA damages in the ACE I/D genotype groups; after incubation, the frequencies of micronuclei were significantly higher in the DD genotype carriers (p < 0.05). To conclude, the study suggests that the ACE I/D polymorphism may contribute to mechanisms and intensity of DNA damages in hypertensive and normotensive individuals.     

SFTG international collaborative study on in vitro micronucleus test II. Using human lymphocytes

This study on the in vitro micronucleus assay, comprising 11 laboratories using human lymphocytes, was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Nine coded substances were assessed for their ability to induce micronuclei in human lymphocytes in vitro, mitomycin C being used as a positive control. Cultures were exposed to the test substances for a short (early or late) time or for a long time, followed by a short or long recovery period, in the presence of cytochalasin B. Each chemical was evaluated, generally in two laboratories, using three treatment schedules at least twice. The data were assessed for acceptability, and then classified as negative, positive or equivocal. Two of seven genotoxic compounds, namely colchicine and bleomycin, clearly induced micronuclei. Reproducible results were difficult to obtain for some substances, which tended to be those acting at specific stages of the cell cycle. Cytosine arabinoside, diethylstilboestrol and 5-fluorouracil were classified as equivocal. Urethane and thiabendazole were classified as negative. The two presumed non-genotoxic compounds, mannitol and clofibrate, did not induce micronuclei. Repeat testing, exposing cells at both an early and late time after mitogenic stimulation, was needed to detect substances classified as equivocal. These results show the importance of achieving sufficient inhibition of nuclear division to avoid the possibility of missing an effect. The evaluation of micronuclei in mononucleated as well as binucleated cells was particularly useful to detect aneugens. There were no false positive results using lymphocytes, indicating a high specificity. It is concluded that the clastogenic or aneugenic potential in vitro of the substances tested was correctly identified in this study, but that refining the protocol to take into account factors such as the stages of the cell cycle exposed to the compound, or the duration of recovery would be likely to improve the sensitivity of detection using lymphocytes.     

Micronucleus formation in cultured human keratinocytes following exposure to mitomycin C and cyclophosphamide

A method is described to investigate the induction of micronuclei in cultured human keratinocytes after short-term exposure to known clastogenic agents. The cytokinesis-block method was applied to facilitate the scoring of micronucleated cells. Mitomycin C, a direct-acting compound, caused a 5-20-fold increase in micronuclei over the controls at the highest concentration tested (1 microgram/ml). Cyclophosphamide, an agent requiring metabolic activation, did not induce the formation of micronuclei in cultured keratinocytes. However, after pretreatment of the keratinocyte cultures with Aroclor 1254 for 72 h, exposure to cyclophosphamide resulted in a 3-fold increase in micronucleus frequency over the controls. No cytogenetic effect of Aroclor 1254 was observed in control experiments.     

The induction of chromosomal damage in rat hepatocytes and lymphocytes II. Alkylation damage and repair of rat-liver DNA after diethylnitrosamine,dimethylnitrosamine and ethyl methanesulphonate in relation to clastogenic effects

Rat-liver DNA alkylation by diethylnitrosamine (DEN), dimethylnitrosamine (DMN) and ethyl methanesulphonate (EMS) was studied in an attempt to relate chromosome-damaging effects of these agents (the formation of micronuclei in hepatocytes; see preceding paper) to specific alkylation patterns. No correlation was observed between the induction of micronuclei and liver DNA N-alkylation, measured as 3- and 7-alkyl-purines. O⁶-Alkylguanine is probably not involved in micronucleus induction because it is lost from DNA too rapidly to explain the much more persistent clastogenic effects. In contrast, both the initial amounts of alkylphosphotriesters and the persistencies of these products roughly paralleled the respective effects on micronucleus induction. The possible involvement of alkylphosphotriesters or other O-alkylation products of comparable stabilities is discussed. Results with DMN suggest that part of the primary DNA methylation damage is converted into a secondary (DNA) lesion and that both the primary and secondary lesion(s) contribute to the process of micronucleus formation.     

A comparative analysis of data on the clastogenicity of 951 chemical substances tested in mammalian cell cultures

A literature review was conducted using original papers published during 1964-1985 on the in vitro clastogenicity of chemical substances. Results of tests on 951 chemical substances were abstracted from over 240 reports to form the database. The evaluation of these data relied on each author's original conclusion on a positive or negative outcome. Of these 951 substances, 447 (47%) were consistently positive either with or without activation; 417 (44%) were negative in the direct test but not tested with metabolic activation systems; 4 were negative but tested only with activation; and 30 (3%) were clearly negative both with and without activation. The remaining 53 substances gave variable results when tested under different experimental protocols or in different cell types, but were positive in at least one test. Although discrepant results were found associated with some cell types, the addition of metabolic activation systems tended to eliminate such variability. No one cell appeared to be superior in response to all clastogens. For screening purposes, the choice of cell may thus depend more on the general usefulness and reliability of a cell type than on a strong response to a particular chemical. However, the use of a suitable metabolic activation system does appear to be of critical importance. The concentration at which clastogenic effects were detected varied extensively for different test substances, ranging from a minimum of 4.3 X 10(-8) to 6.9 X 10(2) mM. Possible mechanisms of action for substances active at only high levels are discussed, but no satisfactory explanation is available at this time. The relevance of tests conducted at concentrations high enough to alter significantly the osmolarity and other culture conditions is considered, and caution urged in the interpretation of test results obtained under physiologically stressful conditions. The clastogenic potential was compared quantitatively using an index of effective concentration (D20) and one which estimates the number of cells with exchange aberrations expected per mg/ml (TR) for data obtained by using a uniform protocol and cultures of Chinese hamster lung (CHL) cells. Both values were distributed over a wide range, demonstrating the variety of genotoxic potential in chemicals. In general, a substance which was active at only high concentrations produced fewer exchange-type aberrations. In vivo activity, as measured by tumourigenic effect and formation of micronuclei in bone marrow, tended to be greater for substances with a D20 below 10(-2) mg/ml and a TR value over 10(3).(ABSTRACT TRUNCATED AT 400 WORDS)     

Genotoxicity biomarkers in M. galloprovincialis as indicators of marine pollutants

Many substances pollute the marine environment. There is today a growing evidence on the increased risk of disease in marine organisms, especially fish, that inhabit contaminated waters. Different types of tumours have been evidenced in fish and shellfish populations. Different short-term biomarkers are available to predict the impact of carcinogens on marine organisms. Their endpoints are different effects at the molecular and cellular level such as gene mutation, chromosome alteration and induction of DNA damage and repair. We have applied two different assays: alkaline elution to measure DNA single strand breaks and micronucleus assay as an index of a chromosomal damage. In order to select an aquatic organism as an indicator of water pollution by carcinogenic agents, we have focused on the mussel. A program of validation of genotoxicity was conducted in aquarium using DMBA. A time-dependence increase of micronuclei was evident after the exposure to 100 ppb/animal. For alkaline elution the effect was 4 times the level of the controls. Experiments in the fields were conducted on adult specimens of Mytilus gdlloprovincialis collected from natural substrates. Our sampling stations were located in the La Spezia gulf, Ligurian sea. Genotoxic effects were evaluated in gill cells. A significant increment of the two parameters in polluted, in comparison with the unpolluted sites has been observed. High frequencies of micronuclei (the highest value was 42 ± 13 with respect to control value 3 ± 2) were scored in mussels from polluted stations. The extent of DNA damage was also relevant with respect to clastogenic damage as revealed by micronucleus test. The greatest value of K (constant of elution) was 8-fold higher with respect to the value of K obtained in the same tissue of mussel from reference areas. Evidence of DNA damage could reflect a recent pollution status, since DNA strand breaks can be rapidly repaired by different mechanisms. On the contrary animals exposed to clastogenic compounds may exhibit elevated micronucleus frequency long after the exposure has ceased. The evaluation of both parameters could provide information of great significance about the pollution status of the water.     

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.     

Establishment of dose-response relationships between doses of Cs-137 γ-rays and frequencies of micronuclei in human peripheral blood lymphocytes

It has been suggested that the yield of micronuclei in human peripheral blood lymphocytes could be used as a biological dosimeter in cases of radiation exposure. In the present study micronuclei were induced in lymphocytes by exposing human blood samples in vitro to various doses of Cs-137 γ-rays. The blood samples were then cultivated using the cytokinesis block method. Coded programs were employed to establish the relationships between the frequencies of micronuclei and various doses of γ-rays. The best fit was obtained by the linear-quadratic model, Y = c + aD + bd², where Y is the yield of micronuclei, D is the dose in Gy and c, a, b, are constants. It seems there is a correlation between the yields of MN in mononuclear cells and the corresponding doses of radiation. Therefore an attempt was made to include these MN in the calculation of the dose-response relationship.     

Effects of deoxyribonucleosides and cell-stimulation on frequencies of ultraviolet-B-induced micronuclei in human peripheral blood lymphocytes

In the present study the involvement of deoxyribonucleotides (dNTPs) in the clastogenicity of ultraviolet-B (UVB) in unstimulated peripheral blood lymphocytes (G(0)-PBLs) was investigated. This was studied by analyzing the frequency of UVB-induced micronuclei (MN), either after adding a cocktail of the four deoxyribonucleosides to the PBLs immediately after exposure to UVB, or by stimulating the cells before exposure. In total, PBLs obtained from two different donors were investigated. For both donors, it could be demonstrated that addition of deoxyribonucleosides to UVB-irradiated G(0)-PBLs resulted in a significant reduction of the clastogenic effect of UVB. A gradual reduction of the clastogenic effect of UVB could also be realized by irradiating PBLs that were progressively more stimulated with the lectin PHA before exposure. The latter finding is explained by upregulation of intracellular pool sizes of dNTPs in stimulated PBLs.     

Clastogenic effects of the fasciolicide drug fasinex on river buffalo lymphocyte cultures in vitro

Fasinex (triclabendazole) has been reported to be an active fasciolocidal agent used in humans and in farm animals. The clastogenic effects of fasinex were tested in lymphocyte cultures of the river buffalo at three final concentrations: 25, 50 and 100 microg/ml. Chromosomal aberrations, sister chromatid exchanges and micronucleus formation are the three cytogenetic parameters used in this study.The results demonstrated that the number of cells with different types of chromosomal aberrations, including chromatid breaks and gaps, isochromatid breaks and gaps and polyploidy, was increased significantly in cultures treated with different doses of fasinex compared to the control. This increase was dose-dependent where there was a positive correlation between increased drug concentration and induction of chromosomal aberrations.The frequency of sister chromatid exchanges and the formation of micronuclei in all lymphocyte cultures treated with different doses of fasinex were increased significantly compared to the control; these increases were also dose-dependent.In conclusion, the three cytogenetic parameters used to evaluate the effect of fasinex revealed that the drug has a strong clastogenic effect on river buffalo lymphocytes in vitro.     

Protection of cellular DNA from γ-radiation-induced damages and enhancement in DNA repair by troxerutin

The effect of troxerutin on γ-radiation-induced DNA strand breaks in different tissues of mice in vivo and formations of the micronuclei were studied in human peripheral blood lymphocytes ex vivo and mice blood reticulocytes in vivo. Treatments with 1 mM troxerutin significantly inhibited the micronuclei induction in the human lymphocytes. Troxerutin protected the human peripheral blood leucocytes from radiation-induced DNA strand breaks in a concentration dependent manner under ex vivo condition of irradiation (2 Gy). Intraperitoneal administration of troxerutin (175 mg/kg body weight) to mice before and after whole body radiation exposure inhibited micronuclei formation in blood reticulocytes significantly. The administration of different doses (75, 125 and 175 mg/kg body weight) of troxerutin 1 h prior to 4 Gy γ-radiation exposure showed dose-dependent decrease in the yield of DNA strand breaks in murine blood leucocytes and bone marrow cells. The dose-dependent protection was more pronounced in bone marrow cells than in blood leucocytes. Administration of 175 mg/kg body weight of the drug (i.p.) 1 h prior or immediately after whole body irradiation of mice showed that the decrease in strand breaks depended on the post-irradiation interval at which the analysis was done. The observed time-dependent decrease in the DNA strand breaks could be attributed to enhanced DNA repair in troxerutin administered animals. Thus in addition to anti-erythrocytic, anti-thrombic, fibrinolytic and oedema-protective rheological activity, troxerutin offers protection against γ-radiation-induced micronuclei formation and DNA strand breaks and enhances repair of radiation-induced DNA strand breaks. (Mol Cell Biochem xxx: 57–68, 2005)     

Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultured cells

In order to better characterize the ochratoxin A (OTA)-induced DNA damage and to further investigate factors which may modulate dose-effect relationships in cells, the induction of micronuclei was studied in V79 Chinese hamster fibroblast cells and in primary cultures of porcine urothelial bladder epithelial cells (PUBEC). OTA was able to induce micronuclei in PUBEC and V79 cells at concentrations below those which were overtly cytotoxic. OTA concentrations between 0.03 and 1 μM caused a dose-dependent increase of micronuclei in V79 cells (up to 3-fold compared to controls); but the lowest tested concentration of 0.01 μM OTA did not induce a higher frequency of micronuclei than in the solvent control, indicative of an apparent threshold. Clear evidence for genotoxic effects was also found in PUBEC cultures treated with OTA concentrations of 1 μM and more, although the dose-effect relationship in PUBEC was more variable for several freshly isolated cell batches, pointing to differences in susceptibility to OTA between bladder cells from different donor animals. The chromosomal genotoxicity of OTA demonstrated in this study is in general accord with previous findings on the induction of clastogenic effects and oxidative DNA damage by OTA. In both cases, the shape of the dose-response curve at very low OTA concentrations supports the existence of a threshold for its genotoxicity. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Retinoic acid-treated pluripotent stem cells undergoing neurogenesis present increased aneuploidy and micronuclei formation

The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naïve cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.     

DNA adducts, strand breaks and micronuclei in mice exposed to styrene by inhalation

Genotoxic and clastogenic effects of styrene were studied in mice. Male NMRI mice were exposed by inhalation to styrene in concentrations of 750 and 1500 mg/m3 for 21, 7, 3 and 1 days (6 h/day, 7 days/week). Followed parameters included styrene in blood, specific styrene oxide (SO) induced DNA adducts, DNA strand breaks and micronuclei. The formation of SO induced 7-SO-guanines and 1-SO-adenines in DNA was analysed from lung tissues by two versions of the 32P-postlabeling technique. In lungs after 21 days of exposure to 1500 mg/m3 the level of 7-SO-guanine was 23.0+/-11.9 adducts/10(8) normal nucleotides, while 1-SO-adenine was detected at the levels of 0.6+/-0.2 adducts/10(8) normal nucleotides. Both 7-SO-guanines and 1-SO-adenines strongly correlated with exposure parameters, particularly with styrene concentration in blood (r=0.875, P=0.0002 and r=0.793, P=0.002, respectively). DNA breaks were measured in peripheral lymphocytes, bone marrow cells and liver cells using comet assay. To discern oxidative damage and abasic sites, endonuclease III was used. In bone marrow of exposed mice slight increase of strand breaks can be detected after 7 days of inhalation. A significant increase was revealed in the endonuclease III-sensitive sites after 21 days of inhalation in bone marrow. In the liver cells inhalation exposure to both concentrations of styrene did not virtually affect either levels of DNA single-strand breaks or endonuclease III-sensitive sites. The inhalation of 1500 mg/m3 of styrene induced significant increase of micronuclei after 7 days of exposure (10.4+/-2.5/1000 cells, i.e. twice higher micronuclei frequency than in controls). After 21 days of inhalation no significant difference between the control group and the two exposed groups was observed. Whether the decrease of micronuclei after 21 days of inhalation was due to the inhibition of cell proliferation caused by styrene or due to the natural elimination of chromatide fragments, remains to be clarified. An interesting link has been found between DNA single-strand breaks in bone marrow and frequencies of micronuclei (r=0.721, P=0.028).     

Induction of in vivo DNA adducts by 4 industrial by-products in the rat-lung-cell system

Benz[a]anthracene (BA), dibenz[a,h]anthracene (DBA), dibenzo[a,i]pyrene (DBP), and dibenz[a,h]acridine (DBAC) are by-products found in many industrial wastes and emissions. Workers in the related occupational settings are potentially exposed to these substances through inhalation. In the present study, induction of DNA adducts in vivo by these chemicals was investigated using ³²P-postlabeling analysis in the rat-lung-cell system. The potency of DNA-adduct inducing activity was also compared to that of two cytogenetic endpoints i.e., sister-chromatid exchange (SCE) and micronucleus formation. Via intratracheal instillation, male CD rats (6/group) were dosed 3 times with BA, DBA, DBP or DBAC in a 24-h interval. Lung cells were enzymatically separated and used to determine the frequency of DNA adducts, SCE and micronuclei. Results show that all 4 test compounds induced DNA adducts, SCEs, and micronuclei in the rat-lung cell in vivo and that the postlabeling DNA adduct assay detected genotoxic activity at lower dose levels than the two cytogenetic assays. These findings suggest that BA, DBA, DBP or DBAC are rat pulmonary genetoxicants and the DNA-adduct assay is more sensitive than SCE or micronucleus assays for detecting the pulmonary genotoxicity of these industrial PAHs in the in vivo rat-lung-cell system.     

Cell division in Lemna minor roots treated with lead

Treatment of Lemna minor L. roots with 15 μM Pb²⁺ supplied as Pb(NO₃)₂ in 50-fold diluted Wang medium caused a progressive reduction in mitotic activity in the root tip. The percentage of dividing nuclei after 1, 6, 12 and 12 h of lead treatment was 6.25, 4.4, 3.4 and 0.3, respectively as compared to 7.1–7.7% in the control. After 6 h of lead treatment the number of cells in metaphase and anaphase was reduced by four- and nine-fold, respectively and after 12 h these phases were not detected. There were 3- and 10-fold fewer cells in telophase after 6 and 24 h, while those in prophase were reduced only in the 24 h treatment (a 30-fold reduction). These effects were associated with an increase in the number of cells exhibiting disturbances including lagging chromosomes, chromosome bridges, micronuclei, and nuclei with more condensed chromatin. The formation of micronuclei in root cells of L. minor cells at a very low dose of lead indicates that roots of this aquatic plant may be more sensitive to lead than those of terrestrial plants.     

Genotoxic effects of the chlorinated hydroxyfuranones 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone and 3,4-dichloro-5-hydroxy-2[5H]-furanone in Tradescantia micronucleus assays

This is the first report of clastogenic effects of chlorinated hydroxyfuranones (CHFs) in plants. Two byproducts of water chlorination, 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) and 3,4-dichloro 5-hydroxy-2[5H]-furanone (MA) induced a dose dependent increase of micronuclei (MN) in pollen mother cells of Tradescantia when doses up to 100 μg MX and 500 μg MA were applied directly to the inflorescences. In contrast, exposure of the stems in aqueous solutions containing up to 1 mg/I MX and 10 mg/I MA did not cause a positive response.