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Genotoxic effect induced by hydrogen peroxide in human hepatoma cells using comet assay.

Benhusein GM, Mutch E, Aburawi S, Williams FM - Libyan J Med (2010)

Bottom Line: Cells were treated with H(2)O(2) at concentrations of 25 µM or 50 µM for 5 min, 30 min, 40 min, 1 h, or 24 h in parallel.Our findings confirm that H(2)O(2) is a typical DNA damage-inducing agent and thus is a good model system to study the effects of oxidative stress.DNA damage in HepG2 cells increased significantly with H(2)O(2) concentration and time of incubation but later decreased likely due to DNA repair mechanisms and antioxidant enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Al-Fateh University for Medical Sciences, Tripoli, Libya.

ABSTRACT

Background: Hydrogen peroxide (H(2)O(2)) is a common reactive oxygen intermediate generated by various forms of oxidative stress.

Aim: The aim of this study was to investigate the DNA damage capacity of H(2)O(2) in HepG2 cells.

Methods: Cells were treated with H(2)O(2) at concentrations of 25 µM or 50 µM for 5 min, 30 min, 40 min, 1 h, or 24 h in parallel. The extent of DNA damage was assessed by the comet assay.

Results: Compared to the control, DNA damage by 25 and 50 µM H(2)O(2) increased significantly with increasing incubation time up to 1 h, but it was not increased at 24 h.

Conclusions: Our findings confirm that H(2)O(2) is a typical DNA damage-inducing agent and thus is a good model system to study the effects of oxidative stress. DNA damage in HepG2 cells increased significantly with H(2)O(2) concentration and time of incubation but later decreased likely due to DNA repair mechanisms and antioxidant enzymes.

No MeSH data available.


Related in: MedlinePlus

Flowchart showing the comet assay for single-cell gel electrophoresis to determine DNA damage (12).
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Figure 0002: Flowchart showing the comet assay for single-cell gel electrophoresis to determine DNA damage (12).

Mentions: Fig. 2 illustrates the comet assay (12). This assay has been adapted from that originally proposed by Singh et al. (13). Partly frosted microscope slides were pre-coated with a smear of 1% low melting-point agarose (LMPA) and allowed to dry for 1 h at 37°C. The slides were then placed on a bed of ice to pre-cool them before adding an agarose sandwich. An aliquot of 170 µl of 1% LMPA was placed onto the slide and immediately covered with a coverslip. Whilst this was setting, 170 µl of the suspended lymphocytes were mixed 1:1 with 2% LMPA. The coverslip was then removed from the initial agarose layer. An aliquot of 170 µl of the agarose/lymphocyte mix was placed on top of the first layer and again covered immediately with a coverslip. Once set, the coverslip was removed and a final layer of 170 µl 1% LMPA was applied to the existing gel to form an agarose sandwich, with the cells in the middle layer. The final coverslip was removed once the gel had set. For each lymphocyte sample, two slides were prepared. At this point the lights were switched off and the remainder of the assay was performed using indirect light.


Genotoxic effect induced by hydrogen peroxide in human hepatoma cells using comet assay.

Benhusein GM, Mutch E, Aburawi S, Williams FM - Libyan J Med (2010)

Flowchart showing the comet assay for single-cell gel electrophoresis to determine DNA damage (12).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3066752&req=5

Figure 0002: Flowchart showing the comet assay for single-cell gel electrophoresis to determine DNA damage (12).
Mentions: Fig. 2 illustrates the comet assay (12). This assay has been adapted from that originally proposed by Singh et al. (13). Partly frosted microscope slides were pre-coated with a smear of 1% low melting-point agarose (LMPA) and allowed to dry for 1 h at 37°C. The slides were then placed on a bed of ice to pre-cool them before adding an agarose sandwich. An aliquot of 170 µl of 1% LMPA was placed onto the slide and immediately covered with a coverslip. Whilst this was setting, 170 µl of the suspended lymphocytes were mixed 1:1 with 2% LMPA. The coverslip was then removed from the initial agarose layer. An aliquot of 170 µl of the agarose/lymphocyte mix was placed on top of the first layer and again covered immediately with a coverslip. Once set, the coverslip was removed and a final layer of 170 µl 1% LMPA was applied to the existing gel to form an agarose sandwich, with the cells in the middle layer. The final coverslip was removed once the gel had set. For each lymphocyte sample, two slides were prepared. At this point the lights were switched off and the remainder of the assay was performed using indirect light.

Bottom Line: Cells were treated with H(2)O(2) at concentrations of 25 µM or 50 µM for 5 min, 30 min, 40 min, 1 h, or 24 h in parallel.Our findings confirm that H(2)O(2) is a typical DNA damage-inducing agent and thus is a good model system to study the effects of oxidative stress.DNA damage in HepG2 cells increased significantly with H(2)O(2) concentration and time of incubation but later decreased likely due to DNA repair mechanisms and antioxidant enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Al-Fateh University for Medical Sciences, Tripoli, Libya.

ABSTRACT

Background: Hydrogen peroxide (H(2)O(2)) is a common reactive oxygen intermediate generated by various forms of oxidative stress.

Aim: The aim of this study was to investigate the DNA damage capacity of H(2)O(2) in HepG2 cells.

Methods: Cells were treated with H(2)O(2) at concentrations of 25 µM or 50 µM for 5 min, 30 min, 40 min, 1 h, or 24 h in parallel. The extent of DNA damage was assessed by the comet assay.

Results: Compared to the control, DNA damage by 25 and 50 µM H(2)O(2) increased significantly with increasing incubation time up to 1 h, but it was not increased at 24 h.

Conclusions: Our findings confirm that H(2)O(2) is a typical DNA damage-inducing agent and thus is a good model system to study the effects of oxidative stress. DNA damage in HepG2 cells increased significantly with H(2)O(2) concentration and time of incubation but later decreased likely due to DNA repair mechanisms and antioxidant enzymes.

No MeSH data available.


Related in: MedlinePlus