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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
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Toxicology)
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 [en]
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: urn:nbn:se:uu:diva-217886ISBN: 978-91-554-8877-2 (print)OAI: oai:DiVA.org:uu-217886DiVA: diva2:694319
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
List of papers
1. High intake of fruit and vegetables is related to low oxidative stress and inflammation in a group of patients with type 2 diabetes
Open this publication in new window or tab >>High intake of fruit and vegetables is related to low oxidative stress and inflammation in a group of patients with type 2 diabetes
Show others...
2007 (English)In: Scandinavian Journal of Food and Nutrition, ISSN 1748-2976, E-ISSN 1748-2984, Vol. 51, no 4, 149-158 p.Article in journal (Refereed) Published
Abstract [en]

Background : Patients with type 2 diabetes have increased levels of oxidative stress and inflammation. A high fruit and vegetable intake may be beneficial. Objective : To study whether fruit and vegetable intake and levels of plasma antioxidants relate to markers of oxidative stress and inflammation in a group of patients with type 2 diabetes. Further, to investigate whether plasma antioxidants are good biomarkers for intake of fruit and vegetables. Design : Patients with type 2 diabetes were studied. Their dietary intake and levels of plasma antioxidants, and markers of oxidative stress and inflammation were analysed. Results : Fruit and vegetable intake was inversely related to oxidative stress. Plasma carotenoids were negatively correlated with inflammation. The plasma levels of -carotene and β-carotene showed strongly positive associations with fruit and vegetable intake. Conclusions : The results suggest that fruit and vegetable intake may decrease oxidative stress and inflammation in this group of patients. An increased intake of fruit and vegetables can therefore be beneficial for patients with type 2 diabetes, since these patients are documented to have raised oxidative stress and inflammation. The study support the usefulness of plasma -carotene and β-carotene as biomarkers for fruit and vegetable intake.

Keyword
antioxidants, fruit, inflammatory cytokine, oxidative stress, type 2 diabetes, vegetables
National Category
Medical and Health Sciences
Research subject
Nutrition
Identifiers
urn:nbn:se:uu:diva-16051 (URN)10.1080/17482970701737285 (DOI)
Available from: 2008-04-11 Created: 2008-04-11 Last updated: 2017-12-08Bibliographically approved
2. Supplementation with a combination of antioxidants does not affect glycaemic control, oxidative stress or inflammation in type 2 diabetes subjects
Open this publication in new window or tab >>Supplementation with a combination of antioxidants does not affect glycaemic control, oxidative stress or inflammation in type 2 diabetes subjects
Show others...
2010 (English)In: Free radical research, ISSN 1071-5762, E-ISSN 1029-2470, Vol. 44, no 12, 1445-1453 p.Article in journal (Refereed) Published
Abstract [en]

The present clinical trial examined the influence of a supplement, containing a combination of antioxidants extracted from fruit, berries and vegetables, on levels of plasma antioxidants (tocopherols, carotenoids and ascorbate), glycaemic control (blood glucose, HbA1c, insulin), oxidative stress biomarkers (F2-isoprostane, malondialdehyd, nitrotyrosine, 8-oxo-7, 8-dihydro-2'-deoxyguanosine, formamidopyrimidine glycosylase sites, frequency of micronucleated erythrocytes) and inflammatory markers (interleukin-6, C-reactive protein, prostaglandin F-metabolite) in type 2 diabetes. Forty subjects were randomly assigned to control, single or double dose group and completed the study. In summary, 12 weeks of antioxidant supplementation did neither affect glycaemic control nor the levels of biomarkers of oxidative stress or inflammation, despite substantially increased plasma concentrations of antioxidants. The absence of an effect may be explained by the selected study subjects with relatively well-controlled diabetes, a high intake of fruit and vegetable and levels of plasma antioxidants, biomarkers of oxidative stress and inflammatory markers comparable to those found in healthy subjects.

Keyword
Antioxidants, supplementation, glycaemic control, oxidative stress, inflammation, diabetes mellitus type 2
National Category
Medical and Health Sciences
Research subject
Nutrition
Identifiers
urn:nbn:se:uu:diva-134116 (URN)10.3109/10715762.2010.515219 (DOI)000283796300007 ()
Available from: 2010-12-02 Created: 2010-11-22 Last updated: 2017-12-12Bibliographically approved
3. Effect of beta-carotene on catechol-induced genotoxicity in vitro: Evidence of both enhanced and reduced DNA damage
Open this publication in new window or tab >>Effect of beta-carotene on catechol-induced genotoxicity in vitro: Evidence of both enhanced and reduced DNA damage
2013 (English)In: Free radical research, ISSN 1071-5762, E-ISSN 1029-2470, Vol. 47, no 9, 692-698 p.Article in journal (Refereed) Published
Abstract [en]

Intake of antioxidants from the diet has been recognized to have beneficial health effects, but the potential benefit of taking antioxidants such as beta-carotene as supplements is controversial. The aim of the present study was to evaluate the potential protective effects of a physiologically relevant concentration (2 mu M) of beta-carotene on the DNA damaging effects of catechol in mouse lymphoma L5178Y cells. Two different exposure protocols were used: simultaneous exposure to beta-carotene and catechol for 3 h; and exposure to catechol for 3 h after 18 h pre-treatment with the vitamin. DNA damage was evaluated using the comet assay (employing one procedure for general damage, and another procedure, which also included oxidative DNA damage). Independent of exposure protocol and procedure for comet assay, beta-carotene did not increase the basal level of DNA damage. However, at the highest concentration of catechol (1 mM), beta-carotene was found to clearly increase the level of catechol-induced DNA damage, especially in the pre-treated cells. Interestingly, an opposite effect was observed at lower concentrations of catechol, but the beta-carotene related reduction of catechol-induced genotoxicity was significant (P < 0.05) only for the procedure including oxidative damage induced by 0.5 mM catechol. Taken together our results indicate that beta-carotene can both reduce and enhance the DNA damaging effects of a genotoxic agent such as catechol. This indicates that it is the level of catechol-induced DNA damage that seems to determine whether beta-carotene should be regarded as a beneficial or detrimental agent when it comes to its use as a dietary supplement.

Keyword
antioxidant, comet assay, hOGG1, mouse lymphoma cells, pro-oxidant
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-206959 (URN)10.3109/10715762.2013.815346 (DOI)000323107900004 ()
Available from: 2013-09-09 Created: 2013-09-09 Last updated: 2017-12-06Bibliographically approved
4. Evaluation of α-tocopherols effect on catechol and o-phenylenediamine induced DNA damage: An in vitro study using two different staining techniques in the comet assay
Open this publication in new window or tab >>Evaluation of α-tocopherols effect on catechol and o-phenylenediamine induced DNA damage: An in vitro study using two different staining techniques in the comet assay
(English)Article in journal (Refereed) Submitted
Abstract [en]

Using the alkaline comet assay, the effect of a physiologically relevant concentration of a-tocopherol (20 mM) on the DNA damaging effects of catechol and o-phenylenedi- amine (OPD) was evaluated in mouse lymphoma L5178Y TK+/- cells. For compara- tive purposes, a third mutagenic agent, 4-nitro-o-phenylenediamine (4-NOPD), was also included in the study. a-Tocopherol was found to be without DNA damaging effects of its own after three hours exposure when tested in concentrations up to 500 mM, and this vitamin was also found to significantly reduce the DNA damage induced by 1 mM catechol. However, a-tocopherol did not reduce the level of DNA damage induced by OPD, indicating that catechol and OPD induce DNA damage by different mechanisms of action. As in a previous study, 4-NOPD did not increase the level of DNA damage, and a-tocopherol did not affect the level of damage when the cells were concomitantly exposed to both agents. The results from a separate ex- periment using different staining techniques for cells that had been exposed to dif- ferent concentrations of catechol, clearly indicated that the more hazardous chemical ethidium bromide could be substituted with the less hazardous chemical GelRedTM in the comet assay.

Keyword
α-tocopherol, catechol, OPD, DNA damage, comet assay, mouse lymphoma cells, GelRed
National Category
Pharmacology and Toxicology
Research subject
Toxicology
Identifiers
urn:nbn:se:uu:diva-211514 (URN)
Available from: 2013-12-03 Created: 2013-11-25 Last updated: 2014-04-29Bibliographically approved
5. 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
Open this publication in new window or tab >>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
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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.

National Category
Other Medical Sciences
Identifiers
urn:nbn:se:uu:diva-211502 (URN)10.1093/mutage/get041 (DOI)000326380000004 ()
Available from: 2013-11-25 Created: 2013-11-25 Last updated: 2017-12-06Bibliographically approved

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