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.