Cultured primary hepatocytes pretreated (protected) with the
iron chelator deferoxamine or the
antioxidant N,N'-diphenyl-p-phenylenediamine (
DPPD) were resistant to the toxicity of 5 microM
naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) during a 180-min exposure. Hepatocytes exposed to
naphthazarin without any protection were abruptly depleted of intracellular
reduced glutathione, and the level of cytosolic Ca2+ was rapidly increased. This was followed by lipid peroxidation, measured as accumulation of
malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNA) intra- and extracellularly; decrease in
ATP levels; destabilization of lysosomes; and finally cell death. The stability of the lysosomal membranes was evaluated by determining retention of the lysosomotropic weak
base acridine orange (AO).
Naphthazarin exposure caused leakage of
protons from the acidic compartment, as indicated by relocalization of AO to the cytosol. Protection of the cell cultures with
deferoxamine or
DPPD prevented destabilization of lysosomes and cell killing. It also reduced the loss of
ATP but did not prevent the depletion of
glutathione or the increase in Ca2+. In cells subjected to
naphthazarin exposure,
DPPD protection also completely inhibited lipid peroxidation, whereas
deferoxamine pretreatment only slightly reduced the intracellular accumulation of MDA and
4-HNA but completely prevented cell
rupture and the leakage of these lipid peroxidation products to the medium that took place in large amounts from unprotected cells exposed to
naphthazarin.
Deferoxamine is taken up by endocytosis and is thus transported to the acidic vacuolar apparatus, whereas the lipophilic
DPPD is rapidly distributed throughout the cells. Inhibiting endocytosis during
deferoxamine pretreatment, by incubating at +4 degrees C or by preexposure to a mixture of the endocytosis-inhibitors
cytochalasin B and
monensin, abolished the protective effect of
deferoxamine. The findings suggest that
naphthazarin-induced cell killing is not caused directly by either
thiol oxidation or an increase in cytosolic free Ca2+, but rather is preceded by lysosomal destabilization, which may be prevented either by inhibition of cellular peroxidation in general or by prevention of
iron-catalyzed oxidative reactions, and involves peroxidation of cellular membranes, energy depletion, and leakage of lysosomal content.
DPPD would protect against cell killing by preventing lipid peroxidation of cellular membranes in general, whereas
deferoxamine seems to allow a limited general cellular peroxidation but specifically prevents peroxidation and fragmentation of lysosomal membranes by chelating intralysosomal
iron and, consequently, leakage of destructive lysosomal contents with ensuing cell
rupture and death. Thus, a certain degree of cellular peroxidation does not appear to be lethal as long as lysosomal membranes are protected, placing lysosomes into a category of cellular loci minora resistentia.