Oxidative stress, fluorescent

Okada et al. examined the effects of TBT on cellular content of glutathione (GSH) in rat thymocites using a flow cytometer and 5-chloromethylfluorescein diacetate, a fluorescent probe for monitoring the change in the cellular content of GSH. TBT at nanomolar concentrations reduced the cellular content of GSH. There is an important implication on the TBT-induced depletion of cellular GSH since GSH has an important role in protecting the cells against oxidative stress and chemical and metal intoxications. TBT-induced decrease in cellular content of GSH in thymocytes may increase the vulnerability of the immune system. ° ... 

Figure 4.7 Changes in intraceiiuiar calcium in cultured rat ventricular myocytes exposed to oxidant stress. Calcium was measured using the fluorescent probe Fura>2. The ratio of the Fura-2 fluorescence measured at 340 and 380 nm excitation is shown and this is proportional to the intracellular calcium concentration. The fast-speed traces shown (note the 3.5 s time-scale) were recorded after various durations of oxidant stress. Myocytes under control conditions (before t = 0) show spontaneous calcium transients. These transients decreased in frequency with oxidant stress until cells failed to show spontaneous activity but continued to maintain a low intracellular calcium.

The above findings are supported in the other studies of the inhibitory effects of flavonoids on iron-stimulated lipid peroxidation. Quercetin was found to be an inhibitor of iron-stimulated hepatic microsomal lipid peroxidation (/50 = 200 pmol I ) [134]. Flavonoids eriodictyol, luteolin, quercetin, and taxifolin inhibited ascorbate and ferrous ion-stimulated MDA formation and oxidative stress (measured by fluorescence of 2,7,-dichlorodihydro-fluorescein) in cultured retinal cells [135]. It should be mentioned that in recent work Heijnen et al. [136] revised the structure activity relationship for the protective effects of flavonoids against lipid peroxidation. 

Cells may show a low level of autofluorescence at 413 nm when irradiated at 324 nm. This fluorescence dramatically increases when d -parinaric acid (159) is incorporated into the cell membrane, either by intercalation or esteriflcation. Exposure to oxidation stress of cells enriched with the 159 fluorescent probe causes diminution of the fluorescence intensity and is directly correlated with formation of lipid hydroperoxides. Addition of antioxidants, such as Vitamin E (21), abates fluorescence diminution. A blanc run of cells enriched with 159 but not subjected to oxidation stress is necessary to follow the degradation of 159 when exposed to UV irradiation. This method was applied to track lipid oxidation during apoptosis and other phenomena, triggered by toxic compounds such as H2O2, f-BuOOH and cumyl hydroperoxide (27)"° 11,424... 

Also, HPLC methods with electrochemical or fluorescent detection are used (H19, M3). In proteins, dityrosine can be estimated by immunochemical methods employing dityrosine-specific antibodies (K5). Measurements of o,o -dityrosine and o-tyrosine levels in rat urine express dityrosine contents in skeletal muscle proteins, and have been proposed as the noninvasive oxidative stress test in vivo. One should be aware, however, that A-formylkynurenine, also formed in protein oxidation, has similar fluorescence properties as dityrosine (excitation 325 nm, emission at 400-450 nm) (G29). Also, oxidation of mellitin when excited at 325 nm produces an increase in fluorescence at 400—450 nm, despite the fact that mellitin does not contain tyrosine. Oxidation of noncontaining Trp residues ribonuclease A and bovine pancreatic trypsin inhibitor with "OH produces loss of tyrosine residues with no increase in fluorescence at 410 nm (S51). There are also methods measuring the increased hydrophobicity of oxidized proteins. Assays are carried out measuring protein binding of a fluorescent probe, 8-anilino-l-naphthalene-sulfonic acid (ANS). Increase in probe binding reflects increased surface hydrophobicity (C7). 

Abstract Cyclosporine A (CsA) therapy is associated with side effects related to oxidative stress. We characterized the reactive oxygen and nitrogen species produced in the extracellular and intracellular compartments of bovine aortic endothelial cells (BAEC) exposed to CsA. CsA induced a dose-dependent increase of the intracellular oxidation of the NO-sensitive fluorescent probe DAF-2/DA. In agreement with this, CsA produced a dose-dependent accumulation of nitrites in the supernatants of BAEC. In contrast, in BAEC treated with CsA, the presence of superoxide anion could only be detected in the intracellular compartment, as measured by the oxidation of dihydroethidium. The formation of peroxynitrite was assessed in the intracellular compartment, by flow cytometry with... 

This same group has also used BODIPY-conjugated lipids and fluoresceinated phosphoethanolamine for similar studies (47,48). As discussed earlier, loss of NAO fluorescence can reflect oxidative stress-induced cardiolipin loss, which is often associated with apoptosis. 

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