Polycyclic aromatic hydrocarbons : DNA damage and cell signaling
Author: Mattsson, Åse
Date: 2008-12-19
Location: Sal Atrium, Nobels väg 12B, Karolinska Institutet
Time: 09.15
Department: Institutet för miljömedicin (IMM) / Institute of Enviromental Medicine
View/ Open:
thesis.pdf (500.8Kb)
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.
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.
List of papers:
I. Hofer T, Badouard C, Bajak E, Ravanat JL, Mattsson A, Cotgreave IA (2005). "Hydrogen peroxide causes greater oxidation in cellular RNA than in DNA." Biol Chem 386(4): 333-7.
Pubmed
View record in Web of Science®
II. Mattsson A, Jernström B, Cotgreave IA, Bajak E (2008). "H2AX phosphorylation in A549 cells induced by the bulky and stable DNA adducts of benzo[a]pyrene and dibenzo[a,l]pyrene diol epoxides." Chem Biol Interact Sep 20: Epub ahead of print.
Pubmed
View record in Web of Science®
III. Mattsson Å, Malmlöf M, Seidel A, Stenius U, Jernström B (2008). "H2AX, Mdm2 and p53 phosphorylation are differently affected by bay- and fjord-region diol epoxides derived from carcinogenic polycyclic aromatic hydrocarbons." Polycyclic Aromatic Compounds. [Accepted]
View record in Web of Science®
Fulltext (DOI)
IV. Mattsson Å, Lundstedt S, Stenius U (2008). "Effects of complex PAH mixtures from contaminated soils on DNA damage signaling in HepG2 cells." [Submitted]
I. Hofer T, Badouard C, Bajak E, Ravanat JL, Mattsson A, Cotgreave IA (2005). "Hydrogen peroxide causes greater oxidation in cellular RNA than in DNA." Biol Chem 386(4): 333-7.
Pubmed
View record in Web of Science®
II. Mattsson A, Jernström B, Cotgreave IA, Bajak E (2008). "H2AX phosphorylation in A549 cells induced by the bulky and stable DNA adducts of benzo[a]pyrene and dibenzo[a,l]pyrene diol epoxides." Chem Biol Interact Sep 20: Epub ahead of print.
Pubmed
View record in Web of Science®
III. Mattsson Å, Malmlöf M, Seidel A, Stenius U, Jernström B (2008). "H2AX, Mdm2 and p53 phosphorylation are differently affected by bay- and fjord-region diol epoxides derived from carcinogenic polycyclic aromatic hydrocarbons." Polycyclic Aromatic Compounds. [Accepted]
View record in Web of Science®
Fulltext (DOI)
IV. Mattsson Å, Lundstedt S, Stenius U (2008). "Effects of complex PAH mixtures from contaminated soils on DNA damage signaling in HepG2 cells." [Submitted]
Issue date: 2008-11-28
Rights:
Publication year: 2008
ISBN: 978-91-7409-265-3
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