Enhanced Mitochondrial Gene Transcript, ATP, Bcl-2 Protein Levels, and Altered Glutathione Distribution in Ethinyl Estradiol-Treated Cultured Female Rat Hepatocytes

Jinqiang Chen*, Michael Delannoy{dagger}, Shelly Odwin*, Ping He*, Michael A. Trush* and James D. Yager*,1

* Department of Environmental Health Sciences, Division of Toxicological Sciences, The Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205–2179; and {dagger} Electron and Confocal Microscopy Laboratory, Department of Physiology, The Johns Hopkins School of Medicine, 625 North Wolfe Street, Baltimore, Maryland 21205

Received April 11, 2003; accepted June 23, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ethinyl estradiol (EE) is a strong promoter and weak hepatocarcinogen in rats. Previously, we demonstrated that EE enhanced the transcript levels of nuclear genome- and mitochondrial genome-encoded genes and respiratory chain activity in female rat liver, and also inhibited transforming growth factor beta (TGFß)-induced apoptosis in cultured liver slices and hepatocytes from female rats. In this study, using cultured female rat hepatocytes, we observed that EE, within 24 h, increased the transcript levels of the mitochondrial genome-encoded genes cytochrome oxidase subunits I, II, and III. This effect was accompanied by increased mitochondrial respiratory chain activity, as reflected by increased mitochondrial superoxide generation, and detected by lucigenin-derived chemiluminescence and cellular ATP levels. EE also enhanced the levels of Bcl-2 protein. Biochemical analyses indicated that EE significantly increased both the levels of glutathione (reduced [GSH] and oxidized [GSSG] forms) per mg protein in mitochondria and nuclei, while the percentage of total glutathione in the oxidized form was not affected. This finding was supported by confocal microscopy. These effects caused by EE may contribute, at least in part, to the EE-mediated inhibition of hepatic apoptosis.

Key Words: ethinyl estradiol; rat hepatocytes; glutathione; mitochondria; apoptosis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ethinyl estradiol (EE), a synthetic estrogen used in oral contraceptives, is a strong tumor promoter and a weak complete carcinogen in rat liver (Liehr, 1997Go). Like several other promoters such as phenobarbital (PB) (Abanobi et al., 1982Go; Barbason et al., 1983Go; Jirtle and Meyer, 1991Go) and some peroxisome proliferators (PPs) (Styles et al., 1991Go; Tanaka et al., 1992Go), EE induces transient growth stimulation (Mayol et al., 1992Go, 1991Go; Yager et al., 1986Go), followed by suppression of basal and induced hepatic growth (mitosuppression) (Yager et al., 1994Go). A common change associated with mitosuppression caused by these promoters is the increased levels of transforming growth factor-beta (TGFß) and mannose-6 phosphate/insulin-like growth factor receptor, to which the latent TGFß binds (Chen et al., 1996Go; Jirtle et al., 1994Go; Rumsby et al., 1994Go). In various liver-derived cells and in cultured hepatocytes, TGFß both inhibits cell growth (Dixon et al., 1999Go; Kimura and Ogihara, 1999Go; Meyer et al., 1990Go; Russell et al., 1988Go) and induces cell death via apoptosis (Bursch et al., 1993Go; Oberhammer et al., 1991Go, 1992Go; Schuster and Krieglstein, 2002Go). These effects of TGFß have been shown to be separable (Brown et al., 1998Go).

It has been shown that apoptosis plays a pivotal role in the modulation of rodent hepatocarinogenesis (Bursch et al., 1984Go; Grasl-Kraupp et al., 1997Go; Schulte-Hermann et al., 1997aGo,bGo, 1998Go). PB and PPs (nafenopin) suppressed both spontaneous and TGFß-induced apoptosis in cultured hepatocytes and livers of rats and mice (Bayly et al., 1994Go; Christensen et al., 1998Go, 1999Go; Perrone et al., 1998Go; Strange and Roberts, 1996Go). Similarly, EE inhibited TGFß-induced apoptosis in cultured, precision-cut liver slices and hepatocytes (Chen et al., 2000Go). However, the mechanism(s) underlying inhibition of hepatic apoptosis by PB, nafenopin, and EE is not clear.

Mitochondria have been shown to have a major role in the regulation of apoptosis (Green and Reed, 1998Go). Previously, we observed that the transcript levels of several nuclear and mitochondrial DNA-encoded mitochondrial respiratory-chain proteins were enhanced in livers of female rats treated in vivo with EE (Chen et al., 1998Go, 1996Go). The increase in transcript levels of mitochondrial genes was followed by increased respiratory-chain activity. Similar effects of EE and estradiol (E2) were observed in human HepG2 cells and shown to be inhibited by a specific anti-estrogen, demonstrating that they are mediated through an estrogen receptor-signaling pathway (Chen et al., 1998Go, 1996Go). The aim of the present study was to provide further characterization of the biochemical and cellular effects of EE on cultured female hepatocytes including the determination of respiratory chain activity, ATP levels, expression of Bcl-2/bax proteins, and the levels and intracellular distribution of glutathione.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals, reagents, and antibodies.
All chemicals used were analytical grade. EE was obtained from Steraloids, Inc. (Newport, RI). All reagents and medium for culture of primary hepatocytes from female rats were obtained from Gibco BRL Life Technologies, Inc. (Gaithersburg, MD) or from Collaborative Research (Waltham, MA). Reduced glutathione (GSH), oxidized glutathione (GSSG), and O-phthalaldehyde (OPT) were purchased from Sigma (St. Louis, MO). Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and Western- and Northern-blot analyses were obtained from BRL Life Technologies and from Bio-Rad (Richmond, CA). Polyclonal antibodies for Bcl-2 and Bax were purchased from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA).

Preparation and culture of female rat hepatocytes of female rats.
Fisher 344 female rats (200–300 g) were used according to the protocols approved by the Animal Care and Use Committee, Johns Hopkins School of Hygiene and Public Health. Hepatocytes were prepared by collagenase perfusion and cultured on collagen-coated, 100-mm Falcon dishes in Chee’s medium as described (Zurlo and Arterburn, 1996Go). Cell viability was determined using trypan blue exclusion and was typically greater than 95%. Each experiment used hepatocytes prepared from a separate rat, and the number of separate experiments and replicate cultures for each set of data is indicated.

Experimental design.
The isolated hepatocytes were inoculated into culture at approximately 8–10 x 106 cells per 100-cm culture dish. The medium was changed after 4 h and again approximately 18 h later, at which point the treatments indicated were begun and continued for 24 h; then the hepatocytes were harvested and assayed by procedures appropriate for the different end points measured. EE was dissolved in ethanol. Control cultures were treated with ethanol, and the final concentration of ethanol in all cultures was 0.05%.

Isolation of total RNA and Northern-blot analysis.
Total RNA was isolated from the hepatocytes using RNAzolTM obtained from TEL-TEST, Inc. (Friendswood, TX) according to the manufacture’s instructions. Preparation of cDNA probes for cytochrome c oxidase subunits I, II, and III (CO I, CO II, and CO III), via polymerase chain reaction (PCR) and Northern-blot analysis, were performed as described previously (Chen et al., 1998Go).

Assay of lucigenin-derived chemiluminescence (LDCL).
Following treatment, LDCL was used to assess intramitochondrial superoxide generation as an indication of mitochondrial respiratory chain activity, using a Berthold LB9505 luminometer as described previously (Chen et al., 1999Go; Li et al., 1999aGo,bGo). Briefly, the cells were removed from the culture dishes, resuspended in 2.5 ml air-saturated phosphate-buffered saline (PBS) buffer (pH 7.4) (approximately 3 x 106 hepatocytes), and assayed intact. The LDCL determination was initiated by adding lucigenin to a final concentration of 10 µM and followed continuously for 30 min in the luminometer. The chemiluminescence detected is expressed as counts per min (cpm). The LDCL data are shown both as the actual chemiluminescent curves generated over the 30-min incubation periods, and in a numerical form based on the integration of the areas under the curve. While differences in actual chemiluminescence cpm do occur among separate experiments, differences among treatments are highly reproducible (Chen et al., 1999Go).

Assay of intracellular ATP levels.
Intracellular ATP levels were determined using a luciferase ATP assay kit (Sigma Chemical Co., St. Louis, MO) according to the manufacture’s instructions. The amount of ATP-driven light produced was measured at 37°C for 2 min using a Berthold LB9505 luminometer. The amount of total cellular protein was determined with protein assay reagents (Bio-Rad) using bovine serum albumin (BSA) as a standard.

Western analysis for Bcl-2/Bax and actin.
Preparation of total protein extracts from hepatocytes and Western analysis for Bcl-2 and Bax were performed as described previously (Ha et al., 1997Go). Briefly, hepatocytes were washed twice with cold 1x PBS, pH 7.4, and harvested by scraping in 1.5 ml 1x PBS and centrifuging at 750 x g for 5 min. The pellets were resuspended in 0.4 ml buffer containing 10 mM Tris (pH 7.5), 1% of nonidet P-40, 10 mM EDTA, plus freshly added 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulflonyl fluoride (PMSF), aprotinin (5 µg/ml), and leupeptin (5 µg/ml), and homogenized with 100 strokes of a l-ml Dounce homogenizer, using a tight pestle. The homogenates were centrifuged at 750 x g for 10 min at 4°C in a microcentrifuge. The supernatant was transferred to a 1.5-ml microcentrifuge tube and stored at -80°C. The amount of protein in the supernatants was determined as described above. Approximately 50 µg of supernatant protein was mixed with 10 µl of 2x SDS loading buffer (100 mM Tris, pH 6.8, 200 mM DTT, 4% SDS, and 0.2% bromophenol blue dye) and boiled for 3 min. The proteins were electrophoretically separated on a 7% SDS–PAGE gel using a mini-gel apparatus (Bio-Rad) at 100 V for 1 h. The gels were electroblotted to nitrocellulose membranes at 25 V overnight at 4°C. Membranes were first probed with goat polyclonal anti-Bcl-2 or anti-Bax and then with anti-mouse IgG conjugated with horseradish peroxidase. Bcl-2 and Bax protein bands were visualized by ECLTM (Amersham/Pharmacia Biotech, Little Chalfont, Buckinghamshire, U.K.).

Isolation of nuclear, mitochondrial, and cytosol fractions and assay of glutathione levels.
Hepatocytes in two 100-mm dishes (about 10 x 106) were harvested and combined. After washing twice with cold 1x PBS, approximately 2 x 106 cells were homogenized with lysis buffer containing 100 mM sodium phosphate (Na2HPO4/NaH2PO4,), pH 7.5, 5 mM EDTA, plus freshly added NP-40 (1%). These crude homogenates were used for the determination of total amount of glutathione, GSH, plus GSSG. The remaining 8 x 106 cells were divided into two equal samples. One was used for isolation of the nuclear fraction, the other for isolation of the mitochondrial and cytosol fractions. The isolation of nuclei was performed as described (Voehringer et al., 1998Go) with some modifications. Briefly, about 4 x 106 cells were homogenized with 50 strokes of a 1-ml Dounce homogenizer in 0.5 ml of silicon oil (dimethyl-polysiloxane-5X, Sigma, St. Louis, MO). The homogenates were layered on the top of 0.5 ml of silicon oil and centrifuged at 1000 rpm in a microcentrifuge for 2 min. The pellets were resuspended in 0.5 ml silicon oil, layered on the top of 0.5 ml of silicon oil, and centrifuged at 1000 rpm for 2 min. Microscopic examination revealed the presence of nuclei. The pellets were dissolved with lysis buffer.

The isolation of the mitochondrial and cytosolic fractions of hepatocytes was performed as described (Chen et al., 1999Go; Pedersen et al., 1978Go). Approximately 4 x 106 cells were homogenized with 100 strokes of a 1-ml Dounce homogenizer in H-medium (0.7 M sucrose, 0.21 M D-mannitol, 0.002 M HEPES, pH 7.4, 0.05% [w/v] BSA). The homogenates were centrifuged twice at 750 x g in a microcentrifuge for 15 min to remove the nuclei. The supernatant was centrifuged at 7500 x g in a microcentrifuge for 15 min to pellet mitochondria. The supernatant was saved and used as the cytosolic fraction. The pellets containing mitochondria were twice resuspended in H-medium and centrifuged at 10,000 x g for 15 min. The pellets were then gently washed twice with H-medium without resuspension and then dissolved with the lysis buffer mentioned above.

The amounts of GSH and GSSG in the nuclear, cytosolic, and mitochondrial fractions were determined as described (Hissin and Hilf, 1976Go). For GSH, 100 µl of sample solution was mixed with 0.8 ml sodium phosphate-EDTA buffer (pH 8.0) and 100 µl of OPT (1 mg/ml), and incubated at room temperature for 15 min. For GSSG, 100 µl of sample was mixed with 0.8 ml 0.1 N NaOH and 100 µl OPT (1 mg/ml), and incubated at room temperature for 15 min. The amount of fluorescent light generated at 350 nm (excitation) and 420 nm (emission) was measured using a CytoFlour Multi-well plate reader (PerSeptive Biosytems, Framingham, MA). The amount of fluorescent light for known amounts of GSH and GSSG was determined simultaneously and used in standard curves. The amount of total glutathione represents the sum of the amount of GSH plus 2x the amount of GSSG. The amount of protein in each fraction was determined as described above. The ratio of total to oxidized glutathione was determined by dividing the total glutathione by 2x the amount of GSSG.

Confocal visualization of mitochondrial GSH.
Mitochondria and GSH were stained with MitoTracker red CM-H2X ROS (reactive oxygen species) and CellTracker green 5-chloromethylfluorescein diacetate (CMFDA) (Molecular Probes, Eugene, OR), respectively, and visualized using a Noran Oz Confocal Laser Scanning Microscope System as described (Voehringer et al., 1998Go). Briefly, the hepatocytes were cultured in collagen-coated 8-well Lab-Tek chambler slides (Nalge Nune International, Naperville, IL) for 15 h and then treated with EE (3 µM) for 24 h. The cells were loaded with 100 nM MitoTracker red CM-H2X ROS and 100 nM CMFDA following the manufacture’s directions. The cells were fixed with 2% paraformaldehyde and mounted with Slowfad (Molecular Probes). The cell samples were visualized using the Noran Oz Confocal laser scanning microscope system, utilizing Intervision software (ver. 6.3) on a silicon graphics INDY R500 platform. A krypton-argon laser (Omnichrome, series 43), exciting at 488 and 568 nm wavelengths, was used to obtain optical sections. Narrow-band emission filters (525 nm and 605 nm) were utilized to eliminate channel cross talk and a 10 µm fixed slit was used to obtain 0.5 µm z-plane sections (as determined by full width half maximum intensity values). The cells were imaged with a 100x oil immersion planar apochromatic objective lens (numerical aperture 1.35) through an Olympus IX-50 inverted microscope.

Statistical analysis of data.
The results presented are from individual experiments using duplicate or triplicate cultures of hepatocytes obtained from an individual rat. The results are representative of at least 3 to 4 independent experiments using hepatocytes prepared from different individual rats. Where indicated, statistical analysis was done using a one-way ANOVA and the differences considered significant when p < 0.05 using a Student-Newman-Keuls method for pair-wide multiple comparisons.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of EE on Transcript Levels of Several Mitochondrial Genes
Cultures of female rat hepatocytes were treated with EE at the indicated concentrations for 24 h, and total RNA was isolated and analyzed by Northern blot. Figure 1AGo shows photographs of autoradiographs and an ethidium bromide-stained gel. The autoradiographs were scanned and the band intensities normalized for 18S + 28S rRNA contents. Figure 1BGo shows the data expressed as fold increase over control. These data show that treatment of rat hepatocytes with EE for 24 h caused increased mRNA levels of CO I, CO II, and CO III. The maximum increase was already attained at 1 µM EE; higher concentrations did not elicit a stronger response.



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FIG. 1. Effects of ethinyl estradiol (EE) on mRNA levels of several mitochondrial genes in cultured hepatocytes. Hepatocytes were prepared from female rats and cultured as described in Materials and Methods. EE, dissolved in ethanol, was added to the cultures at the concentrations indicated. Controls received only ethanol. After 24 h, total RNA was isolated, Northern blotted, and probed with 32P-cDNA probes for the indicated genes. (A) Photographs of autoradiographs of the Northern-blot analyses of transcripts for subunits cytochrome oxidase (CO) I, CO II, and CO III and of the ethidium bromide-stained gels; (B) the mean ratio ± standard deviation of the densitometric scans for autoradiography of transcripts for subunits CO I, CO II, and CO III, corrected for inter-lane differences in mRNA contents represented by the amount of 28S + 18S rRNA and expressed as fold induction relative to control. Each bar represents the mean ± SD (n = 3). Where no error bracket is seen, the SD was too small to be seen on the chart. *Significantly greater than control, p < 0.05.

 
Effects of EE on Mitochondrial Superoxide and ATP Levels
The effects of EE treatment on hepatocyte mitochondrial respiratory chain activity, as reflected by detecting superoxide levels as LDCL and ATP levels, are shown in Figure 2AGo. These data are from one representative experiment; similar results were observed in 2 separate experiments each using hepatocytes from a different rat. Each curve represents the cpm from LDCL, detected every 2.5 min over a 30-min period from a single culture treated with EE for 24 h, as indicated. The data show that EE caused increased LDCL in a concentration-dependent manner. In a separate experiment, LDCL was determined in triplicate EE-treated (3 µM) and control cultures. Total counts due to LDCL were integrated over 30 min. The results, Figure 2BGo, show that EE induced a significant 2-fold increase in LDCL (p < 0.05). In another experiment, we also observed that LDCL levels were increased as early as 12 h after EE treatment (data not shown). Consistent with the increased mitochondrial respiratory chain activity, ATP levels were also significantly increased (p < 0.05) 1.5- to 1.8-fold by EE treatment (Fig. 2CGo). As observed for induction of CO I, II, and III mRNA levels (Fig. 1BGo), the maximum increase was already attained at 1 µM EE; higher concentrations did not elicit a stronger response. Together, these results demonstrate that the increase in transcript levels of mitochondrial genes (Fig. 1Go) is accompanied by an increase in mitochondrial respiratory chain activity and cellular ATP levels.



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FIG. 2. Effects of EE on mitochondrial generation of superoxide and ATP: (A) Effects of various EE concentrations on lucigenin-derived chemiluminescence (LDCL). Each curve represents the LDCL from a single culture treated as indicated. (B) Effects of EE (3 µM) on LDCL. Each bar is the mean ± SD (n = 3) of values representing the integration of the areas under curves expressed as counts per min (cpm). (C) Effects of EE treatment on ATP levels. Each bar represents the mean ± SD (n = 3). *Significantly greater than control, p < 0.05.

 
Effects of EE on Protein Levels of Bcl-2/Bax
Bcl-2 has been shown to block cytochrome c release in association with inhibition of apoptosis (Kluck et al., 1997Go; Yang et al., 1997Go). Figure 3AGo shows a photograph of the Western blot for Bcl-2 (upper), in control and in EE-treated hepatocytes, and for an unknown major protein (lower) that was detected by Ponceau-S dye staining of the same nitrocellulose membrane after electroblotting that was unaffected by treatment. Figure 3BGo shows the densitometric data derived from scans of the Bcl-2 band, normalized for the levels of the unknown protein band and expressed as fold increase over control. Bcl-2 protein levels were increased (compare lanes 1 and 2 to lanes 3–10, Fig. 3AGo) 2- to 2.5-fold (Fig. 3BGo) over those of control. Similar results were observed in a second, separate experiment using hepatocytes from a different rat, and EE did not affect the levels of Bax protein (data not shown).



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FIG. 3. Effects of EE on Bcl-2 protein levels: (A) Western blot and (B) fold increase in Bcl-2 protein caused by EE, relative to controls. Each bar represents the mean ± SD (n = 2 for control and 1 µM EE, n = 3 for 3 and 10 µM EE) of the densitometric scan of the gel.

 
Effects of EE on Glutathione Levels
Over-expression of Bcl-2 in HeLa cells has been associated with increased levels of glutathione in nuclei and mitochondria and with inhibition of apoptosis (Voehringer et al., 1998Go). We examined the effects of 24-h EE treatment on the levels of GSH and GSSG in homogenates, cytosols, mitochondria, and nuclei. Similar results were obtained in a second experiment with hepatocytes from another female rat. In controls, the distribution of protein among the fractions was cytosol, 52%, nuclear, 10%, and mitochondrial, 1.3% (data not shown). Total glutathione represents the sum of the amount of GSH plus 2x GSSG, which were determined separately as described in Materials and Methods. For the nuclear fraction in the 3-µM EE-treatment group, no statistically significant changes in the protein content among the fractions were observed as a result of EE treatment. No significant changes in GSH or GSSG were detected in the homogenate or cytosolic fractions. In the nuclear fraction, small but significant increases were observed in total glutathione at 10 µM EE (1.8-fold), in GSH at (1.5-fold) and in GSSG (1.3-fold), both at 3 µM EE. Similarly, in the mitochondria, small but significant 1.4- to 2.0-fold increases in total glutathione, GSH, and GSSG were seen at 3 and 10 µM EE. The percent glutathione in the oxidized form was calculated. In the homogenate and cytosolic fractions, 50 to 60% of glutathione was oxidized. The nuclear fraction contained somewhat less oxidized glutathione, whereas the amount present in the mitochondrial fraction was lower. No correlation of the percent glutathione in the oxidized form with EE concentration was observed.

Visualization of Glutathione in Mitochondria Using Confocal Microscopy
EE (3 µM)-treated and control hepatocytes were double-stained with CMFDA for GSH and Mitotracker CMX ROS for mitochondria and then visualized under fluorescence confocal laser microscopy, as described in Materials and Methods. The same experiment was performed four times. Each time, 3 to 4 cells were randomly chosen in each group for analysis and similar results were obtained. Figure 4Go shows representative photographs of untreated (upper) and EE-treated (lower) hepatocytes. Figures 4A and 4EGo showed phase-contrast photographs of the hepatocytes; 4B and 4F showed the red staining for mitochondria with Mitotracker red CMX ROS (red channel); 4C and 4G showed the green staining with CMFA for GSH (green channel); and 4D and 4H showed the yellow staining representing colocalization of GSH in mitochondria when overlays were created from the red and green channels. As shown in these photomicrographs, the GSH staining in mitochondria of EE-treated hepatocytes is more intense when compared to that in mitochondria of untreated control hepatocytes. These photomicrographs also indicate that the GSH staining (green) in the nuclei of EE-treated hepatocytes appears to be more intense than that in nuclei of untreated hepatocytes. These observations are consistent with the biochemical results presented in Figure 4Go, which show increased GSH levels in both mitochondria and nuclei of EE-treated hepatocytes.



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FIG. 4. Confocal visualization of glutathione (GSH) in mitochondria and nucleus: upper panel, control hepatocytes; lower panel, EE-treated hepatocytes. A and E are the phase-contrast photomicrographs, B and F show the red staining for mitochondria, C and G show the green staining for GSH, and D and H show the colocalization of mitochondrial and GSH staining. The arrows indicate the nuclei.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As described in the introduction, EE is a strong promoter of hepatocarcinogenesis. The study described here is an aspect of our goal to delineate the molecular, biochemical, and cellular effects caused by EE on hepatocytes that may contribute to its activity as a strong promoter of hepatocarcinogenesis. Previously, we reported that the transcript levels of several mtDNA-encoded mitochondrial genes and respiratory chain activity were enhanced in the livers of EE-treated female rats and in HepG2 cells treated with EE or estradiol, and that these effects were blocked by the antiestrogen ICI182708 (Chen et al., 1998Go, 1999Go, 1996Go). We also found that EE inhibited TGFß-induced apoptosis in cultured, precision-cut liver slices and hepatocytes of female rats (Chen et al., 2000Go).

In the present study, we have investigated several molecular and biochemical alterations caused by EE treatment of cultured female hepatocytes. First, we observed that, within 24 h, EE treatment significantly enhanced the transcript levels of several mtDNA-encoded mitochondrial genes coding for components of the respiratory chain; subunits CO I, CO II, and CO III; respiratory chain activity, as reflected by increased superoxide production detected by LDCL; and cellular ATP levels. These effects were caused by EE within the time frame when EE was previously seen to inhibit TGFß–induced apoptosis (Chen et al., 2000Go). It is unclear whether or not these effects are causally related to EE-mediated inhibition of apoptosis. However, mitochondria play a key role in the regulation of apoptosis by acting as a focal point for a number of signaling pathways that activate the apoptotic process (Green and Reed, 1998Go). Decreased mitochondrial respiratory chain activity, loss of mitochondrial membrane potential, and release of mitochondrial cytochrome c have been associated with apoptotic activation (Green and Reed, 1998Go). E2 has been reported to stabilize mitochondrial function and protect neural cells against the pro-apoptotic action of mutant presenilin-1 (Mattson et al., 1997Go). Thus, the enhanced mitochondrial function in EE-treated hepatocytes may contribute to its inhibition of apoptosis (Chen et al., 2000Go).

Second, we observed that EE increased Bcl-2 protein levels. This is consistent with the reports that E2 increased mRNA levels of Bcl-2 in human MCF-7 cells (Dong et al., 1999Go; Wang and Phang 1995Go) and neurons of adult rat brain (Garcia-Segura et al., 1998Go). A regulatory element within the bcl-2 gene promoter that binds to Sp1 protein and is required for estrogen responsiveness in MCF-7 cells has been identified (Dong et al., 1999Go). It has been reported that increased Bcl-2 mRNA caused by pretreatment of MCF-7 cells with E2 was accompanied by inhibition of apoptosis induced by subsequent exposure to tamoxifen (Wang and Phang, 1995Go). Similarly, overexpression of Bcl-2 was found to inhibit TGFß-induced apoptosis in several human hepatoma cell lines (Huang et al., 1998Go). Furthermore, Bcl-2 and Bcl-XL have also been shown to be involved in the inhibition by PB and PPs of hepatic apoptosis in both cultured hepatocytes and livers of mice (Christensen et al., 1998Go). It has been found that the majority of altered hepatic foci and adenomas induced by chronic treatment with PB and PPs exhibited increased Bcl-2 protein levels compared with surrounding normal hepatocytes (Christensen et al., 1999Go). Inhibition of cytochrome c release by Bcl-2 has been proposed as playing a role in the inhibition of apoptosis (Kluck et al., 1997Go; Yang et al., 1997Go). We have shown that TGFß–induced cytochrome c release is inhibited by EE (Chen et al., 2000Go), and here we have observed that this effect is associated with increased Bcl-2 protein levels. Since cytochrome c release is an early event in the initiation of apoptosis, its inhibition by Bcl-2 could also be one of the mechanistic aspects for EE-mediated inhibition of apoptosis.

Third, we observed that EE enhanced the GSH and GSSG levels in both mitochondria and nuclei. This could be due to the effects of EE on any and/or a combination of the following factors affecting glutathione levels: (1) increased de novo biosynthesis; (2) enhanced transport; and (3) decreased efflux (Fernandez-Checa et al., 1997Go; Lu, 1999Go; Smith et al., 1996Go). Though it is not clear to what extent each factor contributes, the increased mitochondrial respiratory chain activity and ATP levels caused by EE may contribute to glutathione biosynthesis and distribution, since both processes are ATP-dependent (Fernandez-Checa et al., 1997Go; Lu, 1999Go; Smith et al., 1996Go). Bcl-2 is located in mitochondrial and nuclear membranes (Hockenbery et al., 1990Go) and is capable of forming channels in membranes (Schendel et al., 1997Go). Thus, it may be involved in glutathione transport across these membranes. It is possible that the concomitant increased levels of Bcl-2 protein and ATP induced by EE could accelerate the transport of glutathione into mitochondria and nuclei. This is supported by the observation that overexpression of Bcl-2 in HeLa cells brought about increased GSH levels in nuclei and mitochondria (Voehringer et al., 1998Go). Reduced glutathione efflux from mitochondria and nuclei may also contribute to the maintenance of glutathione in these organelles. Glutathione efflux is related to membrane integrity (Fernandez-Checa et al., 1997Go). The opening of the membrane permeability transition pore, a large conductance channel, is regulated by Bcl-2 protein (Kroemer, 1997Go) and mitochondrial function. It was demonstrated (Belzacq et al., 2003Go) that Bcl-2 enhances adenine nucleotide translocator (ANT)-dependent ADP/ATP exchange activity by direct protein-protein interaction. Proapoptotic signals have been shown to inhibit ANT activity through induction of Bax, resulting in the formation of a nonspecific pore leading to the loss of mitochondrial membrane potential (Belzacq et al., 2003Go; Jacotot et al., 2001Go). Thus, the increase in Bcl-2 and ATP levels may also contribute to the maintenance of mitochondrial membrane potential and GSH levels through reduced glutathione efflux.

GSH is the major antioxidant in mitochondria involved in the detoxification of reactive oxygen species (ROS) generated by the mitochondrial respiratory chain (Fernandez-Checa et al., 1997Go). The increased mitochondrial GSH should confer protection for cells against apoptosis induced by ROS. On the other hand, the increased GSH levels in nuclei may also exert protection for cells against oxidative damage to nuclear DNA. In addition, alteration in the nuclear redox status by increasing GSH levels may have effects on gene transcription, and nuclear signal transduction, both before and following an apoptotic stimulus (Voehringer et al., 1998Go). The importance of GSH in protection of cells against apoptosis is suggested by the following observations: (a) induction of apoptosis by anti-fas/APO-1 antibody in human JURKAT T lymphocytes was preceded by a rapid and specific efflux of GSH (van den Dobbelsteen et al., 1996Go); (b) depletion of GSH in neuronal cells caused an initial increase in intracellular ROS followed by a loss of mitochondrial function and then DNA fragmentation (Merad-Boudia et al., 1998Go); (c) overexpression of Bcl-2 in HeLa cells enhanced GSH import into nuclei and mitochondria associated with inhibition of apoptosis (Voehringer et al., 1998Go). Increased ROS has been associated with TGFß-induction of apoptosis in liver cells. The free radical scavenger carboxyfullerene prevented the induction of apoptosis by TGFß in human hepatoma Hep3B cells (Huang et al., 1998Go). In addition, Herrera et al.(2001)Go observed that TGFß enhanced ROS production leading to bcl-xL downregulation, loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase 3. These findings suggest that the EE-induced alterations in glutathione detected in the present study might also contribute to its ability to protect hepatocytes from TGFß–induced apoptosis.

In summary, the results presented in this study indicate that EE enhanced (1) transcript levels of several mitochondrial genes, respiratory chain activity, and ATP levels, (2) Bcl-2 protein levels, and (3) levels of glutathione (GSH and GSSG) in both mitochondria and nuclei. By doing a and b, EE may stabilize and enhance mitochondrial function and inhibit cytochrome c release, whereas the increased ATP and Bcl-2 levels, in turn, may enhance the cross-membrane transport of glutathione into mitochondria and nuclei. Whether or not these effects contribute to EE-mediated inhibition of apoptosis is under investigation.


    ACKNOWLEDGMENTS
 
This research was supported by U.S. PHS grant CA36701. Maintenance and use of shared equipment was supported by NIEHS Center grant P30 ES03819.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (410) 955-0116. Email: jyager{at}jhsph.edu. Back


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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