Annual Report

8. Induction of Superoxide in Glioma Cell Line U87 Stimulated with Lipopolysaccharide and Interferon Gamma: ESR Using a New Flow-type Quartz Cuvette

Hidehiko Nakagawa, Nobuo Ikota, and Toshihiko Ozawa

Keywords: superoxide, nitric oxide , U87 glioma cell, flow-type quartz cuvette, TEMPOL , lipopolysaccharide , interferon-

Superoxide and nitric oxide are endogenous radical species, which are considered to play important roles not only in inflammation as protective factors, but also in signal transduction and in neurodegenerative diseases. Just like immune cells such as macrophages in peripheral tissues, glial cells in the brain are considered to play a role in protecting against infection and oxidative stress. The production of nitric oxide and superoxide is suggested to be induced by treatment with cytokines or endotoxic reagents in several glioma cell lines. However, there have been only a few attempts to measure the induction of superoxide and nitric oxide. In this study, the lines. However, there have been only a few attempts to measure the induction of superoxide and nitric oxide. In this study, the production of superoxide and nitric oxide induced in U87 glioma treated with lipopolysaccharide (LPS) and interferon-(IFN-) was examined by ESR spectroscopy using a newly designed flow-type quartz cuvette without detaching the cells from the culture plate (Fig. 7A,B). ESR spectra of 2,2,6,6-tetramethyl-4-hydroxy-1-piperidinyloxy (TEMPOL) with U87 cells on a quartz culture plate were measured at 15-min intervals and signal intensity was quantified. Without U87 cells, the signal did not decay. The observed rate constant of the signal decay was increased in the case of U87 cells pretreated with LPS and IFN-(Fig.7C). The observed pseudo-first rate constants were calculated to be 3.05 x 10-3 /min/106 cells and 6.14 x 10-3 /min/106 cells in the case of non-treated cells and LPS/IFN--pretreated cells, respectively. This increase of the rate constant by LPS/IFN--pretreated U87 cells was inhibited in the presence of superoxide dismutase (SOD) and catalase (Cat). The observed pseudo-first rate constant was calculated to be 3.39 x 10-3 /min/106 cells in the case of LPS/IFN--pretreated cells with SOD and catalase. From the difference of the observed rate constants in the presence and absence of both SOD and catalase, the signal decay of TEMPOL with superoxide was calculated to be 2.75 x 10-3 /min/106 cells. Since the reaction rate constant of TEMPOL with superoxide has been calculated to be 3.90 x 107 M-1min-1, the concentration of superoxide in LPS/IFN--treated U87 cells was estimated to be 70.5 pM/106 cells.

An ESR spin trapping method using N-dithiocarboxy- sarcosine (DTCS, Fig.7D)-iron complex was ewmployed to measure nitric oxide from U87 cells pretreated with LPS/IFN-for 24 h, but only a weak signal of NO adducts was detected. Furthermore the nitrite and nitrate levels in the medium, measured with a fluorometric detection kit, did not increase for 24 h.

The results obtained in this study suggested that superoxide production by U87 cells was induced by treatment with LPS/IFN- for 24 h. Nitroxide spin probes have been reported to act as a superoxide scavenger or a SOD-mimic. TEMPOL, one of the nitroxide spin probes, was considered to act as a SOD-mimic in the redox mechanism, that is, TEMPOL was readily oxidized by protonated superoxide to oxoammonium cation (Fig. 7E) which in turn oxidized another superoxide to molecular oxygen. According to these reports, TEMPOL catalytically reacted with superoxide, so that the signal intensity of TEMPOL might not decrease, depending on the production of superoxide. However, the decay rate of the TEMPOL signal intensity was considered to reflect the production of superoxide in this study, because the concentration of TEMPOL was much higher than that of superoxide produced in the cells which was essentially spent only in the reaction with TEMPOL. The reaction of the oxoammonium cation with superoxide was considered to be negligible as long as the concentration of TEMPOL was high enough in the system.

In conclusion, it was found that treatment with LPS/IFN- for 24 h induced production of superoxide, but not nitric oxide in a glioma cell line, U87. The work was done by ESR using a superoxide-sensitive spin probe, TEMPOL, and spin-trapping reagent for nitric oxide, Fe-DTCS. The use of the new cuvette designed for adhesive cells and quartz culture plates will be an effective way to measure glioma cells by ESR without their detachment from the plates.

1) Nakagawa, H., Moritake, T., Tsuboi, K., Ikota, N. and Ozawa, T. : FEBS Lett.., 471, 187-190, 2000.
2) Nakagawa, H. , Sumiki, E. , Takusagawa, M., Ikota, N., Matsushima, Y. and Ozawa, T.: Chem. Pharm. Bull., 48, 261-265, 2000.

Fig.7. The structure of the new flow-type quartz cuvette (A); a diagram of the ESR measurement system (B); and ESR signal decay of TEMPOL with U87 glioma cells (C, the time course of the relative signal intensity measured at 15-min intervals). Chemical structures of N - dithiocarboxysarcosine (D) and oxoammonium cation of TEMPOL (E)

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