Oxidative stress in cells is characterized by an accumulation of reactive oxygen species (ROS) and is considered as a major factor in numerous diseases, disorders and aging [4]. ROS can lead to cell death by either necrosis or apoptosis depending on severity of cellular damage by an oxidant injury. Plant polyphenols have currently drawn attention due to their antioxidant activities.

The aim of the study was to examine the effect of quercetin and apigenin on apoptosis and necrosis induction in HeLa cells challenged by H₂O₂. We also try to determine how examined flavonoids affect the redox equilibrium of cells by measurements of antioxidant enzymes, glutathione and ROS levels. Using immunogold electron microscopy we also examined the effect of both flavonoids on localization of Hsp 72 and PARP.

Apoptosis and necrosis identification was determined by staining of the cells with use of the mixture of propidium iodide and Hoechst 33342 [6]. SOD activity was performed by inhibition of adrenaline oxidation assay [8]. CAT activity was performed by decomposition of H₂O₂ [1] and GSH-Per, GSH-Red activities and glutathione level by specific methods [2,3,5]. Free radical level was assessed using NBT assay [7].

In cells exposed to H₂O₂ we observed mainly apoptosis. Meanwhile combined effect of flavonoids and oxidative stress suppressed the apoptosis and cytotoxic effects. SOD activity was greater. Decrease in ROS level was observed in cells pretreated with flavonoids and coincubated with hydrogen peroxide. Increase in total level of glutathione was observed. GSH-R activity was lowered. CAT was overexpressed in cells exposed to apigenin, whereas in quercetin treated cells its activity was lower in all the concentrations. PARP was detected mainly in cytoplasm. There were observed cells with strong PARP- immunopositive reaction. Hsps 72 was located in cytoplasm and nucleus and its distribution differed dependently on the sort of experimental variant.

Our data indicate that flavonoids reduce oxidative stress partly through an inhibition of the generation of ROS production, by increase of SOD and CAT and by raising of the total glutathione level_. Other mechanisms can not be excluded in protective effect of examined flavonoids.

[1] Aebi H. Meth. Enzymol. (1984), 105, 121-126.

[2] Anderson M.E. Meth. Enzymol. (1985), 113, 548-554.

[3] Carlberg I., Mannervick B. Meth. Enzymol. (1985), 113, 484-490.

[4] Davis K.J.A. Oxidative stress: the paradox of aerobic life, in: C. Rice –Evans, B. Halliwell, G.G. Lunt (Eds.), Free Radicals and Oxidative Stress: environment, Drugs and Food additives, Portland Press, London, (1995), pp.1-31.

[5] Flohe I., Gunzler W.A. Meth. Enzymol.(1984), 105, 114-121.

[6] Jankowska A., Skonieczna D., Rommerts F.F.C., Warchoł J.B. Folia Histochem. Cytobiol. (1997), 36, 99-110.

[7] Paździoch-Czochra M., Malarczyk E., Sielewiesiuk J. Cell Biol. Int. (2003), 27, 325-336.

[8] Sun M., Zigman S. Anal. Biochem. (1978), 90, 81-89.

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1. Changes of the cell wall structure as a part of the response system of white rot fungus: Abortiporus biennis to oxidative stress.