| 1. |
- Mattsson, Åse, et al.
(författare)
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Exposure of HepG2 cells to low levels of PAH-containing extracts from contaminated soils results in unpredictable genotoxic stress responses
- 2009
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Ingår i: Environmental and Molecular Mutagenesis. - : John Wiley & Sons. - 0893-6692 .- 1098-2280. ; 50:4, s. 337-348
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Tidskriftsartikel (refereegranskat)abstract
- Contaminated soil is a serious environmental problem, constituting a risk to humans and the environment. Polycyclic aromatic hydrocarbons (PAHs) are often present at contaminated sites. However, risk levels are difficult to estimate because of the complexity of contaminants present. Here, we compare cellular effects of extracts from contaminated soils collected at six industrial settings in Sweden. Chemical analysis showed that all soils contained complex mixtures of PAHs and oxy-PAHs. Western blotting and immunocytochemistry were used to investigate DNA damage signaling in HepG2 cells exposed to extracts from these soils. The effects on phosphorylated Mdm2, p53, Erk, H2AX, 53BP1, and Chk2, cell cycle regulating proteins (cyclin D1 and p21), and cell proliferation were compared. We found that most soil extracts induced phosphorylation of Mdm2 at the 2A10 epitope at low concentrations. This is in line with previous studies suggesting that this endpoint reflects readily repaired DNA-damage. However, we found concentration and time-dependent gamma H2AX and 53BP1 responses that were sustained for 48 hr. These endpoints may reflect the presence of different types of persistent DNA-damage. High concentrations of soil extracts decreased cyclin D1 and increased p21 response, indicating cell cycle arrest. Phosphorylation of Mdm2 at Ser 166, which attenuates the p53 response and is induced by many tumor promoters, was induced in a time-dependent manner and was associated with Erk phosphorylation. Taken together, the PAH extracts elicited unpredictable signaling responses that differed between samples. More polar compounds, i.e., oxy-PAHs, also contributed to the complexity.
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| 2. |
- Mattsson, Åse
(författare)
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Polycyclic aromatic hydrocarbons : DNA damage and cell signaling
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Oxidative stress is a threat to our wellbeing. The formation of reactive oxygen species may result in oxidative lesions in DNA and RNA. When challenging A549 cells with 18O-labeled hydrogen peroxide, we found that RNA was between 14-25 times more sensitive to [18O]-8-oxoGuo formation than [18O]-8-oxodGuo formation in DNA. The A549 cells showed slow turnover rates of adducts in RNA and DNA with half-lifes of approximately 12 h for [18O]-8-oxoGuo in RNA, and 21 h for [18O]-8-oxodGuo in DNA, respectively. Polycyclic aromatic hydrocarbons (PAHs) are widespread mutagenic and carcinogenic environmental contaminants. They require metabolic activation into electrophilic diol epoxides (DEs) to be able to bind to DNA and elicit their biological activity. The bay-region DE of benzo[a]pyrene (BPDE) is more easily removed by nucleotide excision repair (NER) than the fjord-region DE of dibenzo[a,l]pyrene (DBPDE). This could reflect the ability of DBPDE to escape recognition and to investigate this, we studied the affect these DEs have on histone H2AX phosphorylation (gammaH2AX). Human A549 cells were exposed to the DEs for various time and concentration. The results showed that BPDE induced a transient gammaH2AX, while DBPDE exposure resulted in a continuously increasing and persistent gammaH2AX and these data correlated with the known effect on nucleotide excision repair (NER). Thus, the extent of gammaH2AX formation and the persistence was related to both the number of adducts and their structural feature. Further, the gammaH2AX, as well as effects on Mdm2 and p53 were studied in A549 cells in response to the bay-region DEs of chrysene (CDE) and dibenz[a,h]anthracene (DBADE), or the fjord-region DEs of benzo[c]chrysene (B[c]CDE), benzo[g]chrysene (B[g]CDE) and benzo[c]phenanthrene (B[c]PhDE). We found that the fjord-region DEs induced a rapid and concentration-dependent response on Mdm2 2A10 phosphorylation, p53 stabilization and phosphorylation, as well as gammaH2AX, where Mdm2 was the most sensitive marker. The bay-region DEs had less effect on Mdm2 2A10 phosphorylation and induced neither p53 stabilization nor phosphorylation. No gammaH2AX was detected with Western blot in response to bay-region DEs, however, immunostaining revealed reversible gammaH2AX. Also here, the variance between bay- and fjord-region DEs most likely reflect their recognition and handling by NER. PAH contamination of soil at industrial setting constitutes a risk to humans, but the risk is often difficult to estimate due to the complexity of present contaminants. We compared the DNA damage signaling effects in HepG2 cells exposed to PAHs extracted from contaminated soils collected at six different industrial settings in Sweden. Most of the soil extracts induced Mdm2 2A10 phosphorylation at low concentration, which may indicate repairable damage. We found concentration- and time-dependent gammaH2AX and 53BP1 responses, sustaining up to 48 h indicating persistent damage. Effects on cyclin D1 and p21 indicated cell cycle arrest, and phosphorylation of Mdm2 at Ser166, known to attenuate p53 response, was found and was associated with Erk phosphorylation. The PAH extracts elicited unpredictable DNA damage signaling that differed between the samples, and where also more polarcompounds, oxy-PAHs, contributed. We found that established approaches to evaluate carcinogenic potentials of PAH mixtures in contaminated soil are insufficient and call for the development of more sophisticated endpoints.
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| 3. |
- Roos, Åse, et al.
(författare)
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Protocol for Providing Additional Pseudo-Pregnant Recipient Mice for Embryo Transfer and Intra-Uterine Insemination by Plugging in the Middle of the Day
- 2008
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Ingår i: Scandinavian Journal of Laboratory Animal Science. - 0901-3393. ; 35:4, s. 305-310
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Tidskriftsartikel (refereegranskat)abstract
- The fact that 10% of female mice enter oestrus and allow mating in the middle of the day is an old observation that has been more or less forgotten. We here show that this old knowledge can be used to improve the efficacy of both embryo transfer and insemination protocols. The present technical paper shows that rapid re-arrangements of mating cages, to achieve pseudo-pregnant recipients in the middle of the day, can be of great advantage in emergency situations. Such emergency situations occur repeatedly, i.e. when a scientist has forgotten to re-arrange her/his mating cages, and the last important male suddenly has become ill and may die within a few hours. A rapid technique for uterine artificial insemination in mice in such situations is extremely valuable. An artificial intra-uterine insemination requires only a minimum of planning, a minimum of instrumentation and a minimum of surgical training. The artificial insemination must be performed shortly after mating due to rapid constriction of the utero-tubual junction (UTJ). This means that the timing of the insemination is very important. We here show that the success rate for embryo transfers, when using recipients plugged in the middle of the day, was the same as for ordinary overnight mating protocols. In addition, it: should be noted that the success rate (frequency of pregnancies) for uterine inseminations was 55% if using F1 recipients of C57BL/6J (considerable lower if using recipients of inbred C57BL/6J), which is amazingly high, since inseminations in mice is known to be tricky to perform in a reproducible manner.
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