Tetraploidy as a metastable state towards malignant cell transformation within a systemic approach of cancer development.

Tetraploidy, a condition in which a cell has four homologous sets of chromosomes, may be a natural physiological condition or pathophysiological such as in cancer cells or stress induced tetraploidisation. Its contribution to cancer development is well known. However, among the many models proposed to explain the causes, mechanisms and steps of malignant cell transformation, only few integrate tetraploidization into a systemic multistep approach of carcinogenesis. Therefore, we will i) describe the molecular and cellular characteristics of tetraploidy; ii) assess the contribution of stress-induced tetraploidy in cancer development; iii) situate tetraploidy as a metastable state leading to cancer development in a systemic cell-centered approach; iiii) consider knowledge gaps and future perspectives. The available data shows that stress-induced tetraploidisation/polyploidisation leads to p53 stabilisation, cell cycle arrest, followed by cellular senescence or apoptosis, suppressing the proliferation of tetraploid cells. However, if tetraploid cells escape the G1-tetraploidy checkpoint, it may lead to uncontrolled proliferation of tetraploid cells, micronuclei induction, aneuploidy and deploidisation. In addition, tetraploidization favors 3D-chromatin changes and epigenetic effects. The combined effects of genetic and epigenetic changes allow the expression of oncogenic gene expression and cancer progression. Moreover, since micronuclei are inducing inflammation, which in turn may induce additional tetraploidization, tetraploidy-derived genetic instability leads to a carcinogenic vicious cycle. The concept that polyploid cells are metastable intermediates between diploidy and aneuploidy is not new. Metastability denotes an intermediate energetic state within a dynamic system other than the system's state at least energy. Considering in parallel the genetic/epigenetic changes and the probable entropy levels induced by stress-induced tetraploidisation provides a new systemic approach to describe cancer development.

"Micronuclei and Disease" special issue: Aims, scope, and synthesis of outcomes.

The purpose of the "Micronuclei and Disease" special issue (SI) is to: (i) Determine the level of evidence for association of micronuclei (MN), a biomarker of numerical and structural chromosomal aberrations, with risk of specific diseases in humans; (ii) Define plausible mechanisms that explain association of MN with each disease; (iii) Identify knowledge gaps and research needed to translate MN assays into clinical practice. The "MN and Disease" SI includes 14 papers. The first is a review of mechanisms of MN formation and their consequences in humans. 11 papers are systematic reviews and/or meta-analyses of the association of MN with reproduction, child health, inflammation, auto-immune disease, glycation, metabolic diseases, chronic kidney disease, cardiovascular disease, eleven common cancers, ageing and frailty. The penultimate paper focuses on effect of interventions on MN frequency in the elderly. A road map for translation of MN data into clinical practice is the topic of the final paper. The majority of reviewed studies were case-control studies in which the ratio of mean MN frequency in disease cases relative to controls, i.e. the mean ratio (MR), was calculated. The mean of these MR values, estimated by meta-analyses, for lymphocyte and buccal cell MN in non-cancer diseases were 2.3 and 3.6 respectively, and for cancers they were 1.7 and 2.6 respectively. The highest MR values were observed in studies of cancer cases in which MN were measured in the same tissue as the tumour (MR = 4.9-10.8). This special issue is an important milestone in the evidence supporting MN as a reliable genomic biomarker of developmental and degenerative disease risk. These advances, together with results from prospective cohort studies, are helping to identify diseases in which MN assays can be practically employed in the clinical setting to better identify high risk patients and to prioritise them for preventive therapy.

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.

Micronuclei as biomarkers of DNA damage, aneuploidy, inducers of chromosomal hypermutation and as sources of pro-inflammatory DNA in humans.

Micronuclei (MNi) are among the most widely studied biomarkers of DNA damage and chromosomal instability in humans. They originate from chromosome fragments or intact chromosomes that are not included in daughter nuclei during mitosis. The main reasons for their formation are a lack of functional centromere in the chromosome fragments or whole chromosomes or defects in one or more of the proteins of the mitotic system that, consequently, fails to segregate chromosomes properly. Assays have been developed to measure MNi in peripheral blood lymphocytes, red blood cells as well as various types of epithelial cells such as buccal, nasal, urothelial and cervical cells. Some of the assays have been further developed into micronucleus (MN) cytome assays to include additional nuclear anomalies, cell death and nuclear division biomarkers. In addition, the use of molecular probes has been adopted widely for the purpose of understanding the mechanistic origin of MNi. MN assays in humans are used for the purpose of investigating the genotoxic effects of adverse environmental, life-style and occupational factors, genetic susceptibility to DNA damage, and for determining risk of accelerated aging and diseases affected by genomic instability such as developmental defects and cancer. The emerging new knowledge showing that chromosomes trapped in MNi can undergo a high rate of fragmentation and become massively re-arranged have highlighted the possibility that MN formation is not only a biomarker of induced DNA damage but also a mechanism that drives hypermutation. Furthermore, another line of recent research showed that DNA and chromatin leaking from disrupted MNi triggers the innate immune cGAS-STING mechanism that promotes inflammation which can cause a wide-range of age-related diseases if left unresolved. For these reasons, MN assays in humans have become an increasingly important biomarker of disease initiation and progression across all life-stages.

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.

Chemically induced aneuploidy in germ cells. Part II of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

As part of the 7th International Workshops on Genotoxicity Testing held in Tokyo, Japan in November 2017, a workgroup of experts reviewed and assessed the risk of aneugens for human health. The present manuscript is one of three manuscripts from the workgroup and reports on the unanimous consensus reached on the evidence for aneugens affecting germ cells, their mechanisms of action and role in hereditary diseases. There are 24 chemicals with strong or sufficient evidence for germ cell aneugenicity providing robust support for the ability of chemicals to induce germ cell aneuploidy. Interference with microtubule dynamics or inhibition of topoisomerase II function are clear characteristics of germ cell aneugens. Although there are mechanisms of chromosome segregation that are unique to germ cells, there is currently no evidence for germ cell-specific aneugens. However, the available data are heavily skewed toward chemicals that are aneugenic in somatic cells. Development of high-throughput screening assays in suitable animal models for exploring additional targets for aneuploidy induction, such as meiosis-specific proteins, and to prioritize chemicals for the potential to be germ cell aneugens is encouraged. Evidence in animal models support that: oocytes are more sensitive than spermatocytes and somatic cells to aneugens; exposure to aneugens leads to aneuploid conceptuses; and, the frequencies of aneuploidy are similar in germ cells and zygotes. Although aneuploidy in germ cells is a significant cause of infertility and pregnancy loss in humans, there is currently limited evidence that aneugens induce hereditary diseases in human populations because the great majority of aneuploid conceptuses die in utero. Overall, the present work underscores the importance of protecting the human population from exposure to chemicals that can induce aneuploidy in germ cells that, in contrast to carcinogenicity, is directly linked to an adverse outcome.

Targets and mechanisms of chemically induced aneuploidy. Part 1 of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

An aneuploidy workgroup was established as part of the 7th International Workshops on Genotoxicity Testing. The workgroup conducted a review of the scientific literature on the biological mechanisms of aneuploidy in mammalian cells and methods used to detect chemical aneugens. In addition, the current regulatory framework was discussed, with the objective to arrive at consensus statements on the ramifications of exposure to chemical aneugens for human health risk assessment. As part of these efforts, the workgroup explored the use of adverse outcome pathways (AOPs) to document mechanisms of chemically induced aneuploidy in mammalian somatic cells. The group worked on two molecular initiating events (MIEs), tubulin binding and binding to the catalytic domain of aurora kinase B, which result in several adverse outcomes, including aneuploidy. The workgroup agreed that the AOP framework provides a useful approach to link evidence for MIEs with aneuploidy on a cellular level. The evidence linking chemically induced aneuploidy with carcinogenicity and hereditary disease was also reviewed and is presented in two companion papers. In addition, the group came to the consensus that the current regulatory test batteries, while not ideal, are sufficient for the identification of aneugens and human risk assessment. While it is obvious that there are many different MIEs that could lead to the induction of aneuploidy, the most commonly observed mechanisms involving chemical aneugens are related to tubulin binding and, to a lesser extent, inhibition of mitotic kinases. The comprehensive review presented here should help with the identification and risk management of aneugenic agents.

Role of aneuploidy in the carcinogenic process: Part 3 of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

Aneuploidy is regarded as a hallmark of cancer, however, its role is complex with both pro- and anti-carcinogenic effects evident. In this IWGT review, we consider the role of aneuploidy in cancer biology; cancer risk associated with constitutive aneuploidy; rodent carcinogenesis with known chemical aneugens; and chemotherapy-related malignant neoplasms. Aneuploidy is seen at various stages in carcinogenesis. However, the relationship between induced aneuploidy occurring after exposure and clonal aneuploidy present in tumours is not clear. Recent evidence indicates that the induction of chromosomal instability (CIN), may be more important than aneuploidy per se, in the carcinogenic process. Down Syndrome, trisomy 21, is associated with altered hematopoiesis in utero which, in combination with subsequent mutations, results in an increased risk for acute megakaryoblastic and lymphoblastic leukemias. In contrast, there is reduced cancer risk for most solid tumours in Down Syndrome. Mouse models with high levels of aneuploidy are also associated with increased cancer risk for particular tumours with long latencies, but paradoxically other types of tumour often show decreased incidence. The aneugens reviewed that induce cancer in humans and animals all possess other carcinogenic properties, such as mutagenicity, clastogenicity, cytotoxicity, organ toxicities, hormonal and epigenetic changes which likely account for, or interact with aneuploidy, to cause carcinogenesis. Although the role that aneuploidy plays in carcinogenesis has not been fully established, in many cases, it may not play a primary causative role. Tubulin-disrupting aneugens that do not possess other properties linked to carcinogenesis, were not carcinogenic in rodents. Similarly, in humans, for the tubulin-disrupting aneugens colchicine and albendazole, there is no reported association with increased cancer risk. There is a need for further mechanistic studies on agents that induce aneuploidy, particularly by mechanisms other than tubulin disruption and to determine the role of aneuploidy in pre-neoplastic events and in early and late stage neoplasia.

Risks of aneuploidy induction from chemical exposure: Twenty years of collaborative research in Europe from basic science to regulatory implications.

Although Theodor Boveri linked abnormal chromosome numbers and disease more than a century ago, an in-depth understanding of the impact of mitotic and meiotic chromosome segregation errors on cell proliferation and diseases is still lacking. This review reflects on the efforts and results of a large European research network that, from the 1980's until 2004, focused on protection against aneuploidy-inducing factors and tackled the following problems: 1) the origin and consequences of chromosome imbalance in somatic and germ cells; 2) aneuploidy as a result of environmental factors; 3) dose-effect relationships; 4) the need for validated assays to identify aneugenic factors and classify them according to their modes of action; 5) the need for reliable, quantitative data suitable for regulating exposure and preventing aneuploidy induction; 6) the need for mechanistic insight into the consequences of aneuploidy for human health. This activity brought together a consortium of experts from basic science and applied genetic toxicology to prepare the basis for defining guidelines and to encourage regulatory activities for the prevention of induced aneuploidy. Major strengths of the EU research programmes on aneuploidy were having a valuable scientific approach based on well-selected compounds and accurate methods that allow the determination of precise dose-effect relationships, reproducibility and inter-laboratory comparisons. The work was conducted by experienced scientists stimulated by a fascination with the complex scientific issues surrounding aneuploidy; a key strength was asking the right questions at the right time. The strength of the data permitted evaluation at the regulatory level. Finally, the entire enterprise benefited from a solid partnership under the lead of an inspired and stimulating coordinator. The research programme elucidated the major modes of action of aneugens, developed scientifically sound assays to assess aneugens in different tissues, and achieved the international validation of relevant assays with the goal of protecting human populations from aneugenic chemicals. The role of aneuploidy in tumorigenesis will require additional research, and the study of effects of exposure to multiple agents should become a priority. It is hoped that these reflections will stimulate the implementation of aneuploidy testing in national and OECD guidelines.

Validity of the Lymphocyte Cytokinesis-Block Micronucleus Assay (L-CBMN) as biomarker for human exposure to chemicals with different modes of action: A synthesis of systematic reviews.

Recently fourteen systematic reviews applying the same selection and evaluation criteria analyzed the induction of micronuclei in lymphocytes as biomarker for DNA damage induced by human exposure to a given chemical or chemical mixture. The results obtained in the individual reviews were summarized to evaluate the validity of the Cytokinesis-Block-Micronucleus assay in lymphocytes (L-CBMN) and propose recommendations for its use in occupational and environmental exposure studies. All systematic reviews found consistent increases of MN frequencies in exposed subjects versus controls in all genotoxic compounds or group of chemicals investigated, in the following decreasing order: As/Cr/Ni, vinyl chloride, formaldehyde, Hg/Pb/Cd, "miscellaneous", pesticides, cytostatics/antineoplastics, anaesthetic gasses, dust/asbestos/other fibers, polycyclic aromatic hydrocarbons, ethylene oxide, butadiene, styrene and petroleum/derivatives. Two reviews compared the results with the recommended exposure limits. For styrene, MN was found not to be induced under the recommended threshold limit. For vinyl chloride the safe exposure limit based on the L-CBMN data is lower than the current one. The L-CBMN thus appears to be a valid biomarker to assess DNA damage in populations exposed to genotoxic chemicals. Many shortcomings have been reported in assessment of confounding factors, such as lifestyle patterns, in particular diet and the major one the exposure assessment. All these factors together with methodological variables may contribute to the large variability in MN frequencies, also in controls. Information on frequency and origin of MN in more than one tissue (e.g. lymphocytes and buccal cells) in parallel, may provide better understanding of the mechanisms involved. Use of automated MN scoring systems to increase numbers of cells scored and facilitate screening more individuals would increase data reliability and provide information on the link between mutagenicity and carcinogenicity, if the studies are done prospectively. Efforts should be made to unravel the genotoxic effects induced when chronic and/or mixed exposures are involved.

Maternal diet during pregnancy and micronuclei frequency in peripheral blood T lymphocytes in mothers and newborns (Rhea cohort, Crete).

PURPOSE: The study assessed whether diet and adherence to cancer prevention guidelines during pregnancy were associated with micronucleus (MN) frequency in mothers and newborns. MN is biomarkers of early genetic effects that have been associated with cancer risk in adults. METHODS: A total of 188 mothers and 200 newborns from the Rhea cohort (Greece) were included in the study. At early-mid pregnancy, we conducted personal interviews and a validated food frequency questionnaire was completed. With this information, we constructed a score reflecting adherence to the World Cancer Research Fund/American Institute for Cancer Research cancer prevention guidelines on diet, physical activity and body fatness. At delivery, maternal and/or cord blood was collected to measure DNA and hemoglobin adducts of dietary origin and frequencies of MN in binucleated and mononucleated T lymphocytes (MNBN and MNMONO). RESULTS: In mothers, higher levels of red meat consumption were associated with increased MNBN frequency [2nd tertile IRR = 1.34 (1.00, 1.80), 3rd tertile IRR = 1.33 (0.96, 1.85)] and MNMONO frequency [2nd tertile IRR = 1.53 (0.84, 2.77), 3rd tertile IRR = 2.69 (1.44, 5.05)]. The opposite trend was observed for MNBN in newborns [2nd tertile IRR = 0.64 (0.44, 0.94), 3rd tertile IRR = 0.68 (0.46, 1.01)], and no association was observed with MNMONO. Increased MN frequency in pregnant women with high red meat consumption is consistent with previous knowledge. CONCLUSIONS: Our results also suggest exposure to genotoxics during pregnancy might affect differently mothers and newborns. The predictive value of MN as biomarker for childhood cancer, rather than adulthood, remains unclear. With few exceptions, the association between maternal carcinogenic exposures during pregnancy and childhood cancer or early biologic effect biomarkers remains poorly understood.

The lymphocyte cytokinesis block micronucleus test in human populations occupationally exposed to vinyl chloride: A systematic review and meta-analysis.

Vinyl chloride (VC) is widely used in industry in the production of polyvinyl chloride (PVC), which is used to manufacture a large variety of materials. VC was classified as a known (Group 1) human carcinogen by IARC on the basis of increased risk for liver angiosarcoma and hepatocellular cancer, and the carcinogenicity of VC was shown to be mediated by a genotoxic mechanism. Following inhalation, the compound is rapidly absorbed and metabolized in the liver to the electrophilic metabolites chloroethylene-oxide and chloroacetaldehyde, which form DNA adducts that can be processed into point mutations in cancer-related genes detected in humans and rats exposed to VC. A number of genotoxicity biomarkers were applied in workers exposed to VC to detect early biological responses associated with the carcinogenesis process. The present systematic review analyzed the published studies in which the cytokinesis-block micronucleus assay in peripheral lymphocytes (L-CBMN) was applied in VC-exposed subjects. Thirteen out of fifteen retrieved studies performed in China showed increased MN frequencies (FR 1.92-3.98) associated with increased cumulative exposure or employment time. Twofold and more than threefold increases were detected in PVC-exposed workers exposed to a mean of 50ppm of VC in the former Yugoslavia and in South India, respectively. The meta-analysis of MN frequency from six eligible studies confirmed this tendency (pooled MR 2.32 - 95% CI 1.64-3.27). The benchmark dose lower limit for 10% excess risk (BMDL 10) calculated from three studies resulted in an estimated exposure limit of 0.03-0.07mg/m3. Overall the results of this review showed the need for further studies, especially because PVC products from China may contain high levels of uncoupled VCM that could represent a source of exposure to workers and consumers. Moreover, the results underline the importance of re-evaluating the recommended exposure limits using new biomonitoring methods in addition to MN.

Vitamin D insufficient levels during pregnancy and micronuclei frequency in peripheral blood T lymphocytes mothers and newborns (Rhea cohort, Crete).

BACKGROUND & AIMS: Vitamin D deficiency is common among pregnant women and may be associated with several adverse health outcomes including cancer. Micronuclei frequency is a biomarker of early genetic effects and has been used to examine the association between genotoxic exposures and cancer. We examined maternal vitamin D levels during pregnancy in associations with micronuclei frequency in maternal blood and in cord blood. METHODS: 173 mothers and 171 newborns born between 2007 and 2008 in Heraklion (Crete, Greece) were included in the study. Between 14th and 18th weeks of gestation we collected information on maternal diet using food frequency questionnaires (FFQs). We measured maternal serum concentrations of 25-hydroxyvitamin D [25(OH)D] between the first and second trimester of pregnancy. We estimated dietary vitamin D intake using information from FFQ. After delivery we collected cord blood and maternal peripheral blood. We used the cytokinesis-block micronucleus (CBMN) assay to assess the frequencies of micronucleated cells in binucleated T lymphocytes (MNBN). RESULTS: Maternal insufficient serum levels of 25(OH)D (<50 nmol/L) during pregnancy were associated with increased MNBN frequency in cord blood [IRR = 1.32 (95%CI: 1.00, 1.72)]. This increase was higher for newborns with birth weight above the third quartile [≥3.500 kg; IRR = 2.21 (1.26, 3.89)]. Similarly, low levels of dietary vitamin D were associated with increased MNBN frequency in cord blood [middle tertile IRR = 1.08 (0.78, 1.47), lower tertile IRR = 1.51 (1.06, 2.14)]. Insufficient levels of vitamin D were not associated with MNBN in mothers. CONCLUSION: Our results suggest that vitamin D deficiency during pregnancy increases genotoxic risks in newborns. The prevalence of vitamin D deficiency globally is high and it is important to further investigate whether vitamin D supplementation or similar interventions during pregnancy could prevent DNA damage at early stages of life.

Towards a New Paradigm in Nano-Genotoxicology: Facing Complexity of Nanomaterials' Cellular Interactions and Effects.

Changes in paradigm contribute to advances in research. The current paradigms for the evaluation of toxicity of chemicals refer to linear or curvilinear dose-response curves with or without threshold and to surface-dependent induction of oxidative damage for particles. The unique physicochemical properties and biological/genotoxic activity of engineered nanomaterials (NMs) require the development of a new paradigm. Because of their unusual dosimetry and their multiple interactions at NM level (agglomeration/aggregation) and at different cellular and extracellular levels, NMs are likely to have complex modes of action (multiple hits at multiple targets) leading to complex thresholded-non-thresholded dose-response curves. Understanding their cellular targets and their modes of action will contribute to the production of safe-by-design NMs. An integrative, cell-by-cell approach for genotoxic effects should be applied to tackle this emerging paradigm in nano-genotoxicology.

The ex vivo L-CBMN assay detects significant human exposure to butadiene.

1,3-Butadiene (BD), an important industrial chemical used in the production of synthetic rubber and resins and a ubiquitous environmental pollutant, was classified as a human carcinogen by IARC. BD requires metabolic activation to different epoxides that are known to bind to DNA, inducing also DNA-DNA and DNA-protein crosslinks. The DNA damage leading to mutations has been identified as the mode of action of BD. Experimental studies in rodents revealed widely different BD carcinogenic/mutagenic potency in rat and mice, associated to differences in BD metabolism. The available biomonitoring studies in workers occupationally exposed to BD, considering different genetic endpoints, do not allow to reach a conclusion on the BD carcinogenic/mutagenic risk in humans. The present systematic review retrieves and analyzes the published studies on the application of the cytokinesis-block micronucleus assay in peripheral lymphocytes (L-CBMN) of BD exposed subjects. Ten articles were retrieved related to seven studies on BD exposure and one study on exposure to a mixture of compounds in a styrene-butadiene tire manufacturing plant. Four studies carried out in Europe related to heterogeneous groups of workers exposed in BD monomer or polymer manufacturing and processing industries, reporting mean individual exposure levels below 3ppm, failed to find any increase of MN frequency. Three studies, including mixed groups of workers involved in different stages of the production and manufacturing of BD in China, show increased MN frequencies associated with the intensity of the exposure, with a relevant positive response (FR=2.29) when the mean cumulative dose was estimated as 266ppm/year. These results are consistent with the data on the exposure-response curve for total leukemia mortality showing no increase for cumulative exposure less than or equal to 200 BD ppm-years. The L-CBMN assay, measuring both chromosome breakage and chromosome loss, events involved in induction of leukemia, seems to be a promising biomarker for cancer risk induced by BD.

Reprint of "Biomonitoring of genotoxic effects for human exposure to nanomaterials: The challenge ahead".

Exposures to nanomaterials (NMs), with their specific physico-chemical characteristics, are likely to increase over the next years, as their production for industrial, consumer and medical applications is steadily rising. Therefore, there is an urgent need for the implementation of human biomonitoring studies of genotoxic effects after NM exposures in order to monitor and assure safety for workers and the general population. In this review, most commonly used biomarkers of early genetic effects were analyzed for their adequacy after NM exposures. A more in depth analysis of the ex vivo/in vitro lymphocyte MN assay was performed, although, in literature no studies are available using this assay for NM exposures. Therefore, the known factors determining the NMs tissue/cellular targets and the multiplicity of modes of action of NMs were summarized. The main pending questions are whether (1) lymphocytes are a NM target or an adequate surrogate tissue, (2) whether the buccal MN assay might be more suitable for NM exposures via inhalation or ingestion, as buccal cells might be exposed more directly. While the current state-of-the-art does not allow for drawing firm conclusions, major research gaps are identified and some cautious recommendations can be formulated. Therefore in vitro and in vivo studies should be conducted comparing methodologies side-by-side in the same subjects and for different types of NMs. The ex vivo/in vitro MN assay in its automated version, allowing objective analysis of large cohorts and detection of direct and indirect genotoxic effects, remains a valuable candidate for human biomonitoring to NM exposure. Considering the potential cancer risk from exposure to NMs and previous dramatic experiences with too late surveillance of occupational exposures to similar substances (e.g. to asbestos), there is an urgent need to define and implement adequate scientifically sound biomonitoring methods and programme for exposure to NMs.

Biomonitoring of genotoxic effects for human exposure to nanomaterials: The challenge ahead.

Exposures to nanomaterials (NMs), with their specific physico-chemical characteristics, are likely to increase over the next years, as their production for industrial, consumer and medical applications is steadily rising. Therefore, there is an urgent need for the implementation of human biomonitoring studies of genotoxic effects after NM exposures in order to monitor and assure safety for workers and the general population. In this review, most commonly used biomarkers of early genetic effects were analyzed for their adequacy after NM exposures. A more in depth analysis of the ex vivo/in vitro lymphocyte MN assay was performed, although, in literature no studies are available using this assay for NM exposures. Therefore, the known factors determining the NMs tissue/cellular targets and the multiplicity of modes of action of NMs were summarized. The main pending questions are whether (1) lymphocytes are a NM target or an adequate surrogate tissue, (2) whether the buccal MN assay might be more suitable for NM exposures via inhalation or ingestion, as buccal cells might be exposed more directly. While the current state-of-the-art does not allow for drawing firm conclusions, major research gaps are identified and some cautious recommendations can be formulated. Therefore in vitro and in vivo studies should be conducted comparing methodologies side-by-side in the same subjects and for different types of NMs. The ex vivo/in vitro MN assay in its automated version, allowing objective analysis of large cohorts and detection of direct and indirect genotoxic effects, remains a valuable candidate for human biomonitoring to NM exposure. Considering the potential cancer risk from exposure to NMs and previous dramatic experiences with too late surveillance of occupational exposures to similar substances (e.g. to asbestos), there is an urgent need to define and implement adequate scientifically sound biomonitoring methods and programme for exposure to NMs.

Micronucleus frequency in Danish schoolchildren and their mothers from the DEMOCOPHES population.

Micronucleus (MN) frequency is a biomarker for early genetic effects which is often used in human biomonitoring studies. Increased frequency of micronuclei has been associated with high levels of traffic exposure. Further high MN frequency was found predictive for cancer development in several studies of adults. In the present study, the MN frequency in blood samples from the Danish participants of the European pilot project DEMOCOPHES was analysed and related to the area of residence, self-reported and calculated exposure to road traffic as well as to mercury in hair and blood concentrations of persistent organic pollutants and dioxin-like activity measured in the same participants. The MN frequency analysis was performed with the cytokinesis-block micronucleus (CBMN) assay and included 100 children and 119 mothers. We found a significant correlation between mothers and children in the levels of micronuclei in 1000 binucleated T lymphocytes (‰MNBN) and in the proliferation index. Further the levels of ‰MNBN were significantly higher in mothers compared with their children. No significant associations were found for ‰MNBN for traffic related exposure in neither children nor their mothers. In children, a 2.5 times higher micronuclei in mononuclear T lymphocytes were found in children living within 50 m of a busy road, however, this was not found in mothers or in MNBN and the effect of exposure to road traffic on MN frequency needs further investigation. No significant associations were found between MN frequencies and the other biomarkers measured in the same participants.

Outdoor air pollution exposures and micronuclei frequencies in lymphocytes from pregnant women and newborns in Crete, Greece (Rhea cohort).

BACKGROUND: Micronuclei (MN) are biomarkers of early genetic effects that have been used to investigate the association between environmental exposures and cancer. However, few studies have examined the association between environmental exposures during pregnancy and MN in mothers and newborns. OBJECTIVES: We examined MN frequency in maternal blood and in cord blood, in relation to maternal air pollution exposure, and the potential interaction with maternal vitamin C intake and maternal smoking. METHODS: We used the cytokinesis-block micronucleus assay to assess MN frequency per 1000 bi-nucleated T-lymphocytes from 181 mothers and 183 newborns born in 2007-2008 in Heraklion (Crete, Greece). The ESCAPE land-use regression methods were used to estimate annual mean exposure to outdoor air pollution [particulate matter (PM), black carbon, nitrogen dioxide (NO2) and nitrogen oxides (NOx)] at maternal home addresses. Food frequency questionnaires were used to estimate maternal dietary vitamin C intake during pregnancy. Smoking habits were self-reported using questionnaires which were checked by measuring maternal urinary cotinine levels. RESULTS: Exposure to PM2.5 was associated with increased MN frequencies in pregnant women [rate ratio [RR (95%CI)] per 5 µg/m(3)=1.53 (1.02, 2.29)]. This increase was considerably higher among women who did not fulfill the recommended vitamin C dietary allowances [RR=9.35 (2.77, 31.61); n=20]. Exposure to PM2.5-10, PM10, NO2 and NOx were also associated with a higher incidence of MN frequencies in smoker women (n=56). No associations were found for newborns. CONCLUSIONS: We found an association between air pollution, particularly PM2.5, and MN frequency in mothers but not in newborns. This association was more pronounced among women with a lower dietary intake of vitamin C during pregnancy and among women who smoked during pregnancy. While results are clear in mothers, the association between maternal carcinogenic exposures during pregnancy and biomarkers of early biologic effect in the newborn remains poorly understood.

Causes of genome instability: the effect of low dose chemical exposures in modern society.

Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome's integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis.