Low-Level Chemiluminescence of Isolated Hepatocytes

Abstract

1. Oxygenation of isolated hepatocytes leads to an increased emission of low level chemiluminescence and to an accumulation of malondialdehyde, both occurring after a lag phase of about 20 -40 min.2. Spectral analysis of oxygen-induced chemiluminescence of isolated hepatocytes showed three bands at 460,560 and 640 nm, with two shoulders at 525 and 61 5 nm. Singlet molecular oxygen, formed during the free radical process accompanying lipid peroxidation, is identified as the main source of light emission, on the basis of comparison with spectra of singlet oxygen produced in chemical systems [Khan, A. U. und Kasha, M. (1963) J . Am. Chem. Soc. 92,3. Hepatocytes from phenobarbital-pretreated rats, or glutathione-depleted hepatocytes showed a threefold increase in both maximal chemiluminescence intensity and malondialdehyde accumulated, as compared with control cells, whereas the lag phase was not modified by the pretreatments.4. Glutathione-depleted hepatocytes did not show any increase in spontaneous lipid peroxidation as reflected by either malondialdehyde accumulation or chemiluminescence. A dissociation between both parameters was observed on addition of dithioerythritol : chemiluminescence intensity decreased while the malondialdehyde content remained unaltered, 5. It is concluded from these experiments that low-level chemiluminescence emitted from hepatocytes at wavelengths beyond 600 nm ('red band') monitors the steady-state concentration of singlet molecular oxygen, providing a useful tool to examine oxygen-dependent radical damage. Continuous monitoring of singlet oxygen levels affords an advantage over parameters measuring accumulative effects.Low-level chemiluminescence is related to the free radical processes that accompany oxidation of lipids, and specifically to the generation of excited species derived from the interaction of free radicals [I -51. Spontaneous chemiluminescence arising from organs, cells and organelles can give account of endogenous oxidation reactions involving lipids within a physiological frame. However, the count rate for spontaneous chemiluminescence is extremely low, making difficult its characterization. Therefore, efforts are usually made to enhance this spontaneous chemiluminescence by peroxidative conditions, such as NADPH-supplemented microsomal fractions in the absence of autoxidizable substrates [6,7], addition of Fez + or organic hydroperoxides to mitochondria1 preparations [8,9], perfusion of organs with organic hydroperoxides [lo, 1 I] or exposure to hyperbaric oxygen conditions [5].In the present study, we describe the characteristics of chemiluminescence of isolated hepatocytes, as observable in the widely used incubation condition with Krebs-Henseleit buffer, and this is extended to certain conditions, such as glutathione depletion and induction of microsomal enzymes, that favour lipid peroxidation. Hepatocytes, isolated with a technique associated with low peroxidative damage, provide a suitable biological source to evaluate the oxidative events through the chemilu...

Microsomal formation of S-nitrosoglutathione from organic nitrites: possible role of membrane-bound glutathione transferase

“…fed with Altromin standard diet ad libitum, as described previously [16]. Membranes were washed with 100 mM Tris\HCl (pH 7.4) and suspended in a medium containing 0.25 M sucrose, 10 mM Tris\HCl (pH 7.4) and 10 mM MgCl # [15].…”

tert–butylhydroperoxide—Induced toxicity in isolated hepatocytes: Contribution of thiol oxidation and lipid peroxidation

“…Organic hydroperoxides can also interact with the cytochrome P-450 system that catalyzes their cleavage to the corresponding peroxy and alkoxy radicals (6) implicated in the initiation and propagation of lipid peroxidation. In fact, incubation of t-BH with liver microsomes causes extensive lipid peroxidation that can be prevented by the inclusion of cytochrome P-450 inhibitors in the medium (6).…”

The pathophysiological significance of lipid peroxidation in oxidative cell injury

“…These have been reviewed elsewhere (5,6,10) and include the measurement of conjugated dienes that are formed following H -abstraction and rearrangement (see upon reduction of lipid hydroperoxides (23). Other products which can be used for assay are derived from more complex processes and include thiobarbituric acid reactive substance (24,25), fluorescent pigments (26), ethane (27), pentane (281, chemiluminescence (29) and hydroxyalkenals (30). The time course for the formation of some of these products in hepatocytes incubated with 1 mM CCl, is illustrated in Figure 2.…”