Sixth International Conference on Environmental Mutagens in Human Populations

For a while, environment could be defined as "everything except genetic". The recent scientific progress show on the contrary that most of the health impacts of environmental pollutions are mediated through genetic and epigenetic mechanisms. It is now impossible to draw an impervious barrier between these two compartments. Environment and genetic, more particularly mutagenesis, are closely linked and this creates a new form of complexity. What is in stake is the definition of a new paradigm to understand the interactions between pollutants exposures and the onset of human diseases. The new paradigm has to encompass the four preceding ones that have guided the development of scientific research in the field of environmental health. The first one was the time of 'poisons' when Pasteur, Koch and the founders of biochemistry and physiology were thinking in terms of "one pollutants for each disease". The second one was of experimental nature with the development of modern toxicology using animal, tissue or cellular models. Then the epidemiology applied to the observational study of chronic diseases in populations emerged in the 50’s. This was the beginning of the understanding of the multifactorial nature of the determinants of many diseases. Thirty years ago, the US National Academy of Sciences promoted the quantitative risk assessment paradigm. Toxicological and epidemiological knowledge could be synthesized through four formal steps: hazard identification, dose-response relationship analysis, human exposure measurement and finally risk characterization. In this presentation, we will show that for each of these steps, recent advances in research challenge the way to assess the health impact of environmental pollutions. Using examples like bisphenol A, chemical-induced obesity, chlordecone and nanoparticles, we will discuss the desirable characteristics of the fifth paradigm that must integrate many disciplines from mathematics to social sciences.

A growing scientific evidence base shows that exposure to the products of poor combustion, particularly small particles, is responsible for as many as 12 million premature deaths in the world annually. Most well known is the impact of people putting burning material into their mouths—incomplete combustion of tobacco—which is responsible for about half this total. Only relatively recently, however, has the full impact of other sources of incomplete combustion become documented. Based on large international reviews of the evidence coming out this year, it is estimated that outdoor air pollution, primarily combustion-related particles, is responsible for nearly 3 million premature deaths around the world. Although people have traditionally thought of outdoor pollution as an urban phenomenon, recent studies using satellites as well as ground-level monitoring show that rural outdoor air pollution is also a serious problem in many poor countries, including much of Asia. It is also now understood that the smoke from biomass and coal use for cooking in poor countries is responsible for an even greater health burden than that from general outdoor pollution. This household air pollution directly affects 40 percent of the world population. Moving to clean combustion or non-combustion energy sources could therefore have immense health benefits globally.

Statistical treatment of data for human biomonitoring has greatly improved within the last decade, and the most advanced techniques have been translated from the analysis of classic epidemiologic studies to molecular epidemiology. The use of more sophisticated techniques has improved the precision of estimates in human population studies, increasing the reliability of study results. In parallel, the increased popularity of pooled analyses, created the opportunity for a deeper insight into the sources of variability. Large collaborative studies published over the last few decades have revealed that the inter-laboratory and especially the inter-scorer variation are the most important source of variability, setting this heterogeneity as a priority field to address. The recent development of automated systems for chromosome damage scoring is going to dramatically change the level of reliability of these biomarkers. Before introducing these methods, robust standardization studies have to be started, aimed at comparing automated systems in different setting and the overlapping between different company systems. During the presentation we will discuss the list of priorities for systems standardization and the most suitable study design and the statistical analyses to be implemented for addressing these priorities.

Analysis of micronuclei (MN) is widely used for human biomonitoring or in vitro/in vivo genotoxicity testing, and provides a sensitive and relatively easy method to assess genetic damage (Kirsch-Volders et al, 2011). The fact that baseline MN frequencies in cytokinesis-blocked (CB) lymphocytes have been shown to be a predictive biomarker for cancer risk strengthens the importance of the CBMN assay as a reliable method for human biomonitoring of early genetic effects. We showed (Kirsch-Volders and Fenech, 2001) that scoring MN frequencies in binucleated and mononucleated cells enhances the predictive capacity of the assay. However, automation of MN analysis is needed for quicker, more reliable detection while minimizing subjective judgment and scoring, and to allow multi-center cohort analysis for biomonitoring studies. Within the framework of NewGeneris, an EU project, we developed an automated image analysis system for scoring MN in human lymphocytes in collaboration with IMSTAR. The IMSTAR system is based on specific algorithms starting from the cell as a detection unit. The whole detection and scoring process are separated into two distinct steps: in the first step, the cells and nuclei are detected; then, in the second step, the MN are searched for in the detected cells. The fact that our designed software protocol started from the cell as a detection unit, and hence the identification of mono-, bi- and polynucleated cells, and MN in these different sub-populations of cells, allows the assessment of cell proliferation through nuclearity index, which is important for an efficient assessment of mitogen response and cytostasis in human biomonitoring as these are indicative of immune function and cytotoxicity (Decordier et al, 2011). Additional requirements that should be fulfilled for development of an automated MN analysis system include: a) The system should be applicable to the CBMN methodology and accurately distinguish mono, bi- and polynucleated cells; b) Well defined scoring criteria for cell type and MN (eg, Fenech et al, 2003); c) Experienced cytologists to score MN according to the HUMN criteria; d) A standardized slide preparation protocol to obtain uniformity in cell size, cell density, and reproducibility (Decordier et al, 2009); e) Validation of the automated versus manual scoring (Decordier et al, 2009, 2011). This methodology was successfully applied to study different mother-child cohorts within the EU-project NewGeneris (Vande Loock et al, 2011). Funded by the EU Integrated Project NewGeneris, (Contract No FOOD-CT-2005-016320). NewGeneris is the acronym of ‘Newborns and Genotoxic Exposure Risks’ and ECNIS stands for ‘Environmental Cancer Risk, Nutrition and Individual Susceptibility’ (FOOD-CT-2005-513943)

Background and Objective: Electronic waste (e-waste) is an emerging environmental health issue because of its fast accumulation as well as inadequate development in recycling technology. Guiyu, a town in south China, is one of the biggest e-waste recycling centers in the world. E-waste is disassembled and recycled by locals with crude and uncontrolled methods that produce extensive environmental pollutants. The objective of this study is to provide evidence for association between risk to human health and exposure to this e-waste recycling. Methods: Heavy metals of blood were determined by graphite furnace atomic absorption spectrometry. Polybrominated diphenyl ethers (PBDEs), polycyclic aromatic hydrocarbons (PAHs) and Polychlorinated biphenyls (PCBs) of blood were determined by gas chromatography/mass spectrometry in the electron capture negative ionization mode. Questionnaires were used and involved examination and experiments were conducted. All data were analyzed statistically. Results: Guiyu children and/or neonates had significantly elevated blood lead (Pb), cadmium (Cd), chromium (Cr), manganese (Mn), nickel (Ni), PBDEs, PAHs and PCB, and with impairment of neurobehavioral development, temperament alterations, lower forced volume vital capacity (FVC), male neonatal AGD (Anogenital Distance) increment, damage of lymphocyte DNA and changes of antioxidant enzymes activities. Guiyu neonates showed much higher rates of adverse birth outcomes such as fetal death, low birth weight and preterm delivery. Conclusion: Our studies suggest that environmental pollution by improper e-waste process has adversely affected local health, and especially affected children and infant health and development. This kind of exposure to e-waste chemicals may cause long-term adverse outcomes for health.

Aryl Hydrocarbon Receptor (AhR) ligands are ubiquitous endocrine disrupting chemicals (EDCs) used in consumer products, diet, air and water. These chemicals are reproductive toxicants which promote tumor formation in some reproductive model systems but human data are limited. The occurrence of reproductive tumors was exponentially grown during the last three decades especially in Tehran but the underlying risk factors remained unclear. A cross-sectional case control study was conducted on the tissue and serological levels of AhR, sex steroid receptors and 120 lifestyle in relation to exposure to EDCs in 500 premenopausal women with history of endometriosis, uterine leiomyoma, breast fibroadenoma and breast cancer from 2007–2011.Differential levels of AhR, ER, PR ,AR, were determined in mentioned female reproductive tumors . Their association with lifestyle factors was also examined in different female tumors. Logistic regression was used to estimate odds ratios (ORs) and 95 percent confidence intervals (CIs), adjusting for potential risk factors. AhR overexpression in epithelial cells of premenopausal patients emphasized the susceptibility of these cells to environmental induced reproductive disorders. Living near PAHs producing factories, consumption of animal fat, abnormal weight gain, long term (>5yrs) OCP consumption, smoking, severe stress, hormonal deregulations and exposure to other sources of xenoestrogens were correlated with an increased risk of reproductive tumors which were correlated with elevated tissue levels of AhR. Adiposity and abnormal weight gain after 18 years were considered as two major background factors, which may contribute to the levels of endogenous estrogens. It seems that AhR overexpression is affected by exposure to xenoestrogens and by adiposity. Long term exposure to EDCs can increase the tissue levels of AhR and deregulate the expression pattern of sex steroid receptors and other genes in target tissues.

Chronic exposure to environmental toxic chemicals can cause DNA damage, disease and strong selection pressure on the gene pool of exposed populations. A mechanism that can link the exposure and biological consequences is DNA repair activity. Therefore, impairment of the DNA repair function can be a critical mechanism for the development of environmental health problems. In my laboratory, we have developed a Challenge assay to detect functional DNA repair deficiencies. In this assay, lymphocytes from exposed and control subjects are irradiated with X-rays or UV-light to induce DNA damage, thus challenging them to repair the damage. Abnormal repair will result in significantly elevated chromosome aberrations or DNA strand breaks in the Comet assay. Studies around the world have used the assay to indicate that excessive exposure to mutagenic and toxic substances can cause abnormal DNA repair responses. Thus, the risk for the development of health problems is increased. In studies in collaboration with Professor Bersimbaev of Kazakhstan, 3 generations of residents who have been exposed to radioactive fallouts from nuclear bomb testings were studied. The population had generation-based increase in mini-satellite instability and preferential retention of the GSTM1 null genotype. It appears that the chronic exposure to ionizing radiation has caused DNA repair defects and the alteration of the gene pool. The latter suggests that selection pressure can potentially alter the genetic makeup of the future generations. However, no clear cut evidence of such exists among exposed human populations. In summary, these studies indicate that excessive exposure to hazardous substances can cause functional alterations in the DNA repair machinery leading to increase health risk, including the possibility of alteration of gene pool in exposed population

Human risk assessments at low doses, low dose rates and high doses following acute exposure to ionizing radiation are of prime importance in radiation protection. These issues are of continuing importance in respect of social/economic policy relating to the industrial and medical uses of ionizing radiation, and for risk assessment among people occupationally being exposed to low and/or high LET radiation, such as astronauts, pilots, stewardess and nuclear power plant workers, as well as victims of radiation accidents. Consequently, several biological assays were developed and attempts were made to investigate formation of radiation-induced chromosome aberrations and induction of genomic instability in human lymphocytes and fibroblasts. The fluorescence in situ hybridization (FISH) technique using chromosome, chromosome-arm, chromosome region, centromere and telomere-specific DNA libraries has improved the resolution of detecting all classes of radiation-induced chromosomal inter- and intra-changes. Consequently, this has increased significantly the accuracy and detection limit of biological dosimetry. Newly obtained data indicate that (a) Premature chromosome condensation assay (cell fusion assay) is a unique method to be used for immediate dose assessment at low (5cGy) as well as high doses (≥3 Gy) and can accurately discriminate between whole- and partial-body exposure in case of mass casualties and accidental over-exposure to high doses of ionizing radiation, (b) Chemically induced PCC assay has the potential to be applied for biological dosimetry (by analyzing ring-chromosomes) in cases of high doses (> 4 Gy). This assay has been further validated in combination with M-FISH to assess genomic instability of primary tumors, (c) FISH-based translocation assay has the potential to assess acute as well as chronic exposure in cases of accidental as well as occupational exposure to ionizing radiation, either immediately following exposure or retrospectively by defining accumulative effects to red bone marrows. (d) There are distinct fingerprints (such as insertions and complex translocations) for high LET radiation in comparison to low LET radiation. The importance of these findings, their applications in different scenarios of accidental and occupational over-exposure to ionizing radiations, such as Japan atomic bomb survivors, Chernobyl, Istanbul, Mayak and Techa River cohorts, and future perspectives for biological dosimetry will be discussed.

A global concern on the adverse effects of chemicals to human health has led toxicologists, and in particular genetic toxicologists, to identify environmental genotoxins. The conventional method of identification involves techniques such as Ames test, chromosomal aberrations, micronucleus assay, alkaline elution assay, etc. However, these methods are cumbersome, time consuming as well as resource-intensive. During the last two decades, a state-of-the-art technique, the single cell gel electrophoresis (SCGE) /Comet assay, has gained importance as a rapid, visual and sensitive technique for qualitative and quantitative assessment of DNA damage and repair. It is a valuable technique for detection of a variety of DNA damages, which include single and double strand breaks, alkali labile sites and oxidative base damages. Comet assay can be performed on any eukaryotic and some prokaryotic cells and may be used for both in vitro and in vivo screening of geno- and anti-genotoxic potential of chemicals. Its non-invasive nature and requirement of few cells for processing has made this assay widely accepted for monitoring human genotoxicity. Its sensitivity has enabled genetic toxicologists to monitor low-level, long-term exposure to chemicals, thus predicting genetic damage at an early stage. Our studies, in the Indian population, using the alkaline Comet assay have revealed significant gender-related differences in the extent of DNA damage in lymphocytes. DNA damage was also found to vary with eating and smoking habits, age, exercise as well as occupational exposure. In vitro Comet assay studies have also been performed using human peripheral blood lymphocytes and cell lines such as MCF-7, CHO, JM-1 to assess the genotoxic potential of environmental chemicals. The versatility of the Comet assay has indeed proven it to be a Rosetta Stone in the garden of Genetic Toxicology.

National and international efforts to promote research and communicate knowledge into the causes and consequences of damage to the human genome aimed to inform and support measures to ensure a healthy and sustainable environment for future generations. Developed countries have realized the need to regulatory bodies (e.g. FDA, EPA, EMEA, ICH and WHO) to be responsible for approval and administration of drugs, medical devices, cosmetics , food additives and chemicals before marketing their products. Developing countries are building their economy with a focus on industrialization programs at a pace exceeding measures which counteract hazards to both human and environment. Growing na tional industries in the fields of pharmaceuticals, food and petrochemicals will definitely impose a range of hazards to the population and the environment. The challenge facing countries of the gulf region and its neighbours is the lack of a local or regional infrastructure body which focuses on promotion of research and applications of knowledge into genetic toxicology testing, risk assessment, and regulatory policy-making to protect human health and environment. For the last few years attempts by concerned scientists in the region were practiced to fill in gaps and come up with practical solutions to overcome the need for an organization with academic and legislative power to set up regulations and guidelines for the safe marketing, labelling of pharmaceuticals, cosmetics, consumer products, pesticides and chemicals. A proposal for the establishment of a Regional Center of Excellence for Environmental Mutagenesis and Carcinogenesis Research in one of the rich Gulf countries is ready for disclosure. The proposal is based on initiatives of the European safety program REACH (Registration, Evaluation, and Authorization of Chemicals), and the well established regulatory requirements of the Food and Drug Administration, European and Japanese regulatory agencies, which requires manufacturers to conduct testing to identify potential hazards to human health and to the environment, and to submit the test data to regulatory authorities.