ATLA::Alternatives to Laboratory Animals

Volume 25, Number 3

An in vitro system for evaluation of oxidative stress and the effects of antioxidants.

ATLA 25, 279-287, May/June 1997

Benedikte Brogaard and Jørgen Clausen

Department of Life Sciences and Chemistry, Roskilde University, P.O. Box 260, 4000 Roskilde, Denmark

SUMMARY

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 Iymphocytes 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 Iymphocytes to increasing concentrations (up to 100 µM) of the two prooxidants. However, since cellular death occurred at concentrations above 60 µM, only data for exposure to concentrations below 70 µM are presented. When the enzyme activities were expressed per cellular unit (i.e. per gram DNA) 30 µM mitomycin C induced a 30% increase in DT-diaphorase activity. Similarly, a dose-dependent increase (maximum 100% increase) in DT-diaphorase activity was found after exposure to PQ (up to 60 µM). Similar data were obtained when data were related to the total protein. Only a minor increase (11%) in GSH-Px activity was induced by 50 µM mitomycin C, whereas 20-70 µM 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 5 µg 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 160 µg/mI 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 GSH-Px activity.

Keywords: free-radicals, paraquat, mitomycin C, DT-diaphorase, glutathione peroxidase, malondialdehyde, antioxidant, oxidative stress, quercetin, selenium