An In Vitro System for Evaluation of Oxidative Stress and the Effects of Antioxidants

Benedikte Brogaard and Jørgen Clausen

Oxidative stress is defined as an imbalance between prooxidants and antioxidants. There is a need for a simple in vitro method for evaluation of the effects of oxidative stress and the effects of antioxidants. In the present study, we used primary cultures of human lymphocytes exposed to either paraquat (PQ) or mitomycin C, two prooxidants generating two different types of free-radicals formed either by P450-reductase or by DT-diaphorase, respectively. The toxicity was measured by estimation of DT-diaphorase and glutathione peroxidase (GSH-Px) activity, and by estimation of the level of malondialdehyde (MDA) as a function of time and increasing doses of the two prooxidants. The enzyme activities were related to both total DNA content and total protein content of cellular homogenate. All estimations were made by exposing human lymphocytes to increasing concentrations (up to 100μM) of the two prooxidants. However, since cellular death occurred at concentrations above 60μM, only data for exposure to concentrations below 70μM are presented. When the enzyme activities were expressed per cellular unit (i.e. per gram DNA) 30μM mitomycin C induced a 30% increase in DTdiaphorase activity. Similarly, a dose-dependent increase (maximum 100% increase) in DTdiaphorase activity was found after exposure to PQ (up to 60μM). Similar data were obtained when data were related to the total protein. Only a minor increase (11%) in GSH-Px activity was induced by 50μM mitomycin C, whereas 20–70μM PQ induced a 41% increase in GSH-Px activity. Both prooxidants induced more than a doubling in the cellular MDA concentration. These findings demonstrate that both DT-diaphorase and GSH-Px are up-regulated during oxidative stress. However, sensitivity to prooxidant-induced stress seems to depend to some extent on the chemistry of the free-radicals generated. Thus, the single-electron pyridium cations generated by PQ seem to be more toxic than the single-electron semi-quinones generated by mitomycin C. The same cellular system was used to evaluate the effects of antioxidants. Quercetin, a naturally occuring flavone, and selenium (sodium selenite), which is an essential part of glutathione peroxidase, were selected. PQ-induced stress and exposure to 5μg/ml quercetin for 4 hours decreased the MDA level in the medium by 11% and in the cells by 33%. PQ-induced stress and exposure to 160μg/l selenium for 18 hours reduced MDA levels similarly, by 19% in the medium and 14% in the cells. Both antioxidants induced a 50% reduction in GSHPx activity.
You need to register (for free) to download this article. Please log in/register here.

The Effects of Oxidative Stress in In Vitro Cultured Astroglial Cells

Casper Møller Frederiksen and Jørgen Clausen

It has been suggested that glial cells in the central nervous system might function as a buffer and protect neurons and synapses. Associated with such a function, glial cells might be affected in degenerative diseases, for example, Alzheimer’s disease and Parkinson’s disease, due to generation of free-radicals. Free-radicals might be generated during the metabolic
transformation of xenobiotics. The purpose of the present study was to determine whether a xenobiotic (in this case, paraquat), is metabolised in glial cells during the generation of freeradicals. Furthermore, this study determined whether free-radicals can induce DNA fragmentation and whether this fragmentation can be repaired. The data produced indicated that astroglial cells contain P450-reductase which transforms paraquat into a pyridium free-radical. In turn, this causes a dose-dependent DNA fragmentation, as determined by using single-cell gel electrophoresis. The dose-dependent effect was valid up to 80μM paraquat. The oxidative stress induced in the astroglial cells was also associated with a maximum 15% increase in the anti-oxidative enzyme, glutathione peroxidase. After exposure to 40μM paraquat, followed by growth of the cells in a paraquat-free medium, DNA repair was shown to be rather slow, and was only obvious two hours after exposure to paraquat. This might be related the shuttle in which paraquat/P450-reductase is implicated, which causes a protracted generation of free-radicals. The data are discussed in relation to the available literature.
You need to register (for free) to download this article. Please log in/register here.

Oxidative DNA Damage in Human Cells Induced by Paraquat

Helena Petrovská and Mária Dušinská

Oxidative DNA damage was studied after exposing two human transformed cell lines (HeLa and Hep G2) and freshly isolated human peripheral lymphocytes to the herbicide, paraquat. We used the alkaline comet assay, modified by incubating nucleoid DNA with endonuclease III to detect oxidised pyrimidines, and with formamidopyrimidine glycosylase to detect 8-oxo-guanine and ring-opened purines. Paraquat induces both strand breaks and oxidised bases, the amounts of each being dependent on the concentration of paraquat and the cell type exposed. Exposure to lower concentrations of paraquat for 1 hour induced dose-dependent DNA damage in Hep G2 cells and in human peripheral lymphocytes. DNA damage was reduced at higher concentrations. Our results support the finding that paraquat induces oxidative stress but, over a certain concentration range, also stimulates antioxidant protection. Reduction of DNA damage was not found in HeLa cells after exposure for 1 hour or 24 hours. Short-term exposure to paraquat induced a moderate amount of oxidative DNA damage (mainly oxidized pyrimidines) in HeLa cells. Exposure for 24 hours induced a high proportion of oxidised bases and strand breaks. Hep G2 cells showed the greatest number of DNA strand breaks, with no sign of base oxidation.
You need to register (for free) to download this article. Please log in/register here.