Cytogenetic studies of PCB77 on brown trout (<i>Salmo trutta fario</i>) using the micronucleus test and the alkaline comet assay

Polychlorinated biphenyls (PCBs) are stable pollutants, which can be found in almost every compartment of terrestrial and aquatic ecosystems. They are very lipophilic and therefore have the potency of accumulating in the fat stores of animals. The mechanisms by which PCBs exert their adverse effects are still unclear. It is known that PCBs induce some important biotransformation enzymes, but their mutagenic properties are still controversial. The DNA breakage and clastogenic potency of a planar PCB77 (3,3',4,4'-tetrachlorobiphenyl) was determined in vivo in fish, using the single cell gel electrophoresis or comet assay and the micronucleus test, on erythrocytes of the brown trout exposed for 3, 9 and 14 days to initial PCB concentrations of 780 and 918 pg/ml, dissolved in the water, Blood was taken by a caudal puncture and the erythrocytes were either deposited in an agarose gel (0.6%) for the comet assay or smeared directly on slides for the micronucleus test. Five fish were studied per treatment and 50 and 2000 erythrocytes per concentration and per animal were analysed for the comet assay and the micronucleus test respectively. ethyl methanesulphonate (EMS) at a concentration of 25 mg/l water was used as a positive control. Although EMS induced a statistically significant increase of single strand breaks in the comet assay, in neither of the two tests used, were mutagenic effects due to PCB exposure observed

Rabl's model of the interphase chromosome arrangement tested in Chinise hamster cells by premature chromosome condensation and laser-UV-microbeam experiments

In 1885 Carl Rabl published his theory on the internal structure of the interphase nucleus. We have tested two predictions of this theory in fibroblasts grown in vitro from a female Chinese hamster, namely (1) the Rabl-orientation of interphase chromosomes and (2) the stability of the chromosome arrangement established in telophase throughout the subsequent interphase. Tests were carried out by premature chromosome condensation (PCC) and laser-UV-microirradiation of the interphase nucleus. Rabl-orientation of chromosomes was observed in G1 PCCs and G2 PCCs. The cell nucleus was microirradiated in G1 at one or two sites and pulse-labelled with 3H-thymidine for 2h. Cells were processed for autoradiography either immediately thereafter or after an additional growth period of 10 to 60h. Autoradiographs show unscheduled DNA synthesis (UDS) in the microirradiated nuclear part(s). The distribution of labelled chromatin was evaluated in autoradiographs from 1035 cells after microirradiation of a single nuclear site and from 253 cells after microirradiation of two sites. After 30 to 60h postincubation the labelled regions still appeared coherent although the average size of the labelled nuclear area fr increased from 14.2% (0h) to 26.5% (60h). The relative distance dr, i.e. the distance between two microirradiated sites divided by the diameter of the whole nucleus, showed a slight decrease with increasing incubation time. Nine metaphase figures were evaluated for UDS-label after microirradiation of the nuclear edge in G1. An average of 4.3 chromosomes per cell were labelled. Several chromosomes showed joint labelling of both distal chromosome arms including the telomeres, while the centromeric region was free from label. This label pattern is interpreted as the result of a V-shaped orientation of these particular chromosomes in the interphase nucleus with their telomeric regions close to each other at the nuclear edge. Our data support the tested predictions of the Rabl-model. Small time-dependent changes of the nuclear space occupied by single chromosomes and of their relative positions in the interphase nucleus seem possible, while the territorial organization of interphase chromosomes and their arrangement in general is maintained during interphase. The present limitations of the methods used for this study are discussed

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

Development and validation of the <i>in vivo</i> alkaline comet assay for detecting genomic damage in marine flatfish

Biomonitoring is an important subject within environmental sciences. Biomonitoring tests are required to be quick, relatively inexpensive, accurate, and reproducible. No genetic test currently fulfils all of these requirements. The chromosome aberration and sister chromatid exchange tests are very time consuming, the DNA adduct technique is rather expensive, and the micronucleus test has not inconclusively proven its use as a reliable monitoring tool. This work is focused on the validation of the comet assay as a candidate for monitoring marine ecosystems. For the comet assay, this work deals with the effectiveness of tissue dissociation, storage of cells in lysing buffer and in liquid nitrogen, different electrophoretic conditions, neutralisation and fixation of slides, interindividual variation between samples, and responsiveness of four tissue types to ethyl methanesulphonate (EMS). The main conclusions are: (i) dissociation of solid tissues in a phosphate buffer supplemented with 200 mM N-t-butyl-alpha-phenylnitrone provides cells with an acceptable background DNA damage; (ii) freezing of cells or tissues in liquid nitrogen generally leads to an increase in DNA breakage, especially for liver, gill and kidney tissue; (iii) storage of slides in the lysing solution for up to one week gives minor changes in comet tails; (iv) differences in protocols for neutralisation and fixation may influence the results; (v) high intra- and interindividual variations in comets (length and DNA content) may obscure the interpretation of comet results; (vi) blood, gill, liver and kidney all showed a statistically significant increase of DNA damage after exposure to 50 mg EMS/l; (vii) electrophoresis at low voltage for longer periods is to be preferred to high voltage and short electrophoresis times. The simplicity and sensitivity of the comet assay make it an adequate test system for biomonitoring of chronic low level exposure. However, protocols and experimental conditions have to be chosen carefully

Analysis of chromosome positions in the interphase nucleus of Chinese hamster cells by laser-UV-microirradiation experiments

Unsynchronized cells of an essentially diploid strain of female Chinese hamster cells derived from lung tissue (CHL) were laser-UV-microirradiated (=257 nm) in the nucleus either at its central part or at its periphery. After 7–9 h postincubation with 0.5 mM caffeine, chromosome preparations were made in situ. Twenty-one and 29 metaphase spreads, respectively, with partial chromosome shattering (PCS) obtained after micro-irradiation at these two nuclear sites, were Q-banded and analyzed in detail. A positive correlation was observed between the frequency of damage of chromosomes and both their DNA content and length at metaphase. No significant difference was observed between the frequencies of damage obtained for individual chromosomes at either site of microirradiation. The frequency of joint damage of homologous chromosomes was low as compared to nonhomologous ones. Considerable variation was noted in different cells in the combinations of jointly shattered chromosomes. Evidence which justifies an interpretation of these data in terms of an interphase arrangement of chromosome territories is discussed. Our data strongly argue against somatic pairing as a regular event, and suggest a considerable variability of chromosome positions in different nuclei. However, present data do not exclude the possibility of certain non-random chromosomal arrangements in CHL-nuclei. The interphase chromosome distribution revealed by these experiments is compared with centromere-centromere, centromere-center and angle analyses of metaphase spreads and the relationship between interphase and metaphase arrangements of chromosomes is discussed

Commentary: Critical questions, misconceptions and a road map for improving the use of the lymphocyte cytokinesis-block micronucleus assay for in vivo biomonitoring of human exposure to genotoxic chemicals - a HUMN project perspective

Abstract not availableMicheline Kirsch-Volders, Stefano Bonassi, Siegfried Knasmueller, Nina Holland, Claudia Bolognesi, Michael F. Fenec

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology

A novel, integrated in vitro carcinogenicity test to identify genotoxic and non-genotoxic carcinogens using human lymphoblastoid cells

Human exposure to carcinogens occurs via a plethora of environmental sources, with 70–90% of cancers caused by extrinsic factors. Aberrant phenotypes induced by such carcinogenic agents may provide universal biomarkers for cancer causation. Both current in vitro genotoxicity tests and the animal-testing paradigm in human cancer risk assessment fail to accurately represent and predict whether a chemical causes human carcinogenesis. The study aimed to establish whether the integrated analysis of multiple cellular endpoints related to the Hallmarks of Cancer could advance in vitro carcinogenicity assessment. Human lymphoblastoid cells (TK6, MCL-5) were treated for either 4 or 23 h with 8 known in vivo carcinogens, with doses up to 50% Relative Population Doubling (maximum 66.6 mM). The adverse effects of carcinogens on wide-ranging aspects of cellular health were quantified using several approaches; these included chromosome damage, cell signalling, cell morphology, cell-cycle dynamics and bioenergetic perturbations. Cell morphology and gene expression alterations proved particularly sensitive for environmental carcinogen identification. Composite scores for the carcinogens’ adverse effects revealed that this approach could identify both DNA-reactive and non-DNA reactive carcinogens in vitro. The richer datasets generated proved that the holistic evaluation of integrated phenotypic alterations is valuable for effective in vitro risk assessment, while also supporting animal test replacement. Crucially, the study offers valuable insights into the mechanisms of human carcinogenesis resulting from exposure to chemicals that humans are likely to encounter in their environment. Such an understanding of cancer induction via environmental agents is essential for cancer prevention