_:b16348664 . _:b16348646 "Menadione was selected, as we have previously shown that a nongenotoxic agent (extremely low frequency magnetic field) alters cellular responses to a subsequent exposure to menadione [14], [>>15<<]. In addition, expression of cancer-related genes was studied, because altered expression of specific genes might indicate mechanisms involved in maintaining the unstable phenotype over multiple cell generations. Also, F\u00E4lt et al. [16]" . _:b16348654 . _:b16348655 "results" . _:b16348665 . _:b16348666 . _:b16348635 _:b16348640 . . "PMC0" . _:b16348635 _:b16348641 . _:b16348635 _:b16348642 . _:b16348635 _:b16348643 . _:b16348635 _:b16348644 . _:b16348635 _:b16348645 . _:b16348635 _:b16348646 . _:b16348635 _:b16348647 . . . . _:b16348648 "materials and methods" . _:b16348660 "This finding is consistent with dose-response data reported for ionizing radiation-induced GI, i.e., a threshold at relatively low doses and a plateau above it [>>20<<], [21]. As no immediate genotoxicity was observed, the signal that transmits TCDD-induced GI to the later cell generations must be epigenetic by its nature. This is consistent with the current understanding that GI induced by ionizing" . _:b16348653 "The analysis was performed as described by Luukkonen et al. [>>14<<]. Minor modifications on suspension volumes and electrophoresis conditions were:" . . _:b16348635 "introduction" . _:b16348641 . _:b494533191 . . . _:b16348655 _:b16348664 . _:b16348655 _:b16348665 . _:b16348655 _:b16348666 . _:b494533190 . . _:b16348648 _:b16348652 . _:b16348655 _:b16348656 . _:b16348648 _:b16348653 . _:b494533191 "2"^^ . _:b16348655 _:b16348657 . _:b16348648 _:b16348654 . _:b16348655 _:b16348658 . _:b16348655 _:b16348659 . _:b16348655 _:b16348660 . _:b16348648 _:b16348649 . _:b16348648 . _:b494533190 "2"^^ . _:b16348655 _:b16348661 . _:b16348648 _:b16348650 . _:b16348655 _:b16348662 . _:b16348648 _:b16348651 . _:b16348655 _:b16348663 . . _:b16348638 . _:b16348644 . _:b16348655 . _:b16348642 "experimental animals, TCDD evokes a wide range of biological and toxic effects, including reproductive and developmental defects, immunotoxicity, endocrine alterations, thymus atrophy, wasting syndrome, liver toxicity and cancer [8]\u2013[>>11<<]. Practically all of these effects are mediated via the aryl hydrocarbon receptor (AHR), which, upon binding to TCDD, translocates into the nucleus, heterodimerizes with AHR nuclear translocator (ARNT) and binds to dioxin-responsive" . _:b16348636 "Genomic instability (GI) is defined as an increased rate of acquisition of alterations in the genome [>>1<<]. GI can be observed many cell generations later in the progeny of exposed cells as delayed damage, e.g., chromosomal aberrations, mutations, micronuclei or apoptosis." . . . . . . _:b16348664 . _:b16348664 "The gene expression changes also differ from those observed after exposure to ionizing radiation, which causes both immediate and delayed changes in gene expression [>>26<<]. The delayed nature of the changes therefore seems to be a special characteristic for TCDD. In the present study, only a limited number of cancer-related genes were measured, which should be taken into account in interpreting the low" . _:b16348656 . _:b494533197 "2"^^ . _:b16348647 . _:b16348658 "micronucleated erythrocytes were observed only after long-term exposure to very high dose levels that caused overt toxicity, suggesting that induction of MN does not represent a specific genotoxic effect but rather a secondary response [>>7<<]. The findings of the present study are consistent with lack of direct genotoxic effects, as no increase in MN was observed immediately after two days of exposure." . _:b494533196 "2"^^ . _:b494533199 "2"^^ . _:b16348640 "Based on evidence from animal experiments TCDD is a potent tumor promoter, but the tumor-initiating activity is either lacking or the response is weak [6], [>>7<<]. In this study, TCDD was chosen to investigate induction of GI as an agent that is not directly genotoxic. In general, TCDD is a model compound for dioxins, a group of wide-spread, persistent and highly toxic environmental contaminants." . _:b16348647 "Also, F\u00E4lt et al. [>>16<<] found changes in the gene expression pattern of irradiated T-lymphocyte clones cultured for multiple generations after exposure to ionizing radiation." . _:b494533198 "2"^^ . _:b16348657 "TCDD is considered a nongenotoxic carcinogen (also termed epigenetic or non-DNA reactive carcinogen), as it does not appear to cause direct genetic damage [17], [>>18<<]. In rats, increased frequencies of micronucleated erythrocytes were observed only after long-term exposure to very high dose levels that caused overt toxicity, suggesting that induction of MN does not represent a specific genotoxic effect" . _:b16348639 "GI is thought to be a driving force of carcinogenesis in both radiation- and chemical-induced cancer [>>5<<]. A key question for assessing the importance of GI in carcinogenesis is whether also \u201Cnon-genotoxic\u201D carcinogens (agents that do not cause genetic damage in traditional short-term tests) can induce GI." . _:b494533193 "2"^^ . . _:b494533192 "2"^^ . _:b494533195 "2"^^ . . _:b494533194 "2"^^ . . _:b16348662 . _:b16348635 . _:b494533192 . _:b494533191 . _:b16348666 "The findings of Puga et al. indicated a complex response involving multiple cellular processes [>>27<<]. The increased number of changes observed at 8 days in the present study suggests that an even more complex pattern of responses develops in a delayed manner in the progeny of the exposed cells." . _:b16348637 "Exposure to ionizing radiation is the best-known inducer of GI, but also chemical exposures can lead to GI, even though the data are limited [>>2<<]\u2013[4]. GI is thought to be a driving force of carcinogenesis in both radiation- and chemical-induced cancer [5]. A key question for assessing the importance of GI in carcinogenesis is whether also \u201Cnon-genotoxic\u201D carcinogens (agents that do" . _:b494533199 . _:b494533193 . _:b494533194 . _:b494533195 . _:b16348661 . _:b494533196 . _:b16348636 . _:b494533190 . _:b494533197 . _:b494533198 . _:b494533199 . _:b16348637 . . _:b16348661 "This finding is consistent with dose-response data reported for ionizing radiation-induced GI, i.e., a threshold at relatively low doses and a plateau above it [20], [>>21<<]. As no immediate genotoxicity was observed, the signal that transmits TCDD-induced GI to the later cell generations must be epigenetic by its nature. This is consistent with the current understanding that GI induced by ionizing radiation" . _:b16348638 . _:b494533190 . _:b494533191 . _:b16348639 . _:b16348654 "of the six biological pathways involved in transformation and tumorigenesis (Table 1) and has been reported to exhibit good reproducibility and highly comparable results in gene expression measurements with high-density microarrays [>>30<<]. The array also contained controls for RT reaction and PCR reaction as well as a genomic DNA control." . _:b16348662 "This is consistent with the current understanding that GI induced by ionizing radiation is inherited to next cell generations in an epigenetic fashion [>>22<<]\u2013[25]." . . _:b494533197 . _:b16348637 . _:b16348663 . _:b16348657 . _:b16348656 "TCDD is considered a nongenotoxic carcinogen (also termed epigenetic or non-DNA reactive carcinogen), as it does not appear to cause direct genetic damage [>>17<<], [18]. In rats, increased frequencies of micronucleated erythrocytes were observed only after long-term exposure to very high dose levels that caused overt toxicity, suggesting that induction of MN does not represent a specific genotoxic" . . . _:b494533196 . _:b16348660 . _:b16348638 "Exposure to ionizing radiation is the best-known inducer of GI, but also chemical exposures can lead to GI, even though the data are limited [2]\u2013[>>4<<]. GI is thought to be a driving force of carcinogenesis in both radiation- and chemical-induced cancer [5]. A key question for assessing the importance of GI in carcinogenesis is whether also \u201Cnon-genotoxic\u201D carcinogens (agents that do not" . _:b494533199 . _:b16348635 . _:b16348644 . _:b494533198 . _:b16348652 . _:b16348645 "Menadione was selected, as we have previously shown that a nongenotoxic agent (extremely low frequency magnetic field) alters cellular responses to a subsequent exposure to menadione [>>14<<], [15]. In addition, expression of cancer-related genes was studied, because altered expression of specific genes might indicate mechanisms involved in maintaining the unstable phenotype over multiple cell generations. Also, F\u00E4lt et al." . . _:b16348645 . _:b494533193 . _:b494533195 . . . _:b16348646 . _:b494533192 . _:b494533196 . _:b16348647 . _:b494533195 . . _:b16348640 . _:b494533194 . _:b494533197 . "10.1371%2Fjournal.pone.0037895" . _:b16348652 "An alkaline version of the method (pH>13) detecting both DNA single-strand breaks (SSB) and DNA double-strand breaks (DSB) was used as described earlier [>>29<<]. In this method, cells are mixed with agarose, spread on glass microscope slide and lysed by lysis buffer leaving only nuclei on the slide. During electrophoresis, broken DNA fragments migrate away from the nucleus forming a tail that" . _:b16348641 . _:b16348646 . _:b494533194 . _:b16348640 . _:b16348642 . _:b16348644 "The aim of the present study was to investigate TCDD-induced GI in mouse embryonic fibroblasts, which have been utilized earlier to study GI [>>13<<]. For comparison, the cells were exposed also to cadmium chloride, a known genotoxic compound." . _:b16348659 "Delayed increase in MN was also reported in two highly TCDD-exposed women several months after intoxication, when TCDD levels had already decreased [>>19<<]. To the best of our knowledge the present study is the first controlled experimental study showing delayed genetic damage as a result of TCDD exposure. This finding indicates that, although TCDD does not cause direct genotoxicity, it" . _:b16348643 . _:b16348663 "This is consistent with the current understanding that GI induced by ionizing radiation is inherited to next cell generations in an epigenetic fashion [22]\u2013[>>25<<]." . . _:b494533192 . _:b16348652 . . _:b16348653 . _:b16348654 . _:b16348655 . _:b16348639 . _:b16348641 "In experimental animals, TCDD evokes a wide range of biological and toxic effects, including reproductive and developmental defects, immunotoxicity, endocrine alterations, thymus atrophy, wasting syndrome, liver toxicity and cancer [>>8<<]\u2013[11]. Practically all of these effects are mediated via the aryl hydrocarbon receptor (AHR), which, upon binding to TCDD, translocates into the nucleus, heterodimerizes with AHR nuclear translocator (ARNT) and binds to dioxin-responsive" . _:b16348648 . . _:b16348649 . _:b16348643 . . . _:b16348650 . . _:b16348651 . _:b16348650 "The principle of this method is staining first the nuclei of dying cells (i.e. cells with damaged cell membrane) with EMA, lysing the cells, staining the nuclei with SYTOX Green and assorting the nuclei by flow cytometry [>>28<<]." . . _:b16348636 . _:b16348660 . . _:b16348661 . . _:b16348662 . _:b16348645 . _:b16348642 . _:b16348663 . _:b16348656 . . _:b494533193 . _:b16348665 "In a microarray study with human hepatoma HepG2 cells exposed to 10 nM TCDD, 310 out of 5686 known genes were altered by a factor of at least 2.1 immediately after exposure [>>27<<], the proportion of affected genes being close to that observed in the present study (3 out of 84)." . _:b16348657 . _:b494533198 . _:b16348658 . _:b16348649 . . _:b16348659 . _:b16348649 "Micronucleus frequency of cells was analysed by a flow cytometry based assay as described by Luukkonen et al. [>>14<<], with minor changes. In brief, cells were stained with ethidium monoazide bromide (EMA), photoactivated with visible light (light bulb) and the nuclei stained with SYTOX Green dye." . . _:b16348658 . . _:b16348651 "Finally, a flow cytometer (Becton Dickinson FACSCalibur, Becton Dickinson, San Jose, CA) was used in MN analyses according to Bryce et al. [>>28<<] and data were analyzed by CellQuest Software (v." . . _:b16348665 . _:b16348659 . _:b16348643 "all of these effects are mediated via the aryl hydrocarbon receptor (AHR), which, upon binding to TCDD, translocates into the nucleus, heterodimerizes with AHR nuclear translocator (ARNT) and binds to dioxin-responsive elements in DNA [>>12<<]. The best-known effect is the activation of genes for xenobiotic metabolism, such as CYP1A1, but otherwise the TCDD-induced mechanisms in other responses are still largely unknown." . _:b16348666 . . _:b16348635 _:b16348636 . _:b16348635 _:b16348637 . _:b16348635 _:b16348638 . _:b16348650 . _:b16348653 . _:b16348635 _:b16348639 . . _:b16348651 .