Apoptogenic effect of the lipophilic o-naphthoquinone CG 10-248 on rat hepatocytes : light and electron microscopy studies

CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran5,6-dione; CG-NQ), a β-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 μM 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”. Abbreviations: CG-NQ, CG 10-248 o-naphthoquinone; PARP, poly(ADPribose)polymerase; DMFA, dimethylformamide; HRP, horseradish peroxidase. BIOCELL 2003, 27(2): 213-224 ISSN 0327 9545 PRINTED IN ARGENTINA

β-Lapachone cytotoxicity on tumor cells has been often defined as apoptosis (programmed cell death), prompted by the quinone or by its reaction products (Planchon et al., 1995(Planchon et al., , 1999;;Li et al., 1995;Chau et al., 1998;Wuerzberger et al., 1998;Manna et al., 1999;Huang and Pardee, 1999;Shiah et al., 1999;Li et al., 2000;Pink et al., 2000b;Don et al., 2001).Apoptosis production by β-lapachone is expressed by several cellular alterations such as On these grounds, β-lapachone has been proposed for clinical use (Pardee et al., 2002).However, o-naphthoquinone cytotoxicity on tumor cells might imply "undesirable effects" for the human host.Therefore, it seemed of interest to investigate the effect of a single lipophilic o-naphthoquinone on a suitable biological target such as rat hepatocytes.The observations here reported are the first part of a study on the action of CG 2b]pyran 5,6-dione; CG-NQ), another analogue of β-lapachone, on morphology (by light and electron microscopy) and hydrogen peroxide production by hepatocytes.CG-NQ was synthesized by Schaffner-Saba et al. (1984) and has been shown to exert biological activities like β-lapachone on other biological targets (Molina Portela et al., 1996;Stoppani et al., 2000).

Animals
Male Wistar rats (220-250 g) were used in the experiments.Animals were fed a Purina-like rat chow.Protein content of the diet was 23% and included all the essential aminoacids.A consistent light and dark cycle (12 hours) was maintained.

Incubation of hepatocytes
Hepatocyte suspensions were diluted with Hank's-HEPES medium to a concentration of about 3 x 10 6 cells/ ml.Duplicate samples were incubated in Erlenmeyer flasks, with CG-NQ or DMFA.Incubation was performed in a New Brunswick Gyratory Water Bath Shaker, model G76, at 37ºC and at a rate of 90 strokes/min, under carbogen atmosphere.CG-NQ or DMFA was added at zero-time, dissolving the former in DMFA.Controls received the same volume of solvent ("DMFA-samples"), whose concentration remained below 1% (v/v).Samples of incubation mixtures were taken for light or electron microscopy and for measuring H 2 0 2 production.

Electron and light microscopy
After incubation for 4 h, hepatocytes were centrifuged at 3,000 rpm.The pellet was fixed overnight at 4ºC with 1.5% (w/v) glutaraldehyde dissolved in 0.2 M cacodylic acid at pH 7.4-7.6.Pellets were then centrifuged at 3,000 rpm for 15 min and washed with 0.32 M sucrose in the same cacodylic acid buffer solution, in three 15-min washes.For refixation, the same buffer solution supplemented with 1.5% (w/v) osmium tetroxide was applied for two hours at 4ºC.After briefly washing with distilled water, pellets were resuspended for 2 h in 2% (w/v) uranyl acetate and immediately dehydrated for pre-embedding.After embedding, ultrathin sections were stained with 2% (w/v) uranyl acetate in water and afterwards with Reynolds solution.Microphotographs were obtained by means of a C10 Zeiss Electron Microscope using Kodak 4489 Films.For light microscopy, sections were then stained with 0.5% toluidine blue and 0.1% cresyl blue.Observations were performed with an Olympus BX50 microscope equipped with a COOL-SNAP-PRO digital camera, objective 40X.

Results
CG-NQ treatment of hepatocytes produced diverse alterations in cell structure, including the nuclear chromatin, mitochondrion, endoplasmic reticulum and plasma membrane.Such alterations were not equally distributed in all hepatocytes and, therefore, in order to provide a full picture of CG-NQ-induced cell damage, presentation of a number of dissimilar typical hepatocyte samples seems justified.These samples were obtained from similarly incubated hepatocytes (4 h incubation), with 100 µM CG-NQ and the corresponding solvent (DMFA) and are thus strictly comparable.The number of modified hepatocytes was determined throughout and its relative proportion (%) was expressed as a fraction of the total number of hepatocytes examined in each sample (at least 50 cells).
Structural alterations in treated hepatocytes, incubated with 100 µM CGNQ for 4 h, as compared with the corresponding control cells (light microscopy), are depicted in Figure 1.Most hepatocytes (58%) showed mitochondria displaced towards the cell nucleus, 32% presented plasma membrane blebbing and 10% exhib-ited necrosis.At variance with these results, control hepatocytes displayed preserved structure, allowance made for those presenting necrosis (5%).
Figure 2 depicts the preserved ultrastructure of control hepatocytes (electron microscopy), while the inset shows the amplified, preserved ultrastructure of mitochondria and of the endoplasmic reticulum (rough and smooth) in the same hepatocytes.
Figure 3 shows the ultrastructure of (a) CG-NQ treated hepatocyte sample.In the main figure, the more significant alterations were (a) mitochondrial concentration around the nucleus; (b) endoplasmic reticulum (rough and smooth) moved towards the plasma membrane; (c) microvilli scarcity; and (d) incipient blebbing of the plasma membrane.Figure 4 presents a more remarkable picture of hepatocyte damage by CG-NQ.Thus, Figure 4A, depicts a single binucleated hepatocyte with condensed chromatin, bound to the nuclear membrane; most mitochondria were swollen and the plasma membrane showed abundant blebs.In the same picture appear, however, two preserved hepatocytes.Figure 4B includes hepatocytes with their nuclei displaced towards the plasma membrane, abundant aggregated chromatin, swollen mitochondria, vacuolized cytosol and blebs.
Figures 5A-C also illustrate extensive structural damage in CG-NQ-treated hepatocytes.There was cell disintegration with nuclear fragmentation, and large membrane blebs, apoptotic bodies, consisting in pieces of cytosol wrapped in the plasma membrane and containing organelles (mitochondria and endoplasmic reticulum (rough and smooth), as well as swollen mitochondria with condensed matrix.Figure 5D shows the disruption of outer mitochondrial membranes, fragments of which remain preserved.The mitochondrial inner membrane is expanded but preserved, while the matrix appears swollen.Figure E again shows nuclear fragmentation and mitochondrial matrix condensation, that appears dark and small.In other hepatocyte samples, abundant mitochondria were either swollen or condensed, as in Figure 5E.
Hydrogen peroxide production resulting from superoxide dismutation immediately followed CG-NQ addition to hepatocytes and its concentration, increased progressively to reach an effective concentration after 60 min of incubation, which implies possible cytotoxic effects in the subsequent incubation period.Hydrogen   peroxide production was confirmed by adding azide to the hepatocyte suspension.Azide is a specific inhibitor of catalase, the enzyme that decomposes hydrogen peroxide in hepatocytes, thus exerting an antioxidant action.In close agreement with its specific function, azide increased hydrogen peroxide production by hepatocytes, its effect being remarkable during the first 15 or 30 min of incubation, as compared with control values (p ≤ 0.005 or 0.01) for 15 or 30 min incubation time, respectively.

Discussion
The observations described here indicate that, after 4 h incubation of hepatocytes with CG-NQ, significant structural changes occurred.These effects were quite compatible with the apoptotic action of a similar o-naphthoquinone, β-lapachone, in tumor cells, as described in the literature.The structural changes produced by CG-NQ in hepatocytes were not homogenous (Figs.1-5), varying in extent and location.Since hepatocyte samples were all taken from the same incubation mixture at the same incubation time, which was adequate for apoptosis in tumor cells, it must be concluded that CG-NQ action was affected by metabolic peculiarities inherent to cell physiology.
Among the hepatocyte alterations caused by CG-NQ, those of the nucleus seemed particularly crucial to qualify quinone-induced damage as apoptosis.In fact, chromatin alterations are characteristic of β-lapachone induced apoptosis in tumor cells (Planchon et al., 1995(Planchon et al., , 1999;;Li et al., 1995;Wuerzberger et al., 1998;Chau et al., 1998;Shiah et al., 1999;Pink et al., 2000b).These alterations correlate with DNA repair inhibition by βlapachone both in tumor (Boothman et al., 1989) and Trypanosoma cruzi cells (Goijman and Stoppani, 1985).The effects of β-lapachone include DNA depletion, con-densation, laddering and chromatin fragmentation, as described in tumor cells.Similar effects were produced by CG-NQ in hepatocytes (Figs.3-5).These processes may well represent modification of enzymes responsible for DNA repair such as topoisomerases (Boothman et al., 1989;Degrassi et al., 1993;Li et al., 1995;Frydman et al., 1997;Pink et al., 2000a;Shiah et al., 1999) and PARP (Chau et al., 1998;Vanni et al., 1998;Pink et al., 2000a;Don et al., 2001;Fernández Villamil et al., 2001).Taking into account the foregoing, CG-NQ effect on hepatocyte DNA may be explained as a result of (a) direct enzyme inhibition by CG-NQ (e.g.topoisomerases), or (b) by the action of redox activated CG-NQ products including reactive oxygen species (ROS).These reaction products may activate a nuclear signal (e.g.DNA damage) (Walles, 1992) which triggers down-stream apoptosis (Pink et al., 2000b).Damaged cells would then become sensitive to topoisomerase inhibition by the quinone.In addition to these effects, the quinones may inactivate PARP (Pink et al., 2000a;Li et al., 2000;Fernández Villamil et al., 2001) which is involved in DNA repair.The mechanism of PARP inactivation may result from the activation of caspases by the quinone or its reactive derivatives (Planchon et al., 1999;Li et al., 2000) or by a direct action of the quinone (Fernández Villamil et al., 2001).
In close agreement with previous observations using isolated rat liver mitochondria (Lopez et al., 2002), CG-NQ produced significant mitochondrial alterations, including disruption or disappearance of the outer membrane and matrix swelling (Figs.4B, 5D).Mitochondrial alterations were closely related to apoptosis (Zamzami et al., 1995;Vander Heiden et al., 1997).Membrane disruption may depend on inner membrane depolarization by the quinone (Hacker, 1999).Consistent effects were observed with isolated mitochondria incubated with CG-NQ using rhodamine 123 as elec-FIGURE 5. CG-NQ-treated hepatocytes ultrastructure (electron microscopy).A: hepatocytes showing variable degrees of disintegration, with large blebs separating from the cell membrane and producing apoptotic bodies (black arrow head) 3,150X.Bar: 1 cm / 2.11 µm.B: Apoptotic body showing swollen and condensed mitochondria and endoplasmic reticulum (rough and smooth) surrounded by plasma membrane 12,500X.Bar: 1 cm ≡ 0.5 µm.C: showing nucleus fragmented at different sites (black arrow head); abundant swollen and condensed mitochondria; plasma membrane with blebs.3,150X.Bar: 1 cm ≡ 2.1 µm.D: same hepatocyte sample showing partial disruption of mitochondrial outer membrane; preserved inner membrane (one black arrow head) with swollen matrix.Two white arrow heads indicate partially preserved outer mitochondrial membrane.Li, lipid drop.12,500X.Bar: 1 cm ≡ 0.3 µm.E: fragmented nucleus (white arrow head); MIT-CON mitochondria with preserved membranes and condensed matrix; LI, lysosomes.12,500X.Bar: 1 cm ≡ 0.5 µm.Other conditions as in Materials and Methods.trochemical potential indicator (data not shown).In addition to the alterations of mitochondria structure reported here, CG-NQ could also affect mitochondrial functions, specially ATP production, F 0 F 1 -ATPase activity and ion translocation (Lopez et al., 2002).CG-NQ dependent mitochondrial swelling would contribute to apoptosis (Vander Heiden et al., 1997) since it would lead to opening of the mitochondrial permeability pore and the release of cytochrome c, which in turn would contribute to apoptogenesis (Kroemer, 1999;Zamzami et al., 1996).Another interesting action of CG-NQ was the appearance of vacuoles in hepatocyte cytoplasm (Fig. 4B) and the modification of the endoplasmic reticulum, specially its displacement towards the plasma membrane.Vacuole formation is typical of apoptotic cells but the mechanism of vacuolization is not clear (Fearnhead et al., 1995;Henics and Wheatley, 1999).
A peculiar feature of CG-NQ action on hepatocytes was the modification of the plasma membrane (Fig. 1-5).Blebs are protrusions of the plasma membrane that may contain other cell structures such as mitochondria and peroxysomes, constituting the apoptotic body.Another alteration of the plasma membrane was the partial or total disappearance of microvilli.As regards blebbing, studies with cytochalasin D indicate that microfilament structure must be re-ordered to produce blebs and therefore blebbing implies a modification of the cytoskeleton (Thör et al., 1988;Mirabelli et al., 1988;Kondo et al., 1997;Huot et al., 1998;Chen et al., 2000).Hepatocyte modification by naphthoquinones, such as menadione, depends on cytoskeleton modification (active polymerization inhibition) (Thör et al., 1988).Blebbing in hepatocytes is associated to GSH depletion, an effect that occurs after incubation of hepatocytes with lipophilic o-naphthoquinones (Dubin et al., 2002).Loss of microvilli is related to caspase activation, an effect frequently observed after treatment of cells with "reactive oxygen species" generators.
To sum, own results indicate that CG-NQ, a lipophilic o-naphthoquinone, at 100 µM concentration, is capable of inducing apoptosis in rat hepatocytes.These effects raise the question whether similar changes would occur in organisms treated with this and similar o-naphthoquinones such as β-lapachone, which are potential cytostatic agents (Pardee et al., 2002).FIGURE 6.Effect of CG-NQ on H 2 0 2 production by hepatocytes.The reaction medium contained 3 x 10 6 /ml cells.Sodium azide (AZ), control without sodium azide (C).Values are means ± S.E.M. of three duplicate independent measurements.Other conditions as in Materials and Methods.
(a) cell cycle inhibition; (b) nuclear fragmentation and displacement; (c) DNA laddering; (d) DNA depletion; (e) activation or inhibition of topoisomerases I and II; (f) PARP cleavage and inhibition; (g) production of reactive oxygen species (ROS); and (h) modification of cell viability.
Figure A provides an amplified view of the mitochondrion with preserved membranes and a peroxysome, while Figure B shows the endoplasmic reticulum with expanded cisternae and reduced number of ribosomes.