Screening of new antioxidant molecules using flow cytometry.

We present a flow cytometry technique to evaluate the antioxidative properties of molecules on living cells, using a stable murine-murine hybridoma (Mark 3) cell line routinely cultured. Using this technique, intracellular superoxide anions and peroxides were evaluated with dihydrorhodamine (DHR-123) and dichlorofluorescein diacetate (DCFH-DA), respectively. When cells were first incubated for 10 min with either H(2)O(2) or the xanthine (X)/xanthine oxidase (XO) system, this flow cytometric technique was capable of evaluating the oxidative stress on cells. Twenty-one new analogues of ellipticine were synthesized and tested for their antioxidative properties compared to vitamin E and Ebselen used as references. A good statistical reflection of the antioxidative activities of these molecules was achieved by analyzing 35 000 cells in each experiment. Among them, the selenated molecule 18 was found to be 10 times more active than Ebselen but 10 000 times less active than vitamin E. Moreover, eight compounds showed glutathione peroxidase-like activities.

Effects of H2O2 on the growth, secretion, and metabolism of hybridoma cells in culture.

The effect of low concentrations of hydrogen peroxide (H2O2) (5 x 10(-7)-9.5 x 10(-7) M) on cell growth and antibody production was investigated with murine hybridoma cells (Mark 3 and anti-hPL) in culture. Cell growth, measured by flow cytometry with morphological parameters, was significantly stimulated by H2O2 (8 x 10(-7) M) but H2O2 concentration of 7 x 10(-6) M and above increased cell death. H2O2 stimulation of antibody production was nonsignificant. The metabolism of cells treated with 8 x 10(-7) or 1 x 10(-5) M H2O2 was similar to that of the control in terms of glucose and glutamine consumption, lactate and ammonia production, and amino acid concentrations in the medium. The concentrations of lactate dehydrogenase, a marker of cell death, in test and control cells were similar. However, concentrations of intracellular free radicals measured by flow cytometry with dihydrorhodamine 123 (DHR 123) and dichlorofluorescein diacetate (DCFH-DA) as fluorochromes were different. The reactive oxygen species content of cells in 8 x 10(-7) M H2O2 was similar to that of the controls, but there was a sudden, marked production of superoxide anions (detected with DHR 123) and H2O2 or peroxides (detected with DCFH-DA) by cells incubated with 1 x 10(-5) M H2O2 which increased with increasing H2O2 until cell death.