In vitro genotoxicity testing using the micronucleus assay in cell lines, human lymphocytes and 3D human skin models

The toxicological relevance of the micronucleus (MN) test is well defined: it is a multi-target genotoxic endpoint, assessing not only clastogenic and aneugenic events but also some epigenetic effects, which is simple to score, accurate, applicable in different cell types. In addition, it is predictive for cancer, amenable for automation and allows good extrapolation for potential limits of exposure or thresholds and it is easily measured in experimental both in vitro and in vivo systems. Implementation of in vitro micronucleus (IVMN) assays in the battery of tests for hazard and risk assessment of potential mutagens/carcinogens is therefore fully justified. Moreover, the final draft of an OECD guideline became recently available for this test. In this review, we discuss the prerequisites for an acceptable MN assay, including the cell as unit of observation, importance of cell membranes, the requirement of a mitotic or meiotic division and the assessment of cell division in the presence of the test substance. Furthermore, the importance of adequate design of protocols is highlighted and new developments, in particular the in vitro 3D human skin models, are discussed. Finally, we address future research perspectives including the possibility of a combined primary 3D human skin and primary human whole blood culture system, and the need for adaptation of the IVMN assays to assess the genotoxic potential of new materials, in particular nanomaterial

Влияние лечения тиотриазолином на состояние перекисного окисления липидов и уровни иммуновоспалительных и вазоактивных эндотелиальных факторов у больных с хронической сердечной недостаточностью и helicobacter pylori-негативными гастропатиями

Показано, что включение в схему патогенетического лечения больных с ХСН, hp-негативной гастропатией и умеренным или высоким риском сердечно-сосудистых осложнений (ССО) антиоксиданта тиотриазолина сопровождается достоверно более выраженными позитивными эффектами на процессы перекисного окисления липидов и содержание в крови иммуновоспалительных и вазоактивных эндотелиальных факторов по сравнению с динамикой при лечении без использования тиотриазолина, причем степень положительной динамики у больных с высоким риском ССО достоверно больше.Показано, що включення у схему патогенетичного лікування хворих із ХСН, hр-негативною гастропатією і з помірним або високим ризиком серцево-судинних ускладнень (ССУ) антиоксиданта тіотриазоліна супроводжується достовірно більш вираженими позитивними ефектами на процеси перекисного окислення ліпідів та вміст у крові імунозапальних і вазоактивних ендотеліальних факторів порівняно з динамікою при лікуванні без використання тіотриазоліну, причому ступінь позитивної динаміки у хворих із високим ризиком ССУ достовірно більший.It is shown that the use of tiotriazolin in the scheme of pathogenetic treatment of patients with chronic heart failure, Hp-negative gastropathy, and moderate or high risk of cardiovascular complications (CVC) is accompanied by significantly higher positive effect on the processes of lipid peroxidation and the amount of immune inflammation and vasoactive endothelial factors in the blood when compared with the dynamics at treatment without the use of tiotriazolin, the degree of positive dynamics in patients with a high risk of CVC being significantly higher

Micronuclei in cord blood lymphocytes and associations with biomarkers of exposure to carcinogens and hormonally active factors, gene polymorphisms, and gene expression: The NewGeneris cohort

Background: Leukemia incidence has increased in recent decades among European children, suggesting that early-life environmental exposures play an important role in disease development. Objectives: We investigated the hypothesis that childhood susceptibility may increase as a result of in utero exposure to carcinogens and hormonally acting factors. Using cord blood samples from the NewGeneris cohort, we examined associations between a range of biomarkers of carcinogen exposure and hormonally acting factors with micronuclei (MN) frequency as a proxy measure of cancer risk. Associations with gene expression and genotype were also explored. Methods: DNA and protein adducts, gene expression profiles, circulating hormonally acting factors, and GWAS (genome-wide association study) data were investigated in relation to genomic damage measured by MN frequency in lymphocytes from 623 newborns enrolled between 2006 and 2010 across Europe. Results: Malondialdehyde DNA adducts (M1dG) were associated with increased MN frequency in binucleated lymphocytes (MNBN), and exposure to androgenic, estrogenic, and dioxin-like compounds was associated with MN frequency in mononucleated lymphocytes (MNMONO), although no monotonic exposure-outcome relationship was observed. Lower frequencies of MNBN were associated with a 1-unit increase expression of PDCD11, LATS2, TRIM13, CD28, SMC1A, IL7R, and NIPBL genes. Gene expression was significantly higher in association with the highest versus lowest category of bulky and M1dG-DNA adducts for five and six genes, respectively. Gene expression levels were significantly lower for 11 genes in association with the highest versus lowest category of plasma AR CALUX® (chemically activated luciferase expression for androgens) (8 genes), ERα CALUX® (for estrogens) (2 genes), and DR CALUX® (for dioxins). Several SNPs (single-nucleotide polymorphisms) on chromosome 11 near FOLH1 significantly modified associations between androgen activity and MNBN frequency. Polymorphisms in EPHX1/2 and CYP2E1 were associated with MNBN. Conclusion: We measured in utero exposure to selected environmental carcinogens and circulating hormonally acting factors and detected associations with MN frequency in newborns circulating T lymphocytes. The results highlight mechanisms that may contribute to carcinogen-induced leukemia and require further research

Elimination of micronucleated cells by apoptosis after treatment with inhibitors of microtubules

Two major mechanisms responsible for chromosome segregation errors are non-disjunction and chromosome loss, both leading to aneuploidy. Previous studies in our laboratory showed the existence of thresholds for the induction of chromosome non-disjunction and chromosome loss and the induction of apoptosis by microtubule inhibitors. From a mechanistic point of view one can expect that apoptosis contributes to the elimination of cells with premutagenic/mutagenic lesions. If aneuploid cells were eliminated by the induction of apoptosis below the threshold concentrations for chromosome loss and non-disjunction, the defined thresholds would not be applicable to cells unable to undergo apoptosis. The aim of this study was to investigate whether apoptosis was induced directly or indirectly as a response to aberrant chromosome segregation below the thresholds for the induction of chromosome loss and non-disjunction, as previously defined by us. Therefore, human lymphocytes were exposed in vitro to five concentrations of nocodazole and five concentrations of carbendazim representing the threshold concentrations for chromosome non-disjunction and chromosome loss, two concentrations below the lowest threshold and one concentration between the two threshold values. After 48 h exposure to the aneugens, induction of apoptosis was analysed by the annexin-V test. The frequencies of chromosome non-disjunction and chromosome loss were estimated in cytokinesis-blocked human lymphocytes in combination with FISH; this methodology was applied to whole cell cultures as well as to apoptotic and viable cell fractions obtained using magnetic annexin microbead cell sorting. Our results suggest that elimination of aneuploid cells does occur. However, the efficiency of disappearance of micronucleated cells is higher than for cells presenting chromosome non-disjunction. The correlation found between early apoptotic events and micronucleus formation could account, at least in part, for the specific elimination of aneuploid cells.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Lower nucleotide excision repair capacity in newborns compared to their mothers: A pilot study

Recognition of the potential vulnerability of children and newborns and protection of their health is essential, especially regarding to genotoxic compounds. Benzo(a)pyrene B(a)P a commonly found carcinogen, and its metabolite BPDE, are known to cross the placenta. To investigate how well newborns are able to cope with BPDE-induced DNA damage, a recent developed nucleotide excision repair cell phenotype assay was applied in a pilot study of 25 newborn daughters and their mothers, using the Alkaline Comet Assay and taking demographic data into account. Newborns seemed to be less able to repair BPDE-induced DNA damage since lower repair capacity levels were calculated compared to their mothers although statistical significance was not reached. Assessment of repair capacity in combination with genotypes will provide important information to support preventive strategies in neonatal care and to define science based exposure limits for pregnant women and children. © 2013 Elsevier Inc.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

An aphidicolin-block nucleotide excision repair assay measuring DNA incision and repair capacity

The objective of the present study was to develop a cellular phenotype assay for nucleotide excision repair (NER), using benzo[a]pyrene diol epoxide (BPDE) as model mutagen. Since in vitro exposure to BPDE may lead to DNA strand breaks resulting from both direct interaction with DNA and incisions introduced by the repair enzymes, we aimed to discriminate between both types of breaks using the comet assay and quantified the DNA strand breaks after in vitro challenge of peripheral blood mononucleated cells (PBMCs) with BPDE in the presence or absence of the DNA polymerase inhibitor aphidicolin (APC). The assay was performed with a low (0.5 μM) and a high (2.5 μM) BPDE concentration. The individual NER capacity was defined as the amount of DNA damage induced by BPDE in presence of APC, diminished with the damage induced by BPDE and APC alone. First, the assay was applied to a NER-deficient human fibroblast cell line (XPA-/-) to validate the methodology. Lower repair capacity and a higher amount of BPDE-induced DNA adducts were observed for the XPA-/- fibroblasts as compared to the wild-type fibroblasts. Repeated experiments on PBMCs from four donors showed low intra-individual, intra-experimental and inter-assay variation for both concentrations, indicating the reliability of the method. To assess the inter-individual variation, the assay was applied to PBMCs from 22 donors, comparing the repair capacity after exposure to 0.5 μM (N=10) and 2.5 μM (N=12) BPDE. The repair capacity showed a higher inter-individual variation as compared to the intra-individual variation. Moreover, this difference was more pronounced using the low concentration. All these results indicate the adequacy of the method using this low concentration. Further improvement, however, should be recommended by applying the study with low BPDE concentration in a larger population and taking into account the relevant genotypes for NER.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Preterm newborns show slower repair of oxidative damage and paternal smoking associated DNA damage

Newborns have to cope with hypoxia during delivery and a sudden increase in oxygen at birth. Oxygen will partly be released as reactive oxygen species having the potential to cause damage to DNA and proteins. In utero, increase of most (non)-enzymatic antioxidants occurs during last weeks of gestation, making preterm neonates probably more sensitive to oxidative stress. Moreover, it has been hypothesized that oxidative stress might be the common etiological factor for certain neonatal diseases in preterm infants. The aim of this study was to assess background DNA damage; in vitro H2O2 induced oxidative DNA damage and repair capacity (residual DNA damage) in peripheral blood mononucleated cells from 25 preterm newborns and their mothers. In addition, demographic data were taken into account and repair capacity of preterm was compared with full-term newborns. Multivariate linear regression analysis revealed that preterm infants from smoking fathers have higher background DNA damage levels than those from non-smoking fathers, emphasizing the risk of paternal smoking behaviour for the progeny. Significantly higher residual DNA damage found after 15-min repair in preterm children compared to their mothers and higher residual DNA damage after 2 h compared to full-term newborns suggest a slower DNA repair capacity in preterm children. In comparison with preterm infants born by caesarean delivery, preterm infants born by vaginal delivery do repair more slowly the in vitro induced oxidative DNA damage. Final impact of passive smoking and of the slower DNA repair activity of preterm infants need to be confirmed in a larger study population combining transgenerational genetic and/or epigenetic effects, antioxidant levels, genotypes, repair enzyme efficiency/levels and infant morbidity. © The Author 2012.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Evaluation of the apoptogenic potential of hard metal dust (WC-Co), tungsten carbide and metallic cobalt.

The present study aimed at comparing in vitro the apoptogenic properties of metallic cobalt (Co), tungsten carbide (WC) and tungsten carbide-cobalt (WC-Co) in conditions known to cause genotoxicity. Human peripheral blood mononucleated cells were incubated with 2.0-6.0 microg/ml of Co alone or mixed with WC particles and 33.3-100.0 microg/ml WC alone for up to 24 h. Under these culture conditions the majority (60%) of the cobalt metal particles were almost immediately solubilised in the culture medium, while WC remained under the form of particles that were progressively phagocytosed by monocytes. Apoptosis was assessed by Annexin-V staining, flow cytometry and analysis of DNA fragmentation by ELISA. Metallic Co-particles induced apoptosis in vitro. Furthermore, although so far considered as biologically inert, WC particles also induced apoptosis. When compared with its individual components WC-Co displayed an additive apoptotic effect in the DNA fragmentation assay. Apoptosis induced by WC particles was found largely dependent on caspase-9 activation and occurred presumably in monocytes, while that induced by Co involved both caspase-9 and -8 activation. The data suggest that apoptosis induced by the tested WC-Co mixture results from the additive effects of WC apoptosis induced in monocytes and Co-specific apoptosis in both monocytes and lymphocytes. The apoptogenic properties of these metals may be important in the mechanism of lung pathologies induced by the cobalt-containing particles

Simplicity and complexity of genetic susceptibility in the occupational environment

The individual response to physical or chemical stress may vary as a function of the particular gene combination regarding metabolism of chemical mutagens, DNA repair, cell death and cell cycle control. Nowadays, methods for genotyping have become easy to perform and in vitro phenotyping approaches are in development. It is therefore interesting to consider whether these methods assessing genetic susceptibility can be implemented for occupational biomonitoring. A major question is whether genotyping or phenotyping or both has the best predictive value for cancer risk and should be applied. To fully understand the relationship between genotype and phenotype, knowledge about the different factors influencing the expression of a genotype into a phenotype is still missing. In this review we compare advantages and disadvantages of genotyping and phenotyping to assess individual susceptibility and discuss the different parameters modifying the genotype-phenotype relationship. The importance of both approaches is illustrated by a study conducted in our laboratory in workers exposed to low dose ionising radiation. Genotyping for hOGG1, XRCC1 and XRCC3, enzymes involved in base excision and double strand DNA repair was performed, the DNA strand break repair phenotype was assessed by in vitro challenging with γ-rays. The results indicate that hOGG1 and XRCC3 may be predictive for induced mutations after exposure to ionising radiation, and that the in vitro repair phenotype assay might also be a valuable approach to assess individual susceptibility. Additional studies on larger population samples are needed before advising these genetic tests for susceptibility in daily practice.SCOPUS: re.jinfo:eu-repo/semantics/publishe

IN VITRO GENOTOXICITY TESTING USING THE MICRONUCLEUS ASSAY IN CELL LINES, HUMAN LYMPHOCYTES AND 3D HUMAN SKIN MODELS.

The toxicological relevance of the micronucleus (MN) test is well defined: it is a multi-target genotoxic endpoint, assessing not only clastogenic and aneugenic events, but also some epigenetic effects, which is simple to score, accurate, applicable in different celltypes. In addition, it is predictive for cancer, amenable for automation, it allows good extrapolation for potential limits of exposure or thresholds and it is easily measured in experimental both in vitro and in vivo systems. Implementation of in vitro MN (IVMN) assays in the battery of tests for hazard and risk assessment of potential mutagens/carcinogens is therefore fully justified. Moreover, the final draft of its OECD guideline was recently made available. We start this short review by discussing the prerequisites for an acceptable MN assay, including the cell as unit of observation, importance of cell membranes and the requirement of a mitotic or meiotic division and the assessment of cell division in the presence of the test substance. Furthermore, the importance of adequate design of treatment protocols is highlighted and the expected new developments, in particular the in vitro 3D human skin models, are discussed. Finally, we address future research including the possibility of a combined primary 3D human skin and primary human whole blood culture system, and the need for adaptation of the IVMN assays to assess the genotoxic potential of new materials, in particular nanomaterials