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Dioxin induces genomic instability in mouse embryonic fibroblasts.

Korkalainen M, Huumonen K, Naarala J, Viluksela M, Juutilainen J - PLoS ONE (2012)

Bottom Line: Although all responses to TCDD alone were delayed, menadione-induced DNA damage (measured by the Comet assay), was found to be increased directly after a 2-day TCDD exposure, indicating that the stability of the genome was compromised already at this time point.The results suggested a flat dose-response relationship consistent with dose-response data reported for radiation-induced GI.These findings indicate that TCDD, although not directly genotoxic, induces GI, which is associated with impaired DNA damage response.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Health, National Institute for Health and Welfare, Kuopio, Finland. merja.korkalainen@thl.fi

ABSTRACT
Ionizing radiation and certain other exposures have been shown to induce genomic instability (GI), i.e., delayed genetic damage observed many cell generations later in the progeny of the exposed cells. The aim of this study was to investigate induction of GI by a nongenotoxic carcinogen, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mouse embryonic fibroblasts (C3H10T1/2) were exposed to 1, 10 or 100 nM TCDD for 2 days. Micronuclei (MN) and expression of selected cancer-related genes were assayed both immediately and at a delayed point in time (8 days). For comparison, similar experiments were done with cadmium, a known genotoxic agent. TCDD treatment induced an elevated frequency of MN at 8 days, but not directly after the exposure. TCDD-induced alterations in gene expression were also mostly delayed, with more changes observed at 8 days than at 2 days. Exposure to cadmium produced an opposite pattern of responses, with pronounced effects immediately after exposure but no increase in MN and few gene expression changes at 8 days. Although all responses to TCDD alone were delayed, menadione-induced DNA damage (measured by the Comet assay), was found to be increased directly after a 2-day TCDD exposure, indicating that the stability of the genome was compromised already at this time point. The results suggested a flat dose-response relationship consistent with dose-response data reported for radiation-induced GI. These findings indicate that TCDD, although not directly genotoxic, induces GI, which is associated with impaired DNA damage response.

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Effect of TCDD pretreatment on menadione-induced DNA damage and its repair.Comet tail moments were analyzed after TCDD exposure (1, 10 or 100 nM) for 2 days (A) and at the end of 6 days recovery time without exposure (B). After menadione treatment (40 µM) for one hour, cells were allowed to repair menadione-induced DNA damage for 0, 15, or 30 min. Each column represents mean ± SE of 400 Olive tail moments (OTM) in 4 independent experiments (A) or mean ± SE of 300 tail moments of 3 independent experiments (B). The effect of TCDD, tested over all TCDD doses and all three time points, was significant (p = 0.0009) when measured immediately after TCDD exposure, but not at 6 days after the end of exposure. The effect of menadione was significant (p<0.0001) in both cases.
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pone-0037895-g002: Effect of TCDD pretreatment on menadione-induced DNA damage and its repair.Comet tail moments were analyzed after TCDD exposure (1, 10 or 100 nM) for 2 days (A) and at the end of 6 days recovery time without exposure (B). After menadione treatment (40 µM) for one hour, cells were allowed to repair menadione-induced DNA damage for 0, 15, or 30 min. Each column represents mean ± SE of 400 Olive tail moments (OTM) in 4 independent experiments (A) or mean ± SE of 300 tail moments of 3 independent experiments (B). The effect of TCDD, tested over all TCDD doses and all three time points, was significant (p = 0.0009) when measured immediately after TCDD exposure, but not at 6 days after the end of exposure. The effect of menadione was significant (p<0.0001) in both cases.

Mentions: The Comet assay, in contrast to the MN assay, revealed that the stability of the genome was compromised in TCDD-exposed cells already at 2 days. When pretreated cells were challenged with menadione, the resulting DNA damage was increased in TCDD-pretreated cells (Fig. 2a). The overall effect of TCDD was significant at p = 0.0009, and the post tests showed a significant trend (p = 0.003) and that all TCDD groups were significantly (p<0.01) different from the menadione-only exposed group. The effect of menadione alone was significant at p<0.0001. Consistently with the micronucleus data, the TCDD effect showed a flat dose-response relationship between 1 and 100 nM. Interestingly, the increased sensitivity to menadione was not found if the cells were allowed to recover from TCDD for 6 days (Fig. 2b). The level of DNA strand breaks was even slightly (nonsignificantly) lower in the TCDD-exposed cells than in the menadione-only exposed cells at 0 and 15 min after the menadione treatment. It is important to note that the effect of TCDD was observable only as an increased sensitivity to menadione. TCDD alone, without menadione treatment, had no effect in the Comet assay (Fig. 3).


Dioxin induces genomic instability in mouse embryonic fibroblasts.

Korkalainen M, Huumonen K, Naarala J, Viluksela M, Juutilainen J - PLoS ONE (2012)

Effect of TCDD pretreatment on menadione-induced DNA damage and its repair.Comet tail moments were analyzed after TCDD exposure (1, 10 or 100 nM) for 2 days (A) and at the end of 6 days recovery time without exposure (B). After menadione treatment (40 µM) for one hour, cells were allowed to repair menadione-induced DNA damage for 0, 15, or 30 min. Each column represents mean ± SE of 400 Olive tail moments (OTM) in 4 independent experiments (A) or mean ± SE of 300 tail moments of 3 independent experiments (B). The effect of TCDD, tested over all TCDD doses and all three time points, was significant (p = 0.0009) when measured immediately after TCDD exposure, but not at 6 days after the end of exposure. The effect of menadione was significant (p<0.0001) in both cases.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3362596&req=5

pone-0037895-g002: Effect of TCDD pretreatment on menadione-induced DNA damage and its repair.Comet tail moments were analyzed after TCDD exposure (1, 10 or 100 nM) for 2 days (A) and at the end of 6 days recovery time without exposure (B). After menadione treatment (40 µM) for one hour, cells were allowed to repair menadione-induced DNA damage for 0, 15, or 30 min. Each column represents mean ± SE of 400 Olive tail moments (OTM) in 4 independent experiments (A) or mean ± SE of 300 tail moments of 3 independent experiments (B). The effect of TCDD, tested over all TCDD doses and all three time points, was significant (p = 0.0009) when measured immediately after TCDD exposure, but not at 6 days after the end of exposure. The effect of menadione was significant (p<0.0001) in both cases.
Mentions: The Comet assay, in contrast to the MN assay, revealed that the stability of the genome was compromised in TCDD-exposed cells already at 2 days. When pretreated cells were challenged with menadione, the resulting DNA damage was increased in TCDD-pretreated cells (Fig. 2a). The overall effect of TCDD was significant at p = 0.0009, and the post tests showed a significant trend (p = 0.003) and that all TCDD groups were significantly (p<0.01) different from the menadione-only exposed group. The effect of menadione alone was significant at p<0.0001. Consistently with the micronucleus data, the TCDD effect showed a flat dose-response relationship between 1 and 100 nM. Interestingly, the increased sensitivity to menadione was not found if the cells were allowed to recover from TCDD for 6 days (Fig. 2b). The level of DNA strand breaks was even slightly (nonsignificantly) lower in the TCDD-exposed cells than in the menadione-only exposed cells at 0 and 15 min after the menadione treatment. It is important to note that the effect of TCDD was observable only as an increased sensitivity to menadione. TCDD alone, without menadione treatment, had no effect in the Comet assay (Fig. 3).

Bottom Line: Although all responses to TCDD alone were delayed, menadione-induced DNA damage (measured by the Comet assay), was found to be increased directly after a 2-day TCDD exposure, indicating that the stability of the genome was compromised already at this time point.The results suggested a flat dose-response relationship consistent with dose-response data reported for radiation-induced GI.These findings indicate that TCDD, although not directly genotoxic, induces GI, which is associated with impaired DNA damage response.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Health, National Institute for Health and Welfare, Kuopio, Finland. merja.korkalainen@thl.fi

ABSTRACT
Ionizing radiation and certain other exposures have been shown to induce genomic instability (GI), i.e., delayed genetic damage observed many cell generations later in the progeny of the exposed cells. The aim of this study was to investigate induction of GI by a nongenotoxic carcinogen, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mouse embryonic fibroblasts (C3H10T1/2) were exposed to 1, 10 or 100 nM TCDD for 2 days. Micronuclei (MN) and expression of selected cancer-related genes were assayed both immediately and at a delayed point in time (8 days). For comparison, similar experiments were done with cadmium, a known genotoxic agent. TCDD treatment induced an elevated frequency of MN at 8 days, but not directly after the exposure. TCDD-induced alterations in gene expression were also mostly delayed, with more changes observed at 8 days than at 2 days. Exposure to cadmium produced an opposite pattern of responses, with pronounced effects immediately after exposure but no increase in MN and few gene expression changes at 8 days. Although all responses to TCDD alone were delayed, menadione-induced DNA damage (measured by the Comet assay), was found to be increased directly after a 2-day TCDD exposure, indicating that the stability of the genome was compromised already at this time point. The results suggested a flat dose-response relationship consistent with dose-response data reported for radiation-induced GI. These findings indicate that TCDD, although not directly genotoxic, induces GI, which is associated with impaired DNA damage response.

Show MeSH
Related in: MedlinePlus