Towards prevention of aneuploidy-associated cellular senescence and aging: more questions than answers?

The aim of this review is to discuss how aneuploidy contributes to the aging process, and to identify plausible strategies for its prevention. After an overview of mechanisms leading to aneuploidy and the major features of cellular senescence, we discuss the link between (i) aneuploidy and cellular senescence; (ii) aneuploidy and aging; and (iii) cellular senescence and aging. We also consider (i) interactions between aneuploidy, micronuclei, cellular senescence and aging, (ii) the potential of nutritional treatments to prevent aneuploidy-associated senescence and aging, and (iii) knowledge and technological gaps. Evidence for a causal link between aneuploidy, senescence and aging is emerging. In vitro, aneuploidy accompanies the entry into cellular senescence and can itself induce senescence. How aneuploidy contributes in vivo to cellular senescence is less clear. Several routes depending on aneuploidy and/or senescence converge towards chronic inflammation, the major driver of unhealthy aging. Aneuploidy can induce the pro-inflammatory Senescence Associated Secretory Phenotype (SASP), either directly or as a result of micronucleus (MN) induction leading to leakage of DNA into the cytoplasm and triggering of the cGAS-STING pathway of innate immune response. A major difficulty in understanding the impact of aneuploidy on senescence and aging in vivo, results from the heterogeneity of cellular senescence in different tissues at the cytological and molecular level. Due to this complexity, there is at the present time no biomarker or biomarker combination characteristic for all types of senescent cells. In conclusion, a deeper understanding of the critical role aneuploidy plays in cellular senescence and aging is essential to devise practical strategies to protect human populations from aneuploidy-associated pathologies. We discuss emerging evidence, based on in vitro and in vivo studies, that adequate amounts of specific micronutrients are essential for prevention of aneuploidy in humans and that precise nutritional intervention may be essential to help avoid the scourge of aneuploidy-driven diseases.

Aneuploidy, inflammation and diseases.

This review discusses how numerical aneuploidy may trigger inflammation in somatic cells and its consequences. Therefore we: i) summarized current knowledge on the cellular and molecular pathological effects of aneuploidy; ii) considered which of these aspects are able to trigger inflammation; iii) determined the genetic and environmental factors which may modulate the link between aneuploidy and inflammation; iv) explored the rôle of diet in prevention of aneuploidy and inflammation; v) examined whether aneuploidy and inflammation are causes and/or consequences of diseases; vi) identified the knowledge gaps and research needed to translate these observations into improved health care and disease prevention. The relationships between aneuploidy, inflammation and diseases are complex, because they depend on which chromosomes are involved, the proportion of cells affected and which organs are aneuploid in the case of mosaic aneuploidy. Therefore, a systemic approach is recommended to understand the emergence of aneuploidy-driven diseases and to take preventive measures to protect individuals from exposure to aneugenic conditions.

"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.

Inflammatory cytokine storms severity may be fueled by interactions of micronuclei and RNA viruses such as COVID-19 virus SARS-CoV-2. A hypothesis.

In this review we bring together evidence that (i) RNA viruses are a cause of chromosomal instability and micronuclei (MN), (ii) those individuals with high levels of lymphocyte MN have a weakened immune response and are more susceptible to RNA virus infection and (iii) both RNA virus infection and MN formation can induce inflammatory cytokine production. Based on these observations we propose a hypothesis that those who harbor elevated frequencies of MN within their cells are more prone to RNA virus infection and are more likely, through combined effects of leakage of self-DNA from MN and RNA from viruses, to escalate pro-inflammatory cytokine production via the cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING) and the Senescence Associated Secretory Phenotype (SASP) mechanisms to an extent that is unresolvable and therefore confers high risk of causing tissue damage by an excessive and overtly toxic immune response. The corollaries from this hypothesis are (i) those with abnormally high MN frequency are more prone to infection by RNA viruses; (ii) the extent of cytokine production and pro-inflammatory response to infection by RNA viruses is enhanced and possibly exceeds threshold levels that may be unresolvable in those with elevated MN levels in affected organs; (iii) reduction of MN frequency by improving nutrition and life-style factors increases resistance to RNA virus infection and moderates inflammatory cytokine production to a level that is immunologically efficacious and survivable.

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, inflammation and auto-immune disease.

Auto-immune diseases (AUD) are characterized by an immune response to antigenic components of the host itself. The etiology of AUD is not well understood. The available evidence points to an interaction between genetic, epigenetic, environmental, infectious and life-style factors. AUD are more prevalent in women than in men; sex hormones play a crucial role in this sex bias. Micronuclei (MN) emerged as a new player in the induction of AUD, based on the capacity of DNA-sensors to detect self-DNA that leaks into the cytoplasm from disrupted MN and induce the cGAS-STING pathway triggering an innate auto-immune response and chronic inflammation. It was found that inflammation can induce MN and MN can induce inflammation, leading to a vicious inflammation-oxidative-DNA damage-MN-formation-chromothripsis cycle. MN originating from sex chromosome-loss may induce inflammation and AUD. We performed a systematic review of studies reporting MN in patients with systemic or organ-specific AUD. A meta-analysis was performed on lymphocyte MN in diabetes mellitus (10 studies, 457 patients/290 controls) and Behcet's disease (3 studies, 100 patients/70 controls) and for buccal MN in diabetes mellitus (11 studies, 507 patients/427 controls). A statistically significant increase in patients compared to controls was found in the meta-analyses providing an indication of an association between MN and AUD. A 36%-higher mean-MRi in buccal cells (3.8+/-0.7) was found compared to lymphocytes (2.8+/-0.7)(P = 0.01). The meta-MRi in lymphocytes and buccal cells (1.7 and 3.0 respectively) suggest that buccal cells may be more sensitive. To assess their relative sensitivity, studies with measurements from the same subjects would be desirable. It is important that future studies (i) investigate, in well-designed powered studies, the prospective association of MN-formation with AUD and (ii) explore the molecular mechanisms by which chromosome shattering in MN and the release of chromatin fragments from MN lead to the formation of auto-antibodies.

50 years existence and active participation of EEMS (now EEMGS) in the scientific community: A driver of European and international scientific collaborations for the protection of the environment and human health from genome stressors.

EEMS and its successor Society EEMGS have provided a dynamic and successful platform to stimulate research and exchanges among the different actors involved in the protection of the environment and of human health from exposure to genome stressors. It includes basic, translational and applied research projects. This was possible due to the enthusiasm, creativity and support of scientists convinced of the importance of these issues. In the future young scientists will take over with new questions, new challenges, new technologies, new discoveries and new applications. A major challenge is the ethical questions emerging from the impressive potential of present genetic technologies capable of impacting the evolution of nature and humankind. The EEMGS, where academics, regulators and industries meet, should play a central role in these aspects, in particular in support of primary prevention and the establishment of internationally recognized guidelines. Collaboration with colleagues and other teams are of great importance to establish a stimulating open dialogue on scientific questions. However the key issues remain to do careful and rigorous research; to use logic and background knowledge; to define adequate experimental designs; to provide transparency in the protocols; to check repeatability of the results and to combine several statistical approaches in the quest to get to the truth. Among the many challenges ahead, re-evaluation of some key fundamental questions is necessary, such as the interplay between genetics and epigenetics, the existence of specific germ cell mutagens or the identification of the mechanisms leading to mutagen induced diseases. Translational and applied research will further include the development of systemic biomonitoring protocols, if possible in a single biological sample, the redaction of internationally harmonized guidelines but also the organization of platforms between geneticists and physicians open to all actors in the field. The creation of an independent European center to assess risk from exposure to mutagens, in particular in the light of the problematic of global warming might be very helpful.

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.

Epigenetic memory in response to environmental stressors.

Exposure to environmental stressors, toxicants, and nutrient deficiencies can affect DNA in several ways. Some exposures cause damage and alter the structure of DNA, but there is increasing evidence that the same or other environmental exposures, including those that occur during fetal development in utero, can cause epigenetic effects that modulate DNA function and gene expression. Some epigenetic changes to DNA that affect gene transcription are at least partially reversible (i.e., they can be enzymatically reversed after cessation of exposure to environmental agents), but some epigenetic modifications seem to persist, even for decades. To explain the effects of early life experiences (such as famine and exposures to other stressors) on the long-term persistence of specific patterns of epigenetic modifications, such as DNA methylation, we propose an analogy with immune memory. We propose that an epigenetic memory can be established and maintained in self-renewing stem cell compartments. We suggest that the observations on early life effects on adult diseases and the persistence of methylation changes in smokers support our hypothesis, for which a mechanistic basis, however, needs to be further clarified. We outline a new model based on methylation changes. Although these changes seem to be mainly adaptive, they are also implicated in the pathogenesis and onset of diseases, depending on individual genotypic background and types of subsequent exposures. Elucidating the relationships between the adaptive and maladaptive consequences of the epigenetic modifications that result from complex environmental exposures is a major challenge for current and future research in epigenetics.-Vineis, P., Chatziioannou, A., Cunliffe, V. T., Flanagan, J. M., Hanson, M., Kirsch-Volders, M., Kyrtopoulos, S. Epigenetic memory in response to environmental stressors.

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.

Molecular mechanisms by which in vivo exposure to exogenous chemical genotoxic agents can lead to micronucleus formation in lymphocytes in vivo and ex vivo in humans.

The purpose of this review is to summarise current knowledge on the molecular mechanisms by which in vivo exposure to exogenous chemical genotoxins in humans induces micronuclei (MNi) and other nuclear anomalies in lymphocytes in vivo and ex vivo after nuclear division in vitro. MNi originate from acentric chromosome fragments and/or whole chromosomes that are unable to engage with the mitotic spindle and/or fail to segregate properly to the daughter nuclei during anaphase. The lagging fragments or whole chromosomes are surrounded by membrane and become MNi. Acentric fragments are caused by failure of repair or mis-repair of DNA strand breaks which may be induced by chemicals that (i) damage the phosphodiester backbone of DNA, and/or (ii) inhibit the DNA damage response mechanisms or repair of DNA strand breaks and/or (iii) cause DNA replication stress due to DNA adduct or cross-link formation. MNi originating from lagging whole chromosomes may be induced by chemicals that cause defects in centromeres or the mitotic machinery. Mis-repair of chemically-induced DNA breaks may also cause formation of dicentric chromosomes and nucleoplasmic bridges (NPBs) between daughter nuclei in mitosis. NPBs may break and initiate recurring breakage-fusion-bridge cycles and chromosomal instability. The review also explores knowledge on (i) the routes by which lymphocytes in the human body may be exposed to genotoxic chemicals, (ii) kinetics of MNi expression in lymphocytes in vivo and ex vivo in the lymphocyte cytokinesis-block micronucleus (L-CBMN) assay and (iii) current evidence on the efficiency of the L-CBMN assay in detecting in vivo exposure to chemical genotoxins and its concordance with MNi expression in epithelial tissues. The review also identifies important knowledge gaps (e.g. effect of nanomaterials; interactions with nutritional deficiencies etc.) regarding mechanisms by which in vivo chemical genotoxin exposure may cause MNi formation in lymphocytes in vivo and ex vivo in lymphocytes.