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.
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In Vitro Pollen Tube Growth Reveals the Cytotoxic Potential of the Flavonols, Quercetin and Rutin

Fabiana Antognoni, Elisa Ovidi, Anna Rita Taddei, Gabriella Gambellini and Anna Speranza

Flavonols are phytochemicals widely found in commonly consumed foods. In spite of their beneficial effects on human health, however, cytotoxicity and even suspected genotoxicity have also been reported for the flavonol, quercetin. This points to the need for preventive studies to identify any cytotoxic effects associated with pure flavonol intake. This work was performed with the aim of verifying whether a plant-based in vitro system, the pollen tube, could be used to evaluate the cytotoxic potential of exogenous flavonols. Increasing concentrations of the aglycone, quercetin, and its glycoside, rutin, were assayed with regard to tube growth of kiwifruit pollen, determined by applying the pollen tube growth test protocol. This test, based on the photometric quantification of pollen tube mass production in suspension cultures, has already been applied in the sensitive and reliable toxicological evaluation of a wide range of chemicals. Whereas 60–800μM rutin promoted kiwifruit pollen tube elongation, 10–50μM quercetin strongly inhibited growth, and also produced irreversible malformations, such as screw-like tube growth, abnormal vacuolation, alteration of organelle streaming, and nuclear positioning. Thus, the cytotoxic potentials of the two flavonols have been confirmed to differ. Pollen tubes seem to afford a promising test system for a preventive, rapid in vitro biosafety assessment of antioxidant nutritional supplements, without using laboratory animals.
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