Genotoxicity biomarkers in Mytilus galloprovincialis: wild versus caged mussels.

A biomonitoring programme of wild and caged mussels (Mytilus galloprovincialis) was carried out at four selected sites along the Ligurian coast: Cornigliano, Voltri, Zinola, and Sanremo (Italy). Mussels of a very narrow size range were left in situ for 30 days. Adult specimen of mussels from natural substrates were collected in the same areas. Animals from a mussel farm located in La Spezia were used as controls. Micronucleus frequency and DNA single strand breaks, evaluated by alkaline elution, were used as biomarkers of genotoxicity. Mussels were also analyzed for polycyclic aromatic hydrocarbons (PAH) and heavy metals (Hg and Cd). Different gradients of PAH and metal concentrations were detected in tissues of mussels from different samplings sites. A weak correlation was found between single strand breaks and PAH content while MN frequency correlated with Hg concentration (r = 0.28, P < 0.002). A clear distinction between the sites, allowing classification along a pollution gradient (Sanremo < Zinola < Voltri < Cornigliano) was demonstrated by the analysis of genotoxicity parameters. The obtained results suggested that the micronucleus assay compared with DNA damage determination by alkaline elution allow to better discriminate the selected sites. DNA damage expressed as constant of elution (k ml(-1) x 10(3)) ranges from 30 +/- 9.6 to 89.60 +/- 40.10, and micronuclei frequency from 1.78 +/- 1.04 to 24.4 +/- 12.9, in control animals and in mussels from the most polluted site, respectively. Wild mussels accumulated significant concentrations of chemicals and showed a higher induction of chromosomal damage than caged mussels, expressed as micronuclei frequency. Caged mussels showed higher level of DNA damage than wild mussels, probably as a result of recent exposure. DNA damage was higher in September than in May, as opposed to micronuclei frequency being higher in May than in September. Endogenous and exogenous factors, such as change of pollutant input levels or compositions, could be considered the cause of such variability.

Influence of DNA double-strand break rejoining on clonogenic survival and micronucleus yield in human cell lines.

PURPOSE: To examine the role of DNA double-strand break (DSB) rejoining in cell survival and micronucleus yield after 60Co gamma-irradiation. MATERIALS AND METHOD: Thirteen human cell lines (six glioblastoma, five prostate, one melanoma, one squamous cell carcinoma) were irradiated with 60Co gamma-rays to doses of 0-10Gy for cell survival and micronucleus measurements and 0-100Gy for DSB rejoining. Measurements were performed using standard clonogenic, micronucleus and constant-field gel electrophoresis assays. RESULTS: Radioresistance and micronucleus yield were positively correlated (r=0.74, p=0.004). A significant cell type-dependent correlation was demonstrated between total (0-20 h) DSB rejoining and cell survival (r=0.86, p=0.03 for glioblastomas; r=0.79, p=0.04 for other cell lines), with more resistant cell lines showing higher levels of DSB rejoining. No relationship was apparent between fast (0-2 h) or slow (2-20 h) DSB rejoining and clonogenic survival. While there was no relationship between total or slow DSB rejoining and micronucleus yield, a significant and cell type-specific correlation emerged between fast rejoining and micronucleus yield for the glioblastomas (r=0.89, p=0.04) and other cell lines (r=0.76, p=0.04). Cell lines with higher levels of DSB rejoining within 2 h of irradiation showed higher yields of micronuclei. CONCLUSION: Fast DSB rejoining, possibly through interaction with slow DSB rejoining, appears to play an important role in the formation of micronuclei. However, total DSB rejoining reflects intrinsic radiosensitivity. Consideration of differences in DSB rejoining kinetics might contribute to a better understanding of the significance of cell survival and micronucleus data in the clinical and radiation protection setting.

Investigation of genotoxic effect of ultrasound in cases receiving therapeutic ultrasound by using micronucleus method.

In 1991, reported that therapeutic ultrasound (US) did not induce sister chromatid exchanges (SCEs) in patients whereas, in 1984, reported that each of 10 patients exposed to therapeutic US had a statistically significant increase in SCEs. The present study was planned to investigate if there was chromosomal damage resulting from therapeutic US by using a micronucleus (MN) method, and to counter the lack of reports in this area over the past 10 years. A total of 20 female volunteers were included in the study; 10 of them with low back pain (mechanical low back pain and facet syndrome) were treated with US and 10 healthy cases constituted the control group. Patients with low back pain received 10 sessions of US therapy at an intensity of 2 W/cm(2) and a frequency of 1 MHz for 10 min and patients in the control group received sham US therapy for 10 min. Peripheral blood taken before and after the fifth and tenth applications of US therapy was cultured for MN frequencies both for the treatment and the control groups. The scores of MN assessed before the therapy were compared with those at the end of the fifth session and the end of the tenth session in the treatment and the control groups. Pretreatment, end of the fifth session and end of the tenth session MN frequencies were compared between the treatment and the control groups. There was no statistically significant difference in MN frequencies between pretreatment and fifth session or pretreatment and tenth session in both groups. Nor was there any significant difference in the MN frequencies of the treatment and control groups between pretreatment, fifth session and tenth session evaluations. In conclusion, we observed that therapeutic US did not induce increases in MN frequency, which are a sign of cytogenetic damage.

Telomere erosion and chromosomal instability in cells expressing the HPV oncogene 16E6.

Progression to advanced-stage cervical carcinomas is characterized by a recurrent pattern of chromosomal rearrangements. Structural chromosome rearrangements are generated through the fusion of broken chromosome ends. These chromosome breaks may be induced by mutagenic agents such as ionizing radiation, or chromosome ends may be exposed through extensive telomere shortening. The human papilloma virus oncogene 16E6 induces telomerase activity in human keratinocytes, a model system for cervical tumor formation. The present study explores the relationship between 16E6 expression, telomerase activity, and chromosomal instability. We show that the frequency of anaphase bridges is dependent on the level of telomerase activity in 16E6/E7-expressing clones, and is the result of telomere shortening. High frequencies of anaphase bridges, associated with low telomerase activity, correlate with increased chromosome instability. Anaphase bridge formation is also associated with the presence of micronuclei, which are shown to contain unstable chromosomes frequently involved in rearrangements. As anaphase bridges are observed in both high and low telomerase 16E6/E7 clones, but not in hTERT-expressing control clones, expression of 16E6 in these immortalized clones is not sufficient to stabilize shortened telomeres completely. We suggest a model in which HPV-induced tumorigenesis may be dependent on persistent bridge-breakage-fusion cycles that allow for continued genomic rearrangements.

Role of micronucleus-limited DNA in programmed deletion of mse2.9 during macronuclear development of Tetrahymena thermophila.

Extensive programmed DNA rearrangements occur during the development of the somatic macronucleus from the germ line micronucleus in the sexual cycle of the ciliated protozoan Tetrahymena thermophila. Using an in vivo processing assay, we analyzed the role of micronucleus-limited DNA during the programmed deletion of mse2.9, an internal eliminated sequence (IES). We identified a 200-bp region within mse2.9 that contains an important cis-acting element which is required for the targeting of efficient programmed deletion. Our results, obtained with a series of mse2.9-based chimeric IESs, led us to suggest that the cis-acting elements in both micronucleus-limited and macronucleus-retained flanking DNAs stimulate programmed deletion to different degrees depending on the particular eliminated sequence. The mse2.9 IES is situated within the second intron of the micronuclear locus of the ARP1 gene. We show that the expression of ARP1 is not essential for the growth of Tetrahymena. Our results also suggest that mse2.9 is not subject to epigenetic regulation of DNA deletion, placing possible constraints on the scan RNA model of IES excision.

Comparative evaluation of background micronucleus frequencies in domestic mammals.

Exposure of human alimentation-destined animals to toxic substances can be an important risk factor for human health. Mutagenicity tests represent a good method for genotoxic effect evaluation of environmental pollutants. The micronucleus frequency in peripheral blood and bone marrow erythrocytes has been evaluated in four domestic Ungulate species (ox, sheep, swine and horse). For each species two or three groups of animals coming from Italy and other European countries were analysed and the results indicate a relatively low mean frequency of micronucleated erythrocytes (ME), both in peripheral blood and bone marrow. The comparison between these two frequencies in the four species studied shows that the ME frequency in sheep and horse is significantly higher in peripheral blood than in bone marrow, whereas in bovines it is higher in the bone marrow, and in swine the difference is not significant. These results could indicate that in ox and swine the spleen is involved in ME removal from the peripheral circulation, as is known for other species including man. Nevertheless, it cannot be excluded that the same occurs in the other two species, since they exhibit a relatively low peripheral blood ME frequency as well. Further studies on domestic mammals are needed to clarify the spleen function and verify the use of the peripheral blood micronucleus test for genotoxic biomonitoring. At present, the application of the micronucleus test to the bone marrow seems a more suitable method, but, being invasive, it can be used only as a control system of animal hygiene and health, in addition to the routine tests, rather than for biomonitoring.

Anti-genotoxic effects of tea catechins against reactive oxygen species in human lymphoblastoid cells.

Using the cytokinesis-block micronucleus assay in WIL2-NS cells, we investigated the effects of six tea constituents, (-)-epigallocatechin-3-O-gallate (EGCg), (-)-epicatechin-3-O-gallate (ECg), (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (+)-catechin (+C) and gallic acid (GA), on chromosomal damage in two ways; induction by each component on its own and prevention against treatment of reactive oxygen species (ROS). None of the tea constituents induced chromosomal damage at <10 microM. On the other hand, EGCg, EGC, ECg, +C and GA prevented H(2)O(2)-induced chromosomal damage in a dose-dependent manner with a significant effect detected at 1 microM. Chromosomal damage induced by tert-butylhydroperoxide was apparently prevented by EGCg and ECg at 0.3 microM, but not by EGC and GA even at 10 microM, suggesting that the galloyl group linked to flavan-3-ol is needed for the observed protective effect. These results suggest that physiological concentration of tea constituents are not genotoxic but rather anti-genotoxic against ROS, although their preventive effects are slightly different depending on their chemical structure.

Functional consequences of ATM sequence variants for chromosomal radiosensitivity.

The ATM [for ataxia-telangiectasia (A-T) mutated] protein plays a key role in the detection and cellular response to DNA double-strand breaks. Several single-nucleotide polymorphisms (SNPs) have been described in the ATM gene; however, their association with cancer risk or radiosensitivity remains to be fully established. In this study, the functional consequences of specific ATM SNPs on in vitro radiosensitivity, as assessed by micronuclei (MN) formation, were measured in lymphoblastoid cell lines established from 10 breast cancer (BC) patients carrying different ATM missense SNPs, six A-T patients, six A-T heterozygotes (A-T het), and six normal individuals. The BC, A-T het, and A-T cell line groups showed significantly higher mean levels of MN formation after exposure to ionizing radiation (IR) than did the group containing normal cell lines, with similar levels in the BC and A-T het groups. Within the BC lines studied, the group composed of the six carrying the linked 2572T>C (858F>L) and 3161C>G (1054P>R) variants had a higher level of MN after IR exposure compared to that observed in the remaining four BC or in the normal cell lines. This increase was not related to the constitutive ATM mRNA level, which was similar in these BC and the normal cell lines. Our results indicate that alterations in the ATM gene, including the presence of heterozygous mutations and the 2572C and 3161G variant alleles, are associated with increased in vitro chromosomal radiosensitivity, perhaps by interfering with ATM function in a dominant-negative manner.

Inactivation of hCDC4 can cause chromosomal instability.

Aneuploidy, an abnormal chromosome number, has been recognized as a hallmark of human cancer for nearly a century; however, the mechanisms responsible for this abnormality have remained elusive. Here we report the identification of mutations in hCDC4 (also known as Fbw7 or Archipelago) in both human colorectal cancers and their precursor lesions. We show that genetic inactivation of hCDC4, by means of targeted disruption of the gene in karyotypically stable colorectal cancer cells, results in a striking phenotype associated with micronuclei and chromosomal instability. This phenotype can be traced to a defect in the execution of metaphase and subsequent transmission of chromosomes, and is dependent on cyclin E--a protein that is regulated by hCDC4 (refs 2-4). Our data suggest that chromosomal instability is caused by specific genetic alterations in a large fraction of human cancers and can occur before malignant conversion.

The induction of gene mutations and micronuclei by oxiranes and siloranes in mammalian cells in vitro.

Oxiranes and siloranes are candidate molecules for the development of composite materials with low shrinkage. Since some of these molecules are highly reactive, they could lead to adverse biological effects from underlying genetic mechanisms. Therefore, we analyzed the formation of micronuclei (chromosomal aberrations) and the induction of gene mutations (HPRT assay) in mammalian cells. The numbers of micronuclei induced by the oxirane di(cyclohexene-epoxidemethyl)ether (Eth-Ep) at low concentrations (10 micro M) were about five-fold higher than controls. The related compound epoxy cyclohexyl methyl-epoxy cyclo-hexane carboxylate (Est-Ep) was less effective. The activity of diglycidylether of bisphenol A (BADGE) was even lower but similar to the most reactive silorane, di-3,4-epoxy cyclohexylmethyl-dimethyl-silane (DiMe-Sil). No induction of micronuclei was detected in the presence of a rat liver homogenate (S9). Est-Ep and Eth-Ep also induced gene mutations. Our analyses indicated low mutagenic potentials of siloranes; however, some oxiranes induced strong effects at two genetic endpoints.

Micronuclei frequency in children exposed to environmental mutagens: a review.

Cytogenetic monitoring has been traditionally used for the surveillance of populations exposed to genotoxic agents. In recent years sensitivity problems emerged in surveys of populations exposed to low levels of mutagens, and therefore alternative approaches have been explored. Biomonitoring studies in children are a promising field, since because of evident differences in the uptake, metabolism, distribution and excretion of mutagens this population seems to be more susceptible than adults. Further, the effect of major confounders such as cigarettes smoking, occupation, life-style, and dietary factors plays a minor role. Among cytogenetic assays, the micronucleus assay (MN) has several advantages and is increasingly used. A review was then carried out to synthesize the published data on the occurrence of MN in children and adolescents (age range 0-18 years), and to assess the impact of genotoxic exposure on MN frequency. Overall, 20 papers from international literature and 8 Russian papers were included. An effect of age was found within this age range, while the influence of gender on MN frequency was irrelevant. These results were confirmed by the re-analysis of data for 448 children selected from the HUMN database. An effect of chronic and infectious diseases on MN levels has been reported by various authors. Most studies describing the effect of exposure to genotoxic agents (ionizing radiation, chemicals, drugs, environmental tobacco smoke) found an increase of MN in exposed children. The limited number of published papers indicates that the conduct of properly designed studies on the effect of environmental pollutants in children may be difficult. This review confirmed the usefulness of MN assay in biomonitoring studies conducted in children, revealing that in many circumstances investigating children increases the sensitivity of the study, even with low dose exposures.

Spontaneous chromosome loss and colcemid resistance in lymphocytes from patients with myotonic dystrophy type 1.

Myotonic Dystrophy type 1 (DM1) is one of the many inherited human diseases whose molecular defect is the expansion of a trinucleotide DNA sequence. DM1 shares with fragile X syndrome (FMR1), another "unstable triplet syndrome", several molecular features not present in the remaining triplet diseases. As FMR1 is also characterised by chromosome instability at the site of the expanded triplet, lymphocytes from DM1 patients and healthy donors were cultured for micronucleus (MN) analysis, in order to verify if DM1 is also prone to chromosome instability. A FISH analysis was also carried out to detect the presence of centromeric sequences in the observed MN. The data indicate that DM1 patients present a percentage of centromere-positive MN significantly higher than controls, suggesting that chromosome loss is the main mechanism underlying the origin of the increased spontaneous instability. To further assess the proneness to instability of cells of DM1 patients, cultures from patients and controls were treated in vitro with growing concentrations of two different mutagens: colcemid, a "pure" aneugen compound whose target is tubulin, and mytomicin C, a strong clastogen. The results show that the patient group is significantly less sensitive to colcemid. These data, together with FISH analysis, suggest the presence, in DM1 patients, of an already damaged tubulin, which becomes no more sensitive to the effect of colcemid and which could be the main defect underlying the aneugenic effects in DM1.

Micronuclei in peripheral blood lymphocytes as a possible cancer risk biomarker: a cohort study of occupationally exposed workers in Croatia.

AIM: To describe the cohort of Croatian workers monitored for micronuclei in peripheral blood lymphocytes and validate predictive value of micronuclei for the risk of cancer development. METHODS: Between 1985 and 1999, peripheral blood lymphocytes were analyzed with in vitro micronucleus assay in a cohort of 200 subjects occupationally exposed to genotoxic agents. The follow-up for cancer incidence and mortality was performed through the Croatian National Cancer Registry and records of occupational medicine physicians. Micronucleated cell frequency values were compared by Kruskal-Wallis test. RESULTS: The median micronucleated cell frequency value in the cohort was 49 (range, 30-79) per thousand cells. Micronucleated cell frequency was significantly higher in men than in women, which could be attributed to the different distribution of exposures. Micronucleated cell frequency increased with age for both sexes. Smoking habit had no influence on micronucleated cell frequency. The follow-up identified four cases of cancer. Three of them belonged to the highest micronucleated cell frequency tertile. CONCLUSION: Due to a small number of cancer cases, the predictive value of micronuclei for the risk of cancer development in the cohort of Croatian workers was not estimated, but 4 identified cases were more than expected in a similar non-exposed group. The Croatian cohort will contribute to the pooled analysis of the current European study of predictive value of micronuclei for the risk of cancer development.

Does a dose-response relationship reduce sensitivity to hidden bias?

It is often said that an important consideration in judging whether an association between treatment and response is causal is the presence or absence of a dose-response relationship, that is, larger ostensible treatment effects when doses of treatment are larger. This criterion is widely discussed in textbooks and is often mentioned in empirical papers. At the same time, it is well known through both important examples and elementary theory that a treatment may cause dramatic effects with no dose-response relationship, and hidden biases may produce a dose-response relationship when the treatment is without effect. What does a dose-response relationship say about causality? It is observed here that a dose-response relationship may or may not reduce sensitivity to hidden bias, and whether it has or has not can be determined by a suitable analysis using the data at hand. Moreover, a study without a dose-response relationship may or may not be less sensitive to hidden bias than another study with such a relationship, and this, too, can be determined from the data at hand. An example concerning cytogenetic damage among professional painters is used to illustrate.

Micronuclei to detect in vivo chemotherapy damage in a p53 mutated solid tumour.

Apoptosis induction and micronuclei formation were compared following cytotoxic treatments in two rat glioma differing in p53 integrity. In vitro, micronuclei emergence but not apoptosis was linked to the p53 mutated status. In vivo, micronuclei assays were more sensitive to evaluate DNA damage induced by chemotherapy in a p53-mutated solid tumour.

Sister chromatid exchanges and micronuclei analysis in lymphocytes of men exposed to simazine through drinking water.

In some cities of the autonomous community of Extremadura (south-west of Spain), levels of simazine from 10 to 30 ppm were detected in tap water. To analyse the possible effect of this herbicide, two biomarkers, sister chromatid exchanges (SCE) and micronuclei (MN), were used in peripheral blood lymphocytes from males exposed to simazine through drinking water. SCE and MN analysis failed to detect any statistically significant increase in the people exposed to simazine when compared with the controls. With respect to high frequency cells (HFC), a statistically significant difference was detected between exposed and control groups.

An assessment of the genotoxicity of 2-hydroxy-1,4-naphthoquinone, the natural dye ingredient of Henna.

2-Hydroxy-1,4-naphthoquinone (HNQ; Lawsone; CAS 83-72-7) is the principal natural dye ingredient contained in the leaves of Henna (Lawsonia inermis). Published genotoxicity studies on HNQ suggested it was a weak bacterial mutagen for Salmonella typhimurium strain TA98 or was more clearly mutagenic for strain TA 2637, both in the presence of metabolic activation. HNQ was unable to induce sex-linked recessive lethal mutations in Drosophila melanogaster. However, a small increase in micronucleus frequency was reported in the bone marrow of mice at a single mid-range dose level, 24h after intraperitoneal injection. In view of the wide use of Henna hair dyes it was deemed necessary to conduct a thorough investigation, under Good Laboratory Practice conditions, of the genotoxicity of HNQ. HNQ was non-mutagenic in bacterial (Ames test) or mammalian (V79 hprt) assays. It was borderline positive in a mouse lymphoma tk mutation assay and a chromosome aberration test (CHO cells), results that may reflect a similar clastogenic mechanism. Negative in vivo genotoxicity results were noted in the rat hepatocyte in vivo/in vitro UDS test, in peripheral lymphocytes (chromosome aberrations) of rats receiving repeated oral doses of HNQ at the MTD for 28 days, and in mouse and hamster bone marrow chromosome aberration tests. However small, but statistically significant increases in the incidence of bone marrow micronuclei were observed in two out of five tests at 72 h after dosing, but not at 24 or 48 h. There was evidence of haematotoxicity at 72 h, which may have been enhanced by the vehicle (DMSO) used in the positive tests. As erythropoiesis and administration of haematotoxic agents are known to induce small increases in the frequency of bone marrow micronuclei, typically at delayed sampling times, the data suggest that the positive 72 h response produced by HNQ is consistent with stimulation of haematopoiesis subsequent to haematological toxicity of HNQ, and not due to a DNA-reactive mechanism. Overall, the weight of evidence suggests that Henna and HNQ pose no genotoxic risk to the consumer.

Chromosomal aberrations in Crepis capillaris cells detected by FISH.

Crepis capillaris (2n=6) is an excellent plant for the assay of chromosome aberrations after mutagenic treatment. It has simple karyotype: three pairs of morphologically distinct and relatively large chromosomes. The frequency of structural chromosome aberrations and micronuclei in root meristem cells has been used for evaluation of the genotoxicity of chemicals and environmental pollutants. The introduction of fluorescence in situ hybridization method allows more detailed detection and localization of chromosomal rearrangements not only in mitotic but also in interphase nuclei. We demonstrate a few examples of the detection of chromosomal aberrations using rDNA and telomeric sequences as probes for in situ hybridization to C. capillaris chromosomes.