Cisplatin Reduces the Frequencies of Radiotherapy-Induced Micronuclei in Peripheral Blood Lymphocytes of Patients with Gynaecological Cancer: Possible Implications for the Risk of Second Malignant Neoplasms.

Gynaecologic cancers are common among women and treatment includes surgery, radiotherapy or chemotherapy, where the last two methods induce DNA damage in non-targeted cells like peripheral blood lymphocytes (PBL). Damaged normal cells can transform leading to second malignant neoplasms (SMN) but the level of risk and impact of risk modifiers is not well defined. We investigated how radiotherapy alone or in combination with chemotherapy induce DNA damage in PBL of cervix and endometrial cancer patients during therapy. Blood samples were collected from nine endometrial cancer patients (treatment with radiotherapy + chemotherapy-RC) and nine cervical cancer patients (treatment with radiotherapy alone-R) before radiotherapy, 3 weeks after onset of radiotherapy and at the end of radiotherapy. Half of each blood sample was irradiated ex vivo with 2 Gy of gamma radiation in order to check how therapy influenced the sensitivity of PBL to radiation. Analysed endpoints were micronucleus (MN) frequencies, apoptosis frequencies and cell proliferation index. The results were characterised by strong individual variation, especially the MN frequencies and proliferation index. On average, despite higher total dose and larger fields, therapy alone induced the same level of MN in PBL of RC patients as compared to R. This result was accompanied by a higher level of apoptosis and stronger inhibition of cell proliferation in RC patients. The ex vivo dose induced fewer MN, more apoptosis and more strongly inhibited proliferation of PBL of RC as compared to R patients. These results are interpreted as evidence for a sensitizing effect of chemotherapy on radiation cytotoxicity. The possible implications for the risk of second malignant neoplasms are discussed.

Methylglyoxal induces chromosomal instability and mitotic dysfunction in lymphocytes.

Type 2 diabetes is associated with elevated levels of DNA damage, in particular micronuclei (MNi) which are formed by acentric chromosome fragments caused by double-stranded DNA breaks (DSBs), or whole chromosomes which fail to segregate during mitosis. We investigated if methylglyoxal (MGO), a reactive dicarbonyl known to be elevated in type 2 diabetes is capable of increasing chromosomal instability and DNA damage as measured by the cytokinesis block micronucleus cytome (CBMNcyt) assay in B-lymphoblastoid WIL2-NS cells and primary peripheral blood lymphocytes (PBL). We also investigated the level of various dicarbonyl stress biomarkers, including extracellular and intracellular MGO, protein and MGO modifications of DNA. WIL2-NS cells exposed to either MGO or a glyoxalase 1 inhibitor showed increases in MNi and nuclear buds, which were associated with an increase in intracellular MGO. DNA damage in the form of MNi and nucleoplasmic bridges were observed in primary PBL exposed to 10 µM MGO, suggesting low concentrations of MGO may be genotoxic. Furthermore, we showed, using fluorescent in situ hybridisation, that the majority of MNi caused by MGO in WIL2-NS cells were caused by whole chromosome loss events, rather than DSBs. Our data suggest that MGO, a reactive metabolite elevated in type 2 diabetes and other pathologies, can affect genomic integrity by impairing chromosome segregation during mitosis.

Micronuclei, reproduction and child health.

The current review looks for relationships between results from biomarker studies with micronucleus and health effects related to reproduction and children. In adults, an age related increase in MN is well known as well as associations with environmental exposures especially air pollution from traffic and smoking. Literature searches in PubMED and SCOPUS were performed with the following keywords reproduction, children, micronuclei, health effects. In total 162 studies were identified with the keyword children. Concerning children and health and children and environmental exposures, the titles and abstracts of a total of 162 publications were screened for language, inclusion of data from children and selected according to a study selection chart. 9 studies were included for children and health, and 21 studies for children and environmental exposures, with 12 in buccal cells and 9 in lymphocytes. The publications were read and included in tables if data on controls was available. MN frequencies were collected for peripheral blood lymphocytes (PBLs), reticulocytes or buccal cells (BC) and reported as Mean ± SD or Median (IQR). The Mean frequency Ratio, MRi, corresponding to the MN mean for study persons divided by MN mean for control persons was stated as reported in the publication or calculated by us from the data in the publication, where possible. Our systematic analysis revealed a number of positive associations of MN frequencies as a marker of increased health risk in relation to reproduction as well as child health. The majority of studies reported with children concerns exposures of children as well as maternal exposures and newborn health with MN as a biomarker of exposure. Exposure monitoring by MN as biomarker is also reported in studies of school children however most often not related to health effects. The MRis are found in ranges from 1 to 5.5 most studies around 2. As far as MN frequencies in children and exposure are concerned, the MRis range from 0.9 to 5.5, with a range from 1.3-4.9 for lymphocytes and from 1.5 to 2.5 in buccal cells, except for two studies with no differences found between cases and controls. Only one study is available for MRi calculation in reticulocytes with the value of 2.3. These data are supporting MN as a relevant biomarker for children health. However, the data is mostly from small studies with different protocol leaving out the possibility of metanalyses and even statistical comparisons among studies. The actual risk from elevated MNs in children waits large cohort studies with pooled datasets as performed with MN measured in adults. Introduction of buccal cells as non invasive alternative to lymphocytes is increasing and as with the lymphocytes standardised protocols are recommended to enable comparative studies and metaanalyses.

The Genotoxic and Pro-Apoptotic Activities of Advanced Glycation End-Products (MAGE) Measured with Micronuclei Assay Are Inhibited by Their Low Molecular Mass Counterparts.

An association between the cancer invasive activities of cells and their exposure to advanced glycation end-products (AGEs) was described early in some reports. An incubation of cells with BSA-AGE (bovine serum albumin-AGE), BSA-carboxymethyllysine and BSA-methylglyoxal (BSA-MG) resulted in a significant increase in DNA damage. We examined the genotoxic activity of new products synthesized under nonaqueous conditions. These were high molecular mass MAGEs (HMW-MAGEs) formed from protein and melibiose and low molecular mass MAGEs (LMW-MAGEs) obtained from the melibiose and N-α-acetyllysine and N-α-acetylarginine. We have observed by measuring of micronuclei in human lymphocytes in vitro that the studied HMW-MAGEs expressed the genotoxicity. The number of micronuclei (MN) in lymphocytes reached 40.22 ± 5.34 promille (MN/1000CBL), compared to 28.80 ± 6.50 MN/1000 CBL for the reference BSA-MG, whereas a control value was 20.66 ± 1.39 MN/1000CBL. However, the LMW-MAGE fractions did not induce micronuclei formation in the culture of lymphocytes and partially protected DNA against damage in the cells irradiated with X-ray. Human melanoma and all other studied cells, such as bronchial epithelial cells, lung cancer cells and colorectal cancer cells, are susceptible to the genotoxic effects of HMW-MAGEs. The LMW-MAGEs are not genotoxic, while they inhibit HMW-MAGE genotoxic activity. With regard to apoptosis, it is induced with the HMW-MAGE compounds, in the p53 independent way, whereas the low molecular mass product inhibits the apoptosis induction. Further investigations will potentially indicate beneficial apoptotic effect on cancer cells.

Potent aneugenicity of 1-methylpyrene in human cells dependent on metabolic activation by endogenous enzymes.

1-Methylpyrene (1-MP) is a common environmental pollutant and animal carcinogen. After sequential activation by cytochromes P450 and sulfotransferases, it induced gene mutations and micronuclei in mammalian cells. The type of micronuclei formed, entire chromosomes or fragments, was not analysed. In this study, 1-MP and its primary metabolite, 1-hydroxymethylpyrene (1-HMP), were investigated for the induction of centromere-positive and -negative micronuclei in the human hepatoma cell line HepG2 and its derivative C3A, expressing relevant enzymes at higher levels. Under a short-exposure (9 h)/long-recovery regime (2 cell cycles in total), 1-MP and 1-HMP provided negative test results in HepG2 cells. However, they induced micronuclei in C3A cells, the effect being blocked by 1-aminobenzotriazole (inhibitor of cytochromes P450s) and reduced by pentachlorophenol (inhibitor of sulfotransferases). Immunofluorescence staining of centromere protein B in the micronuclei revealed purely clastogenic effects under this regime. Unexpectedly, 1-MP and 1-HMP at concentrations 1/5-1/4 of that required for micronuclei formation led to mitotic arrest and spindle aberrations, as detected by immunofluorescence staining of ß- and γ-tubulin. Following extended exposure (72 h, 2 cell cycles, no recovery), damage to the spindle apparatus and centrosomes was detected at even lower concentrations, with concurrent formation of micronuclei. At low concentrations (1-8 µM 1-MP, 0.25-0.5 µM 1-HMP), the micronuclei induced were unexceptionally centromere-positive. Thus, the chromosome-damaging mechanism of 1-MP was regime and concentration dependent: potently aneugenic under persistent exposure, while clastogenic at higher concentrations following a short-exposure/long-recovery regime. This is a convincing evidence for the existence of metabolic activation-dependent aneugens.

Protective effects of Bidens pilosa on hepatoxicity and nephrotoxicity induced by carbon tetrachloride in rats.

The aim of this study was to assess the protective effects of oral and topical treatment with Bidens pilosa (BP) against carbon tetrachloride (CCl4)- induced toxicity. Fifty-six rats were divided into seven groups: A: CCl4 only; B: CCl4+oral BP; C: CCl4 and topical BP; D: CCl4+oral and topical BP; E: oral BP only; F: negative control; and G: positive control (cyclophosphamide). The animals were treated for 10 weeks. Blood samples were collected for tests of hepatic and renal function, and fragments of the liver, spleen, pancreas, kidney, and intestine were collected for histopathological analyses. Cells from the femoral bone marrow were used for a micronucleus test and 'comet assay'. Statistically significant differences were observed in the levels of gamma-glutamyl transpeptidase (GGT), albumin, urea and creatinine, hepatic inflammation, renal tubular lesion, and inflammation of the intestinal mucosa between the BP-treated groups and untreated group. The median number of micronuclei in group A was 4.00, in group G was 9.00 and in the other groups was 0.00. Group A had the lowest number of cells with a score of 0 and the greatest number with scores of 3 and 4, similar to the results obtained from group G using the 'comet assay'. Thus, BP effectively protected against the toxic effects of CCl4 on the liver, kidney, and intestine and exerted an antimutagenic effect on rats exposed to CCl4.

Low-polar extract from seed of Cleistocalyx nervosum var. paniala modulates initiation and promotion stages of chemically-induced carcinogenesis in rats.

BACKGROUND: Cleistocalyx nervosum var. paniala is a local fruit mainly cultivated in the north of Thailand. Our previous study has reported that the methanol extract of C. nervosum seed presented antimutagenicity in a Salmonella mutation assay. The present study focused on the effect of a low-polar extract of C. nervosum seed on the early stages of diethylnitrosamine (DEN)- and dimethylhydrazine (DMH)-induced carcinogenesis in rats. METHODS: Dried C. nervosum seed powder was extracted using dichloromethane. To study its effect on the initiation stage of carcinogenesis of rats, they were fed with various doses of C. nervosum seed extract (CSE) for 21 days. DEN injection was used to initiate hepatocarcinogenesis and partial hepatectomy was performed to amplify mutated hepatocytes resulting in micronucleated hepatocyte formation. To study the role of CSE on the promotion stage, rats were injected with DEN and DMH to induce preneoplastic lesions and the numbers of glutathione S-transferase placental form (GST-P) positive foci in the liver and aberrant crypt foci (ACF) in the colon were measured. This was followed by CSE administration for 10 weeks. The inhibitory mechanisms of CSE on initiation and promotion stages, including xenobiotic metabolism, cell proliferation and apoptosis, were investigated. RESULTS: The total phenolic content in CSE was 80.34 ± 2.29 mg gallic acid equivalents (GAE) per g of extract and 2,4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone was found to be a major flavonoid. The main terpenoids in CSE were ß-selinene, α-selinene, γ-selinene and o-cymene while 24(Z)-methyl-25-homocholesterol was a major phytosterol. CSE significantly decreased the number of micronucleated hepatocytes in DEN-initiated rats and enhanced the activities of hepatic glutathione S-transferase and UDP-glucuronyltransferase. Furthermore, the formation of preneoplastic lesions in the liver and colon was statistically reduced by CSE. CSE also diminished cell proliferation in the liver and colon indicated by the number of PCNA positive cells. However, CSE did not alter the numbers of apoptotic hepatocytes and colonocytes in DEN- and DMH-initiated rats. CONCLUSIONS: The dichloromethane extract of C. nervosum seed demonstrated chemopreventive effects on chemically-induced carcinogenesis in both initiation and promotion stages in rats. The inhibitory mechanism might be involved in the modulation of hepatic detoxifying enzymes and suppression of hepatocyte and colonocyte proliferation.

Al-Tolerant Barley Mutant hvatr.g Shows the ATR-Regulated DNA Damage Response to Maleic Acid Hydrazide.

ATR, a DNA damage signaling kinase, is required for cell cycle checkpoint regulation and detecting DNA damage caused by genotoxic factors including Al3+ ions. We analyzed the function of the HvATR gene in response to chemical clastogen-maleic acid hydrazide (MH). For this purpose, the Al-tolerant barley TILLING mutant hvatr.g was used. We described the effects of MH on the nuclear genome of hvatr.g mutant and its WT parent cv. "Sebastian", showing that the genotoxic effect measured by TUNEL test and frequency of cells with micronuclei was much stronger in hvatr.g than in WT. MH caused a significant decrease in the mitotic activity of root cells in both genotypes, however this effect was significantly stronger in "Sebastian". The impact of MH on the roots cell cycle, analyzed using flow cytometry, showed no differences between the mutant and WT.

Genotoxicity studies with an ethanolic extract of Kalanchoe pinnata leaves.

Kalanchoe pinnata is a medicinal plant, used mainly in African, Brazilian, and Indian traditional medicine for the treatment of several human disorders. Whole leaf extracts, crude juice of the leaves, and aqueous and organic extracts of the leaves are used. Over the last decade, ethanolic extracts have become the most popular form of Kalanchoe medicinal preparation. In this study, an ethanolic extract of this plant leaf was tested in a battery of standard regulatory genetic toxicology tests. This extract did not induce reverse mutations in the Salmonella/microsome assay but induces a weak genotoxic response in the mouse lymphoma assay and the in vivo micronucleus assay in mice. Our results indicate that this material may cause DNA damage, and its use should be restricted.

Long-term fate of etoposide-induced micronuclei and micronucleated cells in Hela-H2B-GFP cells.

Micronuclei are small nuclear cellular structures containing whole chromosomes or chromosomal fragments. While there is a lot of information available about the origin and formation of micronuclei, less is known about the fate of micronuclei and micronucleated cells. Possible fates include extrusion, degradation, reincorporation and persistence. Live cell imaging was performed to quantitatively analyse the fates of micronuclei and micronucleated cells occurring in vitro. Imaging was conducted for up to 96 h in HeLa-H2B-GFP cells treated with 0.5, 1 and 2 µg/ml etoposide. While a minority of micronuclei was reincorporated into the main nucleus during mitosis, the majority of micronuclei persisted without any alterations. Degradation and extrusion were observed rarely or never. The presence of micronuclei affected the proliferation of the daughter cells and also had an influence on cell death rates. Mitotic errors were found to be clearly increased in micronucleus-containing cells. The results show that micronuclei and micronucleated cells can, although delayed in cell cycle, sustain for multiple divisions.

In vivo kinetics of the genotoxic and cytotoxic activities of cladribine and clofarabine.

The aim of the present in vivo study was to determine the kinetics of the genotoxic and cytotoxic activities of cladribine and clofarabine in mouse normoblasts using flow cytometry. Mice in groups of five were treated with cladribine or clofarabine. Blood samples were obtained from the mouse tails before treatment and every 8 hr posttreatment for 72 hr. These samples were cytometrically scored for micronucleated reticulocytes (RETs), and the percentage of RETs was determined. The results showed that clofarabine and cladribine have early cytotoxic effects that are related to the genotoxic effects reported in previous studies; the drugs have both complex long-lasting genotoxic and cytotoxic kinetic activity, with similar profiles that suggest a relationship between the genotoxic and cytotoxic parameters. The initial genotoxkinetics timing of clofarabine is equivalent to those of difluorodeoxycytidine, likely because both agents inhibit DNA polymerase. Clofarabine shows a higher genotoxic and cytotoxic efficiency than cladribine, in agreement with previous results.

In vitro genomic damage induced by urban fine particulate matter on human lymphocytes.

Urban air pollution represents a global problem, since everyday many mutagenic and carcinogens compounds are emitted into the atmosphere, with consequent adverse health effects on humans and biota. Specifically, particulate matter air pollution was associated with increased risks in human mortality and morbidity. In this paper, we analyse the genomic effects on human lymphocytes of different concentrations of annual Turin PM2.5 extract by an in vitro micronuclei assay. Samplings were collected from an urban meteorological-chemical station positioned in Turin (Italy), one of the most polluted cities in Europe. PM2.5 sampled on filters was used for organic extraction in monthly pools and successively aggregated to produce a mixture representative for a full year PM2.5 collection. Lymphocytes were exposed to four concentrations of PM2.5: 5, 10, 15 and 20 µg/mL and micronuclei, nucleoplasmic bridges and nuclear buds were scored. With respect to controls, PM2.5 significantly increased the frequencies of all analysed biomarkers at all tested concentrations, whereas the CBPI index was significantly reduced only at the concentration of 20 µg/mL. Such in vitro effects can both to stimulate local authorities to adopt efficient measures for air pollution mitigation and to improve human monitoring to detect early precancer lesions.

Ex vivo/in vitro protective effect of myricetin bulk and nano-forms on PhIP-induced DNA damage in lymphocytes from healthy individuals and pre-cancerous MGUS patients.

2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is a central dietary mutagen, produced when proteinaceous food is heated at very high temperatures potentially causing DNA strand breaks. This study investigates the protective potential of a well-researched flavonoid, myricetin in its bulk and nano-forms against oxidative stress induced ex vivo/in vitro by PhIP in lymphocytes from pre-cancerous monoclonal gammopathy of undetermined significance (MGUS) patients and those from healthy individuals. The results from the Comet assay revealed that in the presence of myricetin bulk (10 µM) and myricetin nano (20 µM), the DNA damage caused by a high dose of PhIP (100 µM) was significantly (P < 0.001) reduced in both groups. However, nano has shown better protection in lymphocytes from pre-cancerous patients. Consistent results were obtained from the micronucleus assay where micronuclei frequency in binucleated cells significantly decreased upon supplementing PhIP with myricetin bulk (P < 0.01) and myricetin nano (P < 0.001), compared to the PhIP treatment alone. To briefly determine the cellular pathways involved in the protective role of myricetin against PhIP, we studied gene expression of P53 and ATR kinase (ATM- and Rad3-related), using the real-time PCR technique.

Effects of the DNA repair inhibitors, cytosine arabinoside and 3-aminobenzamide, on the frequency of radiation-induced micronuclei, nuclear buds, and nucleoplasmic bridges.

BACKGROUND: Micronuclei (MN), nuclear bud (NBud), and nucleoplasmic bridge (NPB) are suggested as biomarkers for radiation exposure; however, they have not been extensively studied to understand the underlying mechanisms responsible for their formation. OBJECTIVES: To (1) validate NBud and NPB within the cytokinesis-blocked micronucleus (CBMN) assay as biomarkers for radiation exposure and (2) determine the effects of the DNA repair inhibitors, cytosine arabinoside (Ara C) and 3-aminobenzamide (3-AB) on radiation-induced MN, NBud, and NPB formation. METHODS: Human blood samples were irradiated with 0-3 Gy X-rays and subsequently treated with Ara C and 3-AB. CBMN and chromosome aberration assays were carried out to measure MN, NBud, and NPB and dicentric chromosomes, respectively. RESULTS: The frequency of radiation-induced MN, NBud, and NPB increased in a dose-dependent manner. The frequency of MN, NBud, and NPB was highly and positively correlated with the dicentric chromosome, a standard biomarker for biodosimetry (r > 0.98, p < 0.0001). Furthermore, Ara C increased the frequency of MN, NBud, and NPB, whereas 3-AB increased the frequency of MN and NPB, but not NBud. Further, the potentiating effect of Ara C on the frequency of MN, NBud, and NPB was greater than that of 3-AB. CONCLUSION: Our results validate NBuds and NPBs as independent valuable markers of radiation exposure. Additionally, we suggest that different mechanisms are likely involved in the formation of NBuds and NPBs following X-irradiation; however, additional studies are warranted to better understand the contribution of distinct DNA repair pathways to the formation of radiation-induced damages.

Occupational exposure of dental technicians to methyl methacrylate: Genotoxicity assessment.

Dental technicians may be chronically exposed to methyl methacrylate (MMA), used in the production of dental prostheses. We have studied whether occupational exposure to MMA affects genotoxicity biomarkers such as 8-OHdG formation, comet assay, and buccal micronucleus frequency. MMA exposure was assessed via ambient air analysis. Although no significant differences between exposed and non-exposed individuals were seen with respect to blood genotoxicity measurements, we found a higher level of buccal-cell anomalies in the exposed group.

Evaluation of the genotoxic potential of apoptosis inducers with the γH2AX assay in human cells.

Human risk assessment of genotoxic chemicals is an important area of research. However, the specificity of in vitro mammalian genotoxicity assays is sometime low, as they yield to misleading positive results that are not observe in in vivo studies. Apoptosis can be a confounding factor in the interpretation of the results. Recently, a new strategy for genotoxicity screening, based on the combined analysis of phosphorylated histones H2AX (γH2AX) and H3 (pH3), was proposed to discriminate efficiently aneugenic from clastogenic compounds. However, γH2AX biomarker could also be induce by apoptosis. The aim of the present study was to investigate the specificity of this genotoxic biomarker. For this purpose, we analyzed 26 compounds inducing apoptosis by different mechanism of action, with the γH2AX assay in three human cell lines after 24 h treatment. Most of the tested chemicals were negative in the assay, whatever the cell line tested. The few compounds that generated positive data have also been report positive in other genotoxicity assays. The data presented here demonstrate that the γH2AX assay is not vulnerable to the generation of misleading positive results by apoptosis inducers. Currently, no formal guidelines have been approve for the γH2AX assay for regular genotoxicity studies, but we suggest that this biomarker could be used as a new standard genotoxicity assay.

Micronuclei and disease - Report of HUMN project workshop at Rennes 2019 EEMGS conference.

The "Micronuclei and Disease" workshop was organized by the HUMN Project consortium and hosted by the European Environmental Mutagen and Genomics Society at their annual meeting in Rennes, France, on 23 May 2019. The program of the workshop focused on addressing the emerging evidence linking micronucleus (MN) frequency to human disease. The first objective was to review what has been published and evaluate the level and quality of evidence for the connection between MN frequency and various diseases through all life stages. The second objective was to identify the knowledge gaps and what else needs to be done to determine the clinical utility of MN assays as predictors of disease risk and of prognosis when disease is active. Speakers at the workshop discussed the association of MN frequency with inflammation, infertility, pregnancy complications, obesity, diabetes, cardiovascular disease, kidney disease, cervical and bladder cancer, oral head and neck cancer, lung cancer, accelerated ageing syndromes, neurodegenerative diseases, and a road-map on how to utilise this knowledge was proposed. The outcomes of the workshop indicated that there are significant opportunities for translating the application of MN assays into clinical practice to improve disease prevention and risk management and to inform public health policy.

A new tool for genotoxic risk assessment: Reevaluation of the cytokinesis-block micronucleus assay using semi-automated scoring following telomere and centromere staining.

BACKGROUND: The cytokinesis-block micronucleus (CBMN) assay is an internationally recognized method for measuring DNA damage after exposure to genotoxic agents, as well as a biomarker for DNA repair and chromosomal instability. The high baseline level of micronuclei (MN) in the healthy population has limited the sensitivity and application of the CBMN assay for the follow-up of exposed populations. We reevaluated the sensitivity of the CBNM assay using semi-automated MN scoring following telomere and centromere (TC) staining after in vitro exposure to genotoxic agents (mitomycin or radiation) or aneugenic agents (vinblastine). MATERIALS AND METHODS: Blood samples from 12 healthy donors were exposed to 137Cs at seven doses from 0.1-4 Gy and cultured for 72 h. Cytochalasin B was added at 46 h of culture. The exposure of chemical agents (mitomycin or vinblastine) was performed after 48 h of culture for 3 h. Cytochalasin B was added after treatment and slides were prepared 24 h after. MN was semi-automatically scored following TC staining. Nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) were assessed in a human cell line after TC staining. RESULTS: The introduction TC staining to the scoring of MN not only renders MN scoring more efficient and robust, but also permits discrimination between exposure to clastogenic (MN with only telomere signals) and aneugenic agents (MN with both TC signals). The resulting improvement of MN detection led to an increase in the sensitivity of the CBMN assay following low-dose radiation exposure (0.3 versus 0.1 Gy). Hyperradiosensitivity phenomenon was observed after low dose exposure. A dose-response curve was obtained for up to 4 Gy. In addition, TC staining permits assessment of the nature of NPBs and NBUDs as biomarkers for genotoxicity and chromosomal instability. CONCLUSION: These approaches can be potentially used to follow-up populations exposed to genotoxic agents and assess cancer risk.

Development of an integrated assay in human TK6 cells to permit comprehensive genotoxicity analysis in vitro.

In vitro genetic toxicology assays are used to assess the genotoxic potential of chemicals or mixtures. They measure chromosome damage (e.g., micronucleus [MN] formation) or gene mutation, and different combinations of data generated from such assays are evaluated in concert in order to identify genotoxic hazards. Mode-of-action (MoA) information is also fundamental to understanding any apparent genotoxic response. In view of the importance of these types of data for full characterization of genotoxic potential, we leveraged relevant endpoints already established in the human TK6 cell line to develop a single integrated assay that measures MN formation, gene mutation (at the thymidine kinase locus), and MoA (DNA damage response biomarkers). Several prototypical direct-acting genotoxins (methyl methanesulfonate, mitomycin C, and 4-nitroquinoline 1-oxide), pro-genotoxins (benzo[a]pyrene and cyclophosphamide monohydrate), and one non-DNA reactive genotoxin (vinblastine sulfate) were assessed in the approach and found to elicit genotoxic profiles that were generally consistent with their MoA. In contrast, the non-genotoxic agents D-mannitol and (2-chloroethyl) trimethyl-ammonium chloride induced negligible effects on all endpoints up to a top concentration of 10 mM. Sodium diclofenac, presumed to be non-genotoxic, provoked an induction in the phosphoserine10-H3-positive cell population within a small window of concentrations (0.157-0.314 mM), as well as increases in γH2AX, nuclear p53, and MN at higher concentrations, although it had no effect on the mutation frequency endpoint. G2M cell cycle arrest was also largely observed in cells that exhibited genotoxicity in the in vitro MN assay. The TK6 cell-based integrated assay represents an in vitro approach that permits comprehensive genotoxicity analysis in a human-relevant test system. Moreover, its vis-à-vis nature may facilitate further comprehension of the range of effects that can manifest in human cells in response to DNA-damaging agents.

Multiple-endpoint genotoxicity assay for colon carcinogen 1,2-dimethylhydrazine.

Human risk assessment of the toxic potency of chemicals typically includes genotoxicity assays for predicting carcinogenicity. Gene mutation frequency and chromosomal aberration are two major genotoxicity endpoints in standardized in vitro and in vivo assays. The weight-of-evidence approach in risk assessment is more focused on in vivo assay results; however, animal welfare considerations are aimed at the reduction, replacement, and refinement (3R's) of animal experiments, including a reduction in the number of experimental animals. Proposals to reduce experimental animals in genotoxicity testing include the incorporation of genotoxicity endpoint(s) into other toxicological studies and the combination of two or more assays detecting different genotoxicity endpoints in the same animals. In this study, we used 1,2-dimethylhydrazine as a model chemical of colon carcinogen to assess gene mutation frequency and chromosomal aberration in vivo simultaneously. Specifically, a gene mutation frequency assay was combined with a multiple-organ micronucleus test (peripheral blood, bone marrow, liver, and colon) in F344 gpt delta transgenic rats. Both gpt mutant frequency and micronucleated cell frequency significantly increased in colon and liver but not in bone marrow. Interestingly, we found that the colon carcinogen induced both gene mutations and micronuclei in the targeted colon tissue. Thus, we demonstrated that the mechanism of a carcinogen could be derived from an animal experiment using a lower number of experimental animals as currently recommended. Moreover, a significant increase in mutant frequency in colon and liver was already observed on the first day after treatment completion, as well as on the third day, which is the guideline-recommended period. Thus, this endpoint is compatible with other genotoxicity assays. We confirmed that performing the micronucleus assay in combination with a gene mutation assay in F344 gpt delta transgenic rats is useful to evaluate different genotoxic endpoints simultaneously in the same animals, which reduces the number of experimental animals.