Lucia Migliore

HUman MicroNucleus project: international database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei

Micronucleus (MN) expression in peripheral blood lymphocytes is well established as a standard method for monitoring chromosome damage in human populations. The first results of an analysis of pooled data from laboratories using the cytokinesis‐block micronucleus (CBMN) assay and participating in the HUMN (HUman MicroNucleus project) international collaborative study are presented. The effects of laboratory protocol, scoring criteria, and host factors on baseline micronucleated binucleate cell (MNC) frequency are evaluated, and a reference range of “normal” values against which future studies may be compared is provided. Primary data from historical records were submitted by 25 laboratories distributed in 16 countries. This resulted in a database of nearly 7000 subjects. Potentially significant differences were present in the methods used by participating laboratories, such as in the type of culture medium, the concentration of cytochalasin‐B, the percentage of fetal calf serum, and in the culture method. Differences in criteria for scoring micronuclei were also evident. The overall median MNC frequency in nonexposed (i.e., normal) subjects was 6.5‰ and the interquartile range was between 3 and 12‰. An increase in MNC frequency with age was evident in all but two laboratories. The effect of gender, although not so evident in all databases, was also present, with females having a 19% higher level of MNC frequency (95% confidence interval: 14–24%). Statistical analyses were performed using random‐effects models for correlated data. Our best model, which included exposure to genotoxic factors, host factors, methods, and scoring criteria, explained 75% of the total variance, with the largest contribution attributable to laboratory methods. Environ. Mol. Mutagen. 37:31–45, 2001

Environmental-induced oxidative stress in neurodegenerative disorders and aging

The aetiology of most neurodegenerative disorders is multifactorial and consists of an interaction between environmental factors and genetic predisposition. Free radicals derived primarily from molecular oxygen have been implicated and considered as associated risk factors for a variety of human disorders including neurodegenerative diseases and aging. Damage to tissue biomolecules, including lipids, proteins and DNA, by free radicals is postulated to contribute importantly to the pathophysiology of oxidative stress. The potential of environmental exposure to metals, air pollution and pesticides as well as diet as risk factors via the induction of oxidative stress for neurodegenerative diseases and aging is discussed. The role of genetic background is discussed on the light of the oxidative stress implication, focusing on both complex neurodegenerative diseases (Alzheimers disease, Parkinsons disease, amyotrophic lateral sclerosis) and monogenic neurological disorders (Huntingtons disease, Ataxia telangiectasia, Friedreich Ataxia and others). Emphasis is given to role of the repair mechanisms of oxidative DNA damage in delaying aging and protecting against neurodegeneration. The emerging interplay between environmental-induced oxidative stress and epigenetic modifications of critical genes for neurodegeneration is also discussed.

Oxidative DNA damage in peripheral leukocytes of mild cognitive impairment and AD patients.

It is well established that oxidative stress plays a key role in the degenerative neuronal death and progression of Alzheimers disease (AD), although it is not clear if it is the primary triggering event in the pathogenesis of this disorder. Mild cognitive impairment (MCI) is a clinical condition between normal aging and AD, characterized by a memory deficit without loss of general cognitive and functional abilities. We performed this study by a comet assay analysis to evaluate the level of primary and oxidative DNA damage in two groups of MCI and AD patients, compared to healthy controls. Data showed a significantly higher level of primary DNA damage in leukocytes of AD and also of MCI patients compared to control individuals (average: 2.09+/-0.79 and 2.47+/-1.01, respectively for AD and MCI, versus 1.04+/-0.31 in controls). Moreover, the amount of oxidised DNA bases (both purines and pyrimidines) was significatively higher in the two groups of patients (AD and MCI) compared to controls. Our results give a further indication that oxidative stress, at least at the DNA level, is an earlier event in the pathogenesis of AD.

Genetics, environmental factors and the emerging role of epigenetics in neurodegenerative diseases

In the present review we summarize recent advances in the understanding of the interaction between genetics and environmental factors involved in complex multi-factorial neurodegenerative disorders such as Alzheimers disease (AD), Parkinsons disease (PD) and Amyotrophic Lateral Sclerosis (ALS). The discovery of several genes responsible for the familial forms has led to a better comprehension of the molecular pathways involved in the selective neuronal degeneration which is specific for each of these disorders. However, the vast majority of the cases occurs as sporadic forms, likely resulting from complex gene-gene and gene-environment interplay. Several environmental factors, including, pesticides, metals, head injuries, lifestyles and dietary habits have been associated with increased disease risk or even with protection. Hundreds of genetic variants have been investigated as possible risk factors for the sporadic forms, but results are often conflicting, not repeated or inconclusive. New approaches to environmental health research are revealing us that at the basis there could be chemically induced changes in gene regulation and emphasise the importance of understanding the susceptibility of the human epigenome to dietary and other environmental effects.

Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project.

One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.

Genetics, Cytogenetics, and Epigenetics of Colorectal Cancer

Most of the colorectal cancer (CRC) cases are sporadic, only 25% of the patients have a family history of the disease, and major genes causing syndromes predisposing to CRC only account for 5-6% of the total cases. The following subtypes can be recognized: MIN (microsatellite instability), CIN (chromosomal instability), and CIMP (CpG island methylator phenotype). CIN occurs in 80–85% of CRC. Chromosomal instability proceeds through two major mechanisms, missegregation that results in aneuploidy through the gain or loss of whole chromosomes, and unbalanced structural rearrangements that lead to the loss and/or gain of chromosomal regions. The loss of heterozygosity that occur in the first phases of the CRC cancerogenesis (in particular for the genes on 18q) as well as the alteration of methylation pattern of multiple key genes can drive the development of colorectal cancer by facilitating the acquisition of multiple tumor-associated mutations and the instability phenotype.

Genetic and environmental factors in cancer and neurodegenerative diseases

The aim of this review is to summarise the recent findings in the fields of carcinogenesis and neurodegenerative diseases, the both disorders are characterised by the contribution of different factors including the inheritance of mutated genes, and the exposure to endogenous or exogenous agents during the life. We first analysed the causative genes until now discovered in both processes, then we focused our attention on the role of environmental exposure, susceptibility factors, oxidative stress, apoptosis and aging to the development of such disorders. The genotype at a particular locus may account for an inter-individual susceptibility that can both increase or decrease the risk to develop the pathology especially after the exposure to environmental agents. The mechanism of apoptosis, that is an excellent strategy in order to eliminate damaged cells, seems to be lost during carcinogenesis, while it seems to be involved in the neuronal death in a lot of neurodegenerative disorders. Oxidative stress can both lead to DNA mutations or to the formation of damaged proteins, so being an important risk factor for the initiation and the progression of a disease: in fact it may be one of the causes or can arise as a consequence of a damage caused by other factors increasing then the first damage. It is well established that carcinogenesis is a multi-step process caused by series of successive mutations occurring into a cell and conferring to this cell a growth advantage, so that age is the largest risk factor for cancer in humans. Pathophysiology of neurodegenerative diseases is complex and likely involves multiple overlapping and perhaps redundant pathways of neuronal damage, characterised by the generation of anomalous proteins, often due to mutations in the corresponding gene, and by their subsequent accumulation into or outside specific areas of the brain.

Micronuclei in blood lymphocytes and genetic polymorphism for GSTM1, GSTT1 and NAT2 in pesticide-exposed greenhouse workers.

The frequency of micronuclei (MN) in cultured peripheral lymphocytes was used as a biomarker of genotoxic effects in 34 Italian pesticide-exposed greenhouse workers and 33 unexposed referents matched with the exposed workers for age and smoking habits. The possible influence of the genetic polymorphisms of xenobiotic metabolizing enzymes glutathione S-transferase M1 (GSTM1), T1 (GSTT1), and N-acetyltransferase 2 (NAT2) was also evaluated. To restrict the analysis primarily to cells that have divided once in vitro, MN were scored only in cells showing label after a 42-h incubation with bromodeoxyuridine (BrdU), as detected by immunofluorescence (anti-BrdU technique). Two different concentrations of BrdU (0.5 and 1 microg/ml) were compared. Individual frequencies of micronucleated cells (MNCs) obtained with the two concentrations of BrdU significantly correlated with each other (r=0.55, P<0.001). Higher mean MNCs frequencies (per 1000 cells) were detected among exposed smokers (9.0 at 0.5 microg/ml BrdU and 7.8 at 1 microg/ml BrdU) than in smoking referents (6.3 and 5.9, respectively). In multiple regression analysis controlling for age, sex, smoking and genotypes, a significant elevation of MNC frequency (P=0.004 at 1 microg/ml BrdU; P=0.052 at 0.5 microg/ml BrdU) was observed in greenhouse workers with a work history of extensive pesticide spraying (n=17). Increased MNC frequencies were also associated with ageing at 0.5 microg/ml BrdU, with the GSTM1-positive genotype at both 1 (P=0.028) and 0.5 (P=0.056) microg/ml BrdU in all subjects, and with the NAT2 fast acetylator genotype in smokers at 0.5 microg/ml BrdU (P=0.043). The results indicate that MN rates are increased in greenhouse workers, especially in those involved in pesticide spraying. The GSTM1 positive and NAT2 fast genotypes appear to be associated with elevated MNC frequencies, which contradicts with earlier results on elevated chromosomal aberration rates in GSTM1 null smokers and NAT2 slow subjects.

Micronucleated lymphocytes in people occupationally exposed to potential environmental contaminants: the age effect

This work is part of a research project on 2 groups of tannery workers (i.e., workers employed in the tanning process and those employed in the finishing department), and 2 control groups consisting of individuals paired with each exposed person according to sex, age and smoking habit. The whole study included the evaluation of micronuclei as well as of chromosomal aberrations and sister-chromatid exchanges in peripheral blood lymphocytes. Data on micronucleus analysis in both controls and exposed persons are shown in this paper. There was no statistically significant difference between MN frequencies in the 2 groups of exposed and controls, nor any positive correlation with smoking habit. The effect of age on basal frequency of micronucleated cells clearly emerges in the present study: both controls and exposed show an increase in MN frequency due to age. This could be correlated with a higher sensitivity to breaks, rearrangements or aneuploidogenic events of circulating lymphocytes in aged people.

Concern-driven integrated approaches to nanomaterial testing and assessment - report of the NanoSafety Cluster Working Group 10

Abstract Bringing together topic-related European Union (EU)-funded projects, the so-called “NanoSafety Cluster” aims at identifying key areas for further research on risk assessment procedures for nanomaterials (NM). The outcome of NanoSafety Cluster Working Group 10, this commentary presents a vision for concern-driven integrated approaches for the (eco-)toxicological testing and assessment (IATA) of NM. Such approaches should start out by determining concerns, i.e., specific information needs for a given NM based on realistic exposure scenarios. Recognised concerns can be addressed in a set of tiers using standardised protocols for NM preparation and testing. Tier 1 includes determining physico-chemical properties, non-testing (e.g., structure–activity relationships) and evaluating existing data. In tier 2, a limited set of in vitro and in vivo tests are performed that can either indicate that the risk of the specific concern is sufficiently known or indicate the need for further testing, including details for such testing. Ecotoxicological testing begins with representative test organisms followed by complex test systems. After each tier, it is evaluated whether the information gained permits assessing the safety of the NM so that further testing can be waived. By effectively exploiting all available information, IATA allow accelerating the risk assessment process and reducing testing costs and animal use (in line with the 3Rs principle implemented in EU Directive 2010/63/EU). Combining material properties, exposure, biokinetics and hazard data, information gained with IATA can be used to recognise groups of NM based upon similar modes of action. Grouping of substances in return should form integral part of the IATA themselves.