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Evidence for different mechanisms of action behind the mutagenic effects of 4-NOPD and OPD: the role of DNA damage, oxidative stress and an imbalanced nucleotide pool
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Stockholm University. (Department of Molecular Biosciences)
Stockholm University. (Department of Molecular Biosciences)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
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2013 (English)In: Mutagenesis, ISSN 0267-8357, E-ISSN 1464-3804, Vol. 28, no 6, 637-644 p.Article in journal (Refereed) Published
Abstract [en]

The mutagenicity of 4-nitro-o-phenylenediamine (4-NOPD) and o-phenylenediamine (OPD) was compared using the Mouse Lymphoma Assay (MLA) with or without metabolic activation (S9). As expected, OPD was found to be a more potent mutagen than 4-NOPD. To evaluate possible mechanisms behind their mutagenic effects, the following end points were also monitored in cells that had been exposed to similar concentrations of the compounds as in the MLA: general DNA damage (using a standard protocol for the Comet assay); oxidative DNA damage (using a modified procedure for the Comet assay in combination with the enzyme hOGG1); reactive oxygen species (ROS; using the CM-H(2)DCFDA assay); and the balance of the nucleotide pool (measured after conversion to the corresponding nucleosides dC, dT, dG and dA using high-performance liquid chromatography). Both compounds increased the level of general DNA damage. Again, OPD was found to be more potent than 4-NOPD (which only increased the level of general DNA damage in the presence of S9). Although less obvious for OPD, both compounds increased the level of oxidative DNA damage. However, an increase in intracellular ROS was only observed in cells exposed to 4-NOPD, both with and without S9 (which in itself induced oxidative stress). Both compounds decreased the concentrations of dA, dT and dC. A striking effect of OPD was the sharp reduction of dA observed already at very low concentration, both with and without S9 (which in itself affected the precursor pool). Taken together, our results indicate that indirect effects on DNA, possibly related to an unbalanced nucleotide pool, mediate the mutagenicity and DNA-damaging effects of 4-NOPD and OPD to a large extent. Although induction of intracellular oxidative stress seems to be a possible mechanism behind the genotoxicity of 4-NOPD, this pathway seems to be of less importance for the more potent mutagen OPD.

Place, publisher, year, edition, pages
2013. Vol. 28, no 6, 637-644 p.
National Category
Other Medical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-211502DOI: 10.1093/mutage/get041ISI: 000326380000004OAI: oai:DiVA.org:uu-211502DiVA: diva2:666943
Available from: 2013-11-25 Created: 2013-11-25 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Effects of Antioxidants and Pro-oxidants on Oxidative Stress and DNA Damage using the Comet Assay: Studies on Blood Cells from Type 2 Diabetes Subjects and Mouse Lymphoma Cells
Open this publication in new window or tab >>Effects of Antioxidants and Pro-oxidants on Oxidative Stress and DNA Damage using the Comet Assay: Studies on Blood Cells from Type 2 Diabetes Subjects and Mouse Lymphoma Cells
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Diet and oral supplements comprise two distinct sources of antioxidants known to prevent oxidative stress. Beneficial effects from antioxidants have been seen for patients at risk for type 2 diabetes.

The aim of this thesis was to evaluate the positive effects of antioxidants against oxidative stress and DNA damage in type 2 diabetes subjects. We also used antioxidants as tools to determine the mechanisms behind genotoxicity induced by mutagenic pro-oxidative agents in mouse lymphoma cells. Several techniques were used to measure oxidative stress and DNA damage, but the main technique used was alkaline comet assay.

The results showed that the fruit and vegetable intake was inversely related to oxidative stress in type 2 diabetes subjects. However, oral supplementary intake of 20 antioxidants did not decrease oxidative stress biomarkers.

In studies on mouse lymphoma cells, using the alkaline comet assay, DNA damage was induced by catechol and o-phenylenediamine (OPD), while 4-nitro-o-phenylenediamine (4-NOPD) induced only oxidative damage, showing different mechanisms of action behind the mutagenicity of the compounds. Also, oxidative stress was induced by catechol and 4-NOPD, whereas imbalances in the nucleotide pool were seen after exposure to OPD or 4-NOPD. Addition of antioxidants together with these pro-oxidants showed that β-carotene was able to reduce DNA damage at low concentrations of catechol, but increased DNA damage at high concentration. In comparison, addition of α-tocopherol slightly decreased catechol-induced DNA damage at all concentrations of catechol. However, no effect of α-tocopherol was seen on OPD-or 4-NOPD-induced DNA damage.

In conclusion, antioxidants from fruits and vegetables, but not from oral supplements, reduced oxidative stress in type 2 diabetes patients, suggesting fruits and vegetables being a healthier source for antioxidant-intake, as compared to oral supplements. Different mechanisms of action for mutagenic pro-oxidants were shown in mouse lymphoma cells, introducing the nucleotide pool as an interesting target for oxidative stress. Reduction of catechol-induced DNA damage by β-carotene or α-tocopherol was shown, with a pro-oxidative action of β-carotene at high concentration of catechol, Interestingly, α-tocopherol was not able to decrease OPD- or 4-NOPD-induced DNA damage, supporting different mechanisms of action behind the genotoxicity from the three pro-oxidants.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 78 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 185
Keyword
metabolic syndrome, fruit and vegetable intake, plasma antioxidants, beta-carotene, alpha-tocopherol, inflammation, oxidative DNA damage, lipid peroxidation, mouse lymphoma assay, ROS, nucleotide pool, viability, DNA dye
National Category
Pharmacology and Toxicology
Research subject
Toxicology
Identifiers
urn:nbn:se:uu:diva-217886 (URN)978-91-554-8877-2 (ISBN)
Public defence
2014-03-28, A1:107a, Biomedical center, Husargatan 3, Uppsala, 09:15 (Swedish)
Opponent
Supervisors
Available from: 2014-03-06 Created: 2014-02-05 Last updated: 2014-04-29

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Åsgård, RikardHellman, Björn

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