ABSTRACT
CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione; CG-NQ), a beta-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 microM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on reactive oxygen species.
Subject(s)
Humans , Male , Apoptosis/drug effects , Hepatocytes/drug effects , Naphthoquinones/pharmacology , Naphthoquinones/toxicity , Apoptosis/physiology , Cells, Cultured , Chromatin/drug effects , Chromatin/pathology , Cell Surface Extensions/drug effects , Cell Surface Extensions/pathology , Cell Surface Extensions/ultrastructure , DNA Fragmentation/drug effects , DNA Fragmentation/physiology , Hepatocytes/metabolism , Hepatocytes/ultrastructure , Microscopy, Electron , Intracellular Membranes/drug effects , Intracellular Membranes/pathology , Intracellular Membranes/ultrastructure , Microvilli/drug effects , Microvilli/pathology , Microvilli/ultrastructure , Mitochondria/drug effects , Mitochondria/pathology , Mitochondria/ultrastructure , Hydrogen Peroxide/metabolism , Rats , Rats, Wistar , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/pathology , Endoplasmic Reticulum/ultrastructureABSTRACT
Our original aim was to determine whether dBcAMP-induced activation of cultured astrocytes affected the course of subsequent viral infection. After 2 h exposure of 2-day-old first subculture of mouse astrocytes to dBcAMP 1 mM, cell monolayers grown in glass coverslips of Leighton tubes were inoculated with 10(3) PFU of Theiler virus-GDVII strain (TMEV-GDVII). At 9 days post-infection (pi), viral infectivity persisted in supernatants from dBcAMP-treated cultures, but was no longer detectable in non-stimulated controls. The relatively spared astroglial monolayer at day 1 pi, hardly affected by progressive viral cytolytic effect, was chosen for immunolabeled cell count, whether by viral antigen or GFAP. To this end, 20 fields for each coverslip were digitalized at 250x final magnification. In dBcAMP treated cultures, viral antigen(+) cells were fewer and lower in percentage versus infected cultures lacking stimulation. As regards GFAP staining, stimulation or infection per se induced a greater number and percentage of labeled astrocytes. According to morphometric characterization, such increase was due to a greater number of process-bearing astrocytes. It may be concluded that, regardless of previous dBcAMP treatment, early TMEV-GDVII infection enhanced immunocytochemical and morphological differentiation in cultured astrocytes.