The Role of Hepatocellular Oxidative Stress in Kupffer Cell Activation during 1,2-Dichlorobenzene-Induced Hepatotoxicity

Husam S. Younis*, Alan R. Parrish{dagger} and I. Glenn Sipes*,1

* Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721; and {dagger} Department of Medical Pharmacology and Toxicology, Texas A&M University System Health Science Center, College Station, Texas 77843

Received May 16, 2003; accepted July 20, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1,2-Dichlorobenzene (1,2-DCB), an industrial solvent, is a known hepatotoxicant. Two oxidative events in the liver contribute to 1,2-DCB-induced liver injury: an initial hepatocellular oxidative stress, followed by oxidant stress associated with an inflammatory response. We hypothesize that the initial hepatocellular oxidative event triggers molecular and cellular processes within hepatocytes that lead to the production of factors that contribute to Kupffer cell (KC) activation and upregulation of the inflammatory cascade. To investigate the molecular effects of 1,2-DCB, primary cultures of Fischer-344 (F-344) and Sprague-Dawley (SD) rat hepatocytes were incubated with 1,2-DCB (3.6–12.4 µmol) and examined for enhanced DNA-binding activity of the oxidant-sensitive transcription factors activator protein–1 (AP-1), nuclear factor-kappa B (NF-{kappa}B), and electrophile responsive element (EpRE), and production and release of the chemokine cytokine-induced neutrophil chemoattractant (CINC). In F-344 rat hepatocytes, the activities of AP-1 and NF-{kappa}B were increased by as much as 3-fold by 6 h of 1,2-DCB treatment, when compared to control. Nuclear translocation of EpRE was also enhanced by 3-fold and occurred 2 h following 1,2-DCB treatment. These events were greater in F-344 than in SD rat hepatocytes incubated with 1,2-DCB. Moreover, F-344 rat hepatocytes produced and released CINC following incubation with 1,2-DCB, but SD rat hepatocytes did not. Lastly, conditioned media from 1,2-DCB-treated F-344 rat hepatocytes stimulated KC activity as determined by enhanced NF-{kappa}B-binding activity and increased nitric oxide production. Collectively, these data suggest that the mechanisms of 1,2-DCB-induced hepatotoxicity involve intercellular communication whereby compromised hepatocytes may signal KC activation via the production and release of oxidant-sensitive chemokines and cytokines.

Key Words: Kupffer cell; Fischer-344 rat; Sprague-Dawley rat; 1,2-dichlorobenzine.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1,2-Dichlorobenzene (1,2-DCB) is an industrial solvent primarily used in the synthesis of herbicides. 1,2-DCB is a potent centrilobular hepatotoxicant in the Fischer-344 (F-344) rat, but causes much less liver injury in the Sprague-Dawley (SD) rat. Bioactivation of 1,2-DCB by hepatic cytochrome P450 enzymes is required to initiate the events that ultimately lead to hepatic injury (NTP, 1985Go; Gunawardhana et al., 1993Go; Stine et al., 1991Go). Phagocytic cells, especially Kupffer cells (KC), also contribute greatly to the hepatotoxicity of 1,2-DCB. For example, inhibition of KC activity by gadolinium chloride (GdCl3) or methyl palmitate (MP) decreased 1,2-DCB mediated elevations in alanine aminotransferase (ALT) activity as well as the extent of necrosis (Gunawardhana et al., 1993Go; Hoglen et al., 1998Go). Moreover, KC isolated from 1,2-DCB-treated F-344 rats produce exaggerated amounts of superoxide anion (Hoglen et al., 1998Go). The production of reactive oxygen species correlates with the formation of conjugated dienes and 4-hydroxynonenal in livers of F-344 rats at 16 and 24 h following 1,2-DCB administration, which is greater than that found in SD rats (Hoglen et al., 1998Go; Younis et al., 2000Go). KC inhibition with GdCl3 reduced the markers of lipid peroxidation and suggested that KC-derived reactive oxygen species and subsequent peroxidation of lipid membranes may be a mechanism by which these cells contribute to the progression of 1,2-DCB-mediated hepatocellular necrosis.

The role of KC in chemical-induced liver injury has been documented for other hepatotoxicants such as carbon tetrachloride, hypervitaminosis A, cadmium, and acetaminophen (Decker, 1990Go; Kuester et al., 2002Go; Laskin and Pedino, 1995Go; Laskin et al., 1986Go; Sipes et al., 1989Go). Although it is clear that KC activation contributes to the hepatotoxicity of chemicals, the mechanism(s) by which these macrophages become activated remains to be characterized. Cytokines such as tumor necrosis factor alpha (TNF-{alpha}) and cytokine-induced neutrophil chemoattractant (CINC) factor have been shown to activate KC and neutrophils, respectively, in the setting of liver injury (Dong et al., 1998Go; Horbach et al., 1997Go; Spitzer et al., 1996Go; Yamada et al., 1999Go). The sources of cytokines and chemokines in the liver to trigger the activation of KC and recruitment of inflammatory cells may include both parenchymal and nonparenchymal cells. Although the capacity for hepatocytes to produce and secrete cytokines and chemokines is low when compared to macrophages and endothelial cells, hepatocytes have been shown to produce and secrete TNF-{alpha} and CINC following exposure to hydrogen peroxide (Horbach et al., 1997Go), heavy metals (Dong et al., 1998Go), and during conditions of ischemia (Kataoka et al., 2002Go). The processes that mediate cytokine/chemokine production and release by hepatocytes are unclear but may involve oxidative stress (Horbach et al., 1997Go).

An intracellular oxidative stress occurs in hepatocytes following 1,2-DCB treatment. This pro-oxidant state is dependent on the bioactivation of 1,2-DCB and is independent of KC activity (Younis et al., 2000Go). Evidence for an intracellular oxidative event is the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) as soon as 1 h following 1,2-DCB treatment. Interestingly, the extent of GSSG production in F-344 rats is much greater than that produced by SD rats. Therefore, the differences in susceptibility between these rat strains to 1,2-DCB may be due to hepatocellular oxidative stress. This work will test the hypothesis that hepatocellular oxidative stress leads to the transcription and translation of genes in hepatocytes whose products (e.g., cytokines/chemokines) ultimately result in the activation of KC and the subsequent inflammatory cascade. Specifically, nuclear factor-kappa B (NF-{kappa}B), activator protein–1 (AP-1) and electrophile responsive element (EpRE) transcription factor-binding activity and the production of TNF-{alpha} and CINC will be determined in F-344 and SD rat hepatocytes treated with 1,2-DCB. The difference in susceptibility between F-344 and SD rats was utilized to determine the importance of oxidative stress and redox-sensitive molecular and cellular events in 1,2-DCB-induced hepatotoxicity. The results of this study provide a contributing mechanism by which hepatocytes may activate KC and the subsequent inflammatory cascade that is responsible for 1,2-DCB induced liver injury.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
1,2-DCB (HPLC grade) was purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI). All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO).

Animals.
Male F-344 (161–190 g, 8–9 weeks old) and SD (175–199 g, 8–9 weeks old) rats were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). The animals were housed in hanging wire cages in an AAALAC-approved facility and received humane care. All animals had free access to food (Harlan Teklan 4% mouse/rat diet, Harlan Teklan, Indianapolis, IN) and water and were allowed at least one week of acclimation prior to use.

Isolation of primary cultured hepatocytes from rats.
Hepatocytes were isolated from rats using a two-step in situ liver perfusion as previously described by McQueen et al. (1989)Go. Briefly, animals were anesthetized with pentobarbital (50 mg/kg, ip) and the portal vein was exposed. Hank’s balanced salt solution (Life Technologies, Grand Island, NY) was perfused through the portal vein for 4 min at 40 ml/min, followed by Williams Medium E (WME: Life Technologies, Grand Island, NY) containing 0.25mg/ml collagenase B (Boehringer Mannheim, Mannheim, Germany) for 6.5 min at 20 ml/min. After sacrifice, the liver was removed from the animal and massaged through gauze to obtain a suspension of cells. The resulting hepatic cell suspension was centrifuged (4 min at 500 rpm). The pelleted hepatocytes were resuspended in WME supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and the concentration of cells was determined. Cell viability exceeded 90% for all experiments. Approximately 2 x 106 cells were plated onto T-25 tissue-culture flasks (Falcon, Plymouth, UK) and allowed to adhere. After 2 h, the medium was replaced with serum-free-WME and the hepatocyte monolayers were ready for treatment.

Treatment of primary cultured hepatocytes.
For dose-response and time-course studies, hepatocytes were exposed to 1,2-DCB (3.6, 7.1, and 12.4 µmol in dimethyl sulfoxide [DMSO]) or DMSO vehicle for 0 to 13 h at 37°C in an atmosphere of 5% CO2:95% air. Dosing solutions were placed in a glass boat that was positioned at the neck of a sealed T-25 flask. 1,2-DCB was allowed to vaporize in the flask and to partition from the gas phase into the culture medium (Younis et al., 2000Go). By one h of treatment, the concentration of 1,2-DCB in the media was 147 µM with the 3.6 µmol dose, 230 µM for the 7.1 µmol dose, and 277 µM for the 12.4 µmol dose. At selected times, cells and media were harvested for determination of lactate dehydrogenase (LDH) activity, an indicator of toxicity. LDH activity was assessed using an LDH enzymatic kit (Sigma Chemical Company, St. Louis, MO)

For determination of GSSG concentrations, hepatocytes were exposed to either DMSO, 1,2-DCB (3.6 µmol in DMSO), 1,4-dichlorobenzene (1,4-DCB: 3.6 µmol in DMSO), or L-buthionine sulfoxamine (BSO: 2 mM) for 0, 1, 3.5, or 6.5 h. The cells were harvested in 1 ml 3.5% perchloric acid (PCA) and kept frozen at -80°C until analysis. In other studies, hepatocyte monolayers were treated with catalase (4800 U/ml) concurrently with 1,2-DCB or DMSO exposure.

The phosphorylation activity of JNK in hepatocytes was determined following 0.5, 1, and 2 h of incubation with 1,2-DCB (3.6 and 7.1 µmol). Determination of transcription factor-binding activity was assessed 2, 4, and 6 h following 1,2-DCB treatment. The production and release of cytokines were determined by hepatocytes after 12 and 24 h of incubation with 1,2-DCB, DMSO, BSO, or 1,4-DCB, as described above. The hepatocytes were scraped with lysis buffer (0.5 ml) and stored at 4°C. In other studies, F-344 rats were treated with 1,2-DCB (3.6 mmol/kg, ip) or corn oil vehicle (2 ml/kg) in vivo for 6 h before isolation of hepatocytes. These hepatocytes (2 x 106) were allowed to adhere to T-25 flasks for 2 h and then were incubated with WME. Following 12 or 24 h of incubation, media and cell lysates were harvested for analyses, as described above for cytokines.

To determine if hepatocytes released factors that modulate KC, hepatocytes were incubated at 37°C with 1,2-DCB (3.6 µmol) in an atmosphere of 5% CO2:95% air. Other hepatocyte incubations received no treatment while some received only DMSO vehicle. After 24 h of incubation, media from each treatment group were harvested, centrifuged (400 rpm for 5 min at 37°C) and supernatants (i.e., conditioned media) incubated with KC.

Isolation and treatment of primary cultured KCs from F-344 rats.
KC were isolated as previously described by Hoglen et al. (1998)Go. Briefly, male F-344 rats were anesthetized with pentobarbital (50 mg/kg, ip). The liver was then perfused with Geyes Balanced Salt Solution (GBSS) for 4 min (20 ml/min) at 37°C. This was followed by perfusion of an enzyme digest containing 47.9U/ml collagenase type 2 (Worthington Biomedical Corp., Lakewood, NJ) and 1.3 mg/ml protease dissolved in GBSS for 7.5 min (20 ml/min). The liver was then harvested, sliced with a razor, and incubated in 90 ml of an enzyme digest (32.5 U/ml collagenase type 2, 0.6mg/ml protease and Dnase I 11 µg/ml) for 30 min at 37°C. Following centrifugation (500 rpm for 5min) nonparenchymal cells were harvested by collecting the supernatant and then concentrated by centrifugation at 2000 rpm for 10 min. The pelleted nonparenchymal cells including KC were collected in a syringe and loaded onto an elutriator (Beckman J6-C equipped with a JE 5.0 rotor/Sanderson cell) at 2500 rpm and 22°C. KC were elutriated with GBSS containing 0.1% BSA once the flow rate surpassed 33 ml/min. The cells in the fraction were centrifuged (2000 rpm for 10min) and the pellet was resuspended in RPMI containing 10% bovine calf serum. Cell viability exceeded 90% for all experiments. KC were plated on 6-well plates at a density of 2 x 106 cells/well and incubated as described above for 24 h. KC were then incubated with hepatocyte-conditioned media (2 ml/well) from 1,2-DCB, DMSO or control-treated hepatocytes. KC also were treated with lipopolysaccharide (LPS) at 100 ng/ml as positive control for NF-{kappa}B activation. In other studies, conditioned media from 1,2-DCB-treated hepatocytes was incubated with anti-CINC antibody (1:250, Peptide International, Louisville, KY) followed by Protein A conjugated to sepharose beads, to immunoprecipitate and remove CINC from the media prior to incubation with KC. Conditioned media from 1,2-DCB-treated hepatocytes was also incubated with only Protein A conjugated to sepharose beads to serve as a control. After 24 h of incubation, cells were harvested for nuclear proteins to determine NF-{kappa}B-binding activity.

Determination of GSSG.
The concentration of GSSG in hepatocytes was determined at 0, 1, 3.5, and 6.5 h following treatment with 1,2-DCB. The cells were scraped in 1 ml 3.5% perchloric acid (PCA) and kept frozen at -80°C until analysis. GSSG was derivatized and analyzed using a method described by Fariss and Reed (1987)Go. Briefly, 50 ml of internal standard, {gamma}-glutamyl glutamate (1 mM), 50 ml of bathophenanthrolinedisulfonic acid (10 mM), and 100 ml of 70% PCA were added to 0.5 ml of hepatocyte suspensions. The mixtures were frozen at -80°C for 1.5 h. Samples were then thawed and centrifuged at 14,000 rpm for 5 min. To a 0.5-ml aliquot from each sample, 50 ml of thiol stabilizer (iodonic acid [1 M] in bromocresol purple [0.2 mM]) and 0.48 ml of base solution (2 M potassium hydroxide in 2.4 M potassium bicarbonate) were added. After a 10-min incubation at room temperature (25°C) in the dark, 1 ml of 1% fluorodinitrobenzene in ethanol was added. The samples were stored at 4°C in the dark for 24 h and then filtered (0.22 µm) prior to HPLC analysis. GSSG standards ranging from 0 to 8 µM were prepared and analyzed along with hepatocyte samples.

Samples were analyzed on a Beckman System Gold HPLC system (San Ramon, CA) using a reverse-phase amino column (Rainin, Woburn, MA) with ultraviolet detection set at 395 nm and at a flow rate of 1 ml/min. The mobile phase consisted of 80% methanol in water and 0.5 M ammonium acetate containing 64% methanol in water. A gradient was utilized to elute GSSG as described by Fariss and Reed (1987)Go.

Determination of nuclear transcription factor-binding activity.
NF-{kappa}B- AP-1-, and EpRE-bnding activity were determined using the electrophoretic mobility shift analysis as described by Dignam et al. (1983)Go. Briefly, hepatocytes (2 x 106 cells) or KC (10–12 x 106 cells) were lysed by homogenization and nuclei were collected at 2000 x g. Nuclear proteins were extracted by incubation with buffer for 1 h on ice and the protein concentration was determined by the Coomassie-Plus protein assay reagent (Pierce, Rockford, IL.). Nuclear protein extract (2.5–5 µg) was incubated with double-stranded, [32P]-labeled oligonucleotide in extraction buffer. After 0.5 h, the reaction mixture was loaded on 5% nondenaturing polyacrylamide gels. Gels were dried and exposed to Kodak X-OMAT film for autoradiography. The following oligos (double stranded) were used: AP-1: 5'-CgCTTgATgAgTCAgCCgGAA-3', NF-{kappa}B: 5'-AgTTgAggggACTTTCCCAggC-3', EpRE: 5'-TTTCTgCTTAgTCATTgTCTT-3'. The rat quinone reductase gene (RNQO1) was used to determine the binding activity of EpRE, which contains a sequence for this transcription factor in its promoter region. Bands were quantified using a phosphoimager. Data are expressed as fold increase above DMSO control.

Determination of TNF-{alpha} and CINC.
CINC and TNF-{alpha} were determined in hepatocytes treated with 1,2-DCB or DMSO vehicle using Western-blot analysis. Briefly, proteins from media were concentrated using YM-3 Centriplus centrifugation filter systems (Millipore Corp., Bedford, CT). Proteins were separated from concentrated media and cell lysates using SDS–PAGE followed by transfer to a nitrocellulose membrane (Stamer et al., 1994Go). The membrane was incubated with rabbit monoclonal antibody against rat CINC-1 protein (Peptide International, Louisville, KY) or rabbit polyclonal antibody against mouse TNF-{alpha} (Genzyme, Cambridge, MA). Following a washing period, goat antirabbit horseradish peroxidase antibody (Amersham Life Science, Arlington Heights, IL.) was incubated with the membrane. Finally, the membrane was incubated with substrate and immunoreactive proteins were visualized using autoradiography.

Determination of JNK.
JNK was determined by the method of Hibi et al.(1993)Go. F-344 and SD rat hepatocytes were treated with 1,2-DCB or DMSO for 0.5 to 2 h as previously described, and cytosolic proteins were harvested following homogenization of the cells. JNK was precipitated from the cytosolic fraction with a GST-c-Jun (1–79) fusion protein. This complex was incubated with [32P]-labeled oligonucleotide and the reaction mixture was loaded on 10% polyacrylamide gels. The gels were dried and exposed to Kodak X-OMAT film for autoradiography. Bands were quantified using a phosphoimager as previously described.

Determination of CXC receptor-2 expression.
KC were isolated from F-344 rats as previously mentioned and plated onto 1-cm coverslips in 6-well plates. Immunohistochemistry was performed as described by Fujino et al.(2000)Go. Briefly, the cells were fixed onto to the cover slips using methanol/acetone (7:3) and blocked with 2%BSA in PBS. The coverslips were then incubated with mouse monoclonal anti-human CXC Receptor 2 (CXCR2) primary antibody (1:500; Biosource International, Santa Cruz, CA) overnight at 4°C. Following a wash with PBS, antirabbit biotinylated secondary antibody (1:500) was placed on the cover slips for 1 h and then Cy-5 fluorescent antibody conjugated to streptoavin (1:500) was incubated for an additional 1 h at room temperature. The cover slips were fixed to slides and stored at 4°C in the dark until analysis. Confocal microscopy was used for visualization.

Determination of nitrite.
Nitrite was measured in KC media, as a marker of nitric oxide, by the method of Green et al. (1982). Briefly, in a 96-well plate 90 µl of sulfanilamide and 90 µl of N-1-naphylethylenediamine were added to 90 µl of sample or nitrite standards (1–15 µmol). The samples and standards were read at 570 nm using a Dynatech MR5000 plate reader (Dynatech Laboratories, Chantilly, VA).

Statistical analysis.
The data are expressed as mean ± standard error of the mean (SEM). To compare F-344 and SD rat strains with respect to 1,2-DCB and DMSO treatments, the data were analyzed using two-way analysis of variance (ANOA) followed by Student Newman-Kuels or Bonferroni’s post-hoc tests. One-way ANOVA and a post-hoc test were performed for all other analyses. Differences were considered significant if p <= 0.05. All statistical analyses were performed using SigmaStat statistical software for Windows, 1.0 (Jandel Scientific, San Rafael, CA).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytotoxicity of 1,2-DCB in Hepatocytes Isolated from Naive F-344 and SD Rats
The cytotoxicity of 1,2-DCB to hepatocytes, as assessed by leakage of LDH into media, was characterized with respect to concentration and time of incubation. Large elevations in LDH release were apparent by 1.5 h of incubation of hepatocytes with the highest concentration of 1,2-DCB (12.4 µmol) (Fig. 1Go). At lower concentrations of 1,2-DCB, elevations in LDH activity did not occur until later time points of incubation. At any concentration or time of incubation with 1,2-DCB, the extent of LDH release was equivalent for F-344 and SD rat hepatocytes.



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FIG. 1. Time-course and dose-response relationships for the cytotoxicity of 1,2-DCB in hepatocytes isolated from F-344 and SD rats: Hepatocytes isolated from naive (A) F-344 and (B) SD rats were maintained in monolayer cultures for 2 h, then incubated with 1,2-DCB (3.6–12.4 µmol) or DMSO for various lengths of time (1.5–13 h). Cytotoxicity was assessed by the percent of LDH released in the media from that of total LDH (i.e., media and hepatocellular LDH concentrations). Data are expressed as mean ± SEM; n = 3 for F-344 rats and n = 4 for SD rats.

 
Concentrations of GSSG in F-344 and SD Rat Hepatocytes Incubated with 1,2-DCB
Hepatocytes were incubated with 1,2-DCB (3.6 µmol) for up to 6.5 h to determine if 1,2-DCB induces oxidative stress within these cells as marked by enhanced production of GSSG. A subtoxic dose of 1,2-DCB was chosen to rule out the contribution of cytotoxicity to the production of GSSG. In these studies, the formation of GSSG significantly increased in F-344 rat hepatocytes 1 h after 1,2-DCB exposure, rising 13% above DMSO control (Fig. 2Go; 40.5 ± 1.1 vs. 34.3 ± 0.9 nmol/106 cells). GSSG concentrations remained elevated at 3.5 and 6.5 h. No increases in GSSG were observed in SD rat hepatocytes incubated with the same dose of 1,2-DCB. Pretreatment of F-344 rat hepatocytes with catalase inhibited the increase in GSSG concentrations associated with 1,2-DCB. Incubation of cells with BSO or 1,4-DCB did not result in elevated GSSG concentrations. However, BSO did deplete glutathione in both F-344 and SD hepatocytes to the same degree (data not shown).



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FIG. 2. GSSG concentrations in primary cultured rat hepatocytes following incubation with 1,2-DCB: Hepatocytes isolated from naive F-344 and SD rats were maintained in monolayer cultures for 2 h, then incubated with 1,2-DCB (3.6 µmol), 1,4-DCB (3.6 µmol), BSO (2 mM), or DMSO vehicle for 1–6.5 h. Catalase (CAT, 4800 U/ml) was concomitantly incubated with 1,2-DCB-treated cells. GSSG concentrations were determined by an HPLC method using a UV detector (395 nm). Data are expressed as mean ± SEM; n = 3 for F-344 rats and n = 4 for SD rats; *significant difference from all other groups.

 
Activation of AP-1, NF-{kappa}B, and EpRE Transcription Factors in Hepatocytes
To determine the molecular consequences of 1,2-DCB-induced oxidative stress, the binding activities of AP-1, NF-{kappa}B, and EpRE were examined in hepatocytes of F-344 and SD rats (Fig. 3Go). Enhanced binding activity of NF-{kappa}B was significantly increased in hepatocytes of F-344 rats incubated for 2 h with 1,2-DCB and was greatest at 6 h of treatment (Fig. 3Go and Table 1Go). In contrast, NF-{kappa}B-binding activity was not significantly increased in SD rat hepatocytes incubated with 1,2-DCB (Table 1Go). Elevations of AP-1-binding activity occurred in both F-344 and SD rat hepatocytes. The increases in AP-1 activity between the rat strains were not significantly different from each other. Enhanced DNA-binding activity of EpRE to the RNQO1 consensus sequence was observed in F-344 rat hepatocytes following incubation with 1,2-DCB. Binding of EpRE increased significantly by 4.0- and 2.8-fold above control after 6 h of incubation with 3.6 and 7.1 µmol of 1,2-DCB, respectively (Table 1Go). For SD rat hepatocytes, there were no significant increases in EpRE-binding activity with 1,2-DCB treatment.



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FIG. 3. Transcription factor-binding activity in primary cultured rat hepatocytes following incubation with 1,2-DCB: Hepatocytes isolated from naive F-344 and SD rats were maintained in monolayer cultures for 2 h, then incubated with 1,2-DCB (3.6b and 7.1c µmol) or DMSOa for 2–6 h. The transcription factors were assessed using electrophoretic mobility shift assay (EMSA): Representative autoradiograph of NF-{kappa}B-binding activity in F-344 rat hepatocytes. FP, free probe.

 

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TABLE 1 Summary Table of Results of Binding Activities of AP-1, NF-{kappa}B, and EpRE Examined in Hepatocytes of F-344 and SD Rats
 
Enhanced c-Jun Phosphorylation by JNK
Since the binding activity of AP-1 was increased, the activity of JNK, an upstream regulator of AP-1, was examined in hepatocytes treated with 1,2-DCB. Statistically significant increases in JNK phosphorylation activity occurred as early as 1 h following incubation of F-344 and SD rat hepatocytes with 1,2-DCB (3.6 and 7.1 µmol) (Fig. 4Go and Table 2Go). These increases in JNK phosphorylation activity were similar for F-344 and SD rat hepatocytes.



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FIG. 4. JNK phosphorylation activity in primary cultured rat hepatocytes following incubation with 1,2-DCB: Hepatocytes isolated from naive F-344 and SD rats were maintained in monolayer cultures for 2 h, then incubated with 1,2-DCB(3.6b and 7.1c µmol), or DMSOa for 0.5–2 h. JNK phosphorylation activity was determined using GST-c-jun (1–79) to precipitate JNK. Representative autoradiograph of GST-c-jun phosphorylation by JNK in F-344 rat hepatocytes.

 

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TABLE 2 Summary Table of Results of JNK Activity F-344 and SD Rat Hepatocytes Incubated in with 1,2-DCB
 
Protein Expression of CINC and TNF-{alpha}
The amount of CINC protein was significantly increased in the media of F-344 rat hepatocytes following incubation with 1,2-DCB (3.6 µmol) by 2.2-fold at 12 h and 1.6-fold at 24 h of incubation (Fig. 5Go). There were no significant increases in the amounts of CINC protein in hepatocyte cell lysates of 1,2-DCB treated cells. There were no elevations in CINC release into media or in cell lysates of F-344 hepatocytes incubated with 1,4-DCB or BSO. In SD rat hepatocytes, there were no increases in CINC in media following 12 h of incubation with 1,2-DCB as compared to DMSO control. By 24 h there was a 1.5-fold increase in CINC in both the media and cell lysate of SD rat hepatocytes incubated with 1,2-DCB as compared to control, but these elevations were not significantly different from control. A comparison of the relative increases in CINC between F-344 and SD rat hepatocytes revealed that the elevations in this protein were significantly greater in F-344 rat hepatocytes following incubation with 1,2-DCB at 12 h. The expression of CINC protein in naive hepatocytes of F-344 and SD rats was not different from that of DMSO-treated hepatocytes (data not shown).



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FIG. 5. CINC protein expression in primary cultured rat hepatocytes: Hepatocytes isolated from naive F-344 and SD rats were maintained in monolayer cultures for 2 h, then incubated with 1,2-DCB (3.6 µmol) or DMSO for 12 or 24 h. In other studies, F-344 rats were administered 1,2-DCB (3.6 mmol/kg) or corn oil vehicle in vivo for 6 h, and then hepatocytes were isolated and incubated for 12 or 24 h. CINC was determined by Western-blot analysis and visualized by chemiluminescence. Representative autoradiograph of a Western blot for CINC from F-344 rat hepatocytes: The lane designated as CINC was loaded with 20 ng synthetic rat CINC protein (8 kD) that is present at the 9 kD molecular weight marker. M, media; C, cell lysate.

 
CINC was also produced from hepatocytes after in vivo exposure to 1,2-DCB. F-344 rats were treated with 1,2-DCB (3.6 mmol/kg, in corn oil) for 6 h, after which hepatocytes were isolated and incubated ex vivo. After 24 h of incubation, there was increased amounts of CINC in the media and cell lysates of these hepatocyte cell cultures (Table 3Go). The production and release of CINC by these hepatocytes was not due to a loss of plasma membrane integrity since the levels of LDH were not altered from control (data not shown).


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TABLE 3 Summary Table of Results of Amounts of CINC in the Media and Cell Lysates of Hepatocyte Cell Cultures
 
The protein levels of TNF-{alpha} were not altered in F-344 or SD rat hepatocytes treated with 1,2-DCB in both in vitro and in vivo studies following 12 or 24 h of culture (data not shown).

Expression of CXC Receptor 2 in KCs
Immunohistochemical analysis revealed that CXCR2 was expressed by naive KC isolated from F-344 rats. The labeling for CXCR2 was only observed when fixed KC were incubated with primary antibody (Fig. 6Go). The background labeling of the secondary antibodies was minimal.



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FIG. 6. CXC receptor 2 protein expression by KC: KC were isolated from naive F-344 rats and fixed to coverslips with methanol/acetone. (A) KC were incubated with 2% bovine serum albumin or (B) primary CXCR2 antibody (dissolved in 2% bovine serum albumin) and visualized with Cy-5 secondary antibody using confocal microscopy; n = 3.

 
Activation of KCs
Conditioned media from 1,2-DCB-treated hepatocytes were incubated with naive KC from F-344 rats to determine if hepatocytes can signal KC for activation assessed by NF-{kappa}B-binding activity and nitric-oxide production. KC incubated with conditioned media from 1,2-DCB-treated hepatocytes had increased NF-{kappa}B-binding activity and nitric oxide production (Figs. 7AGo and 7CGo). NF-{kappa}B activity was not elevated in KC incubated with DMSO, control media (i.e., nonconditioned) or control media containing 1,2-DCB (data not shown). LPS, as a positive control, enhanced NF-{kappa}B-binding activity when added to conditioned media of DMSO-treated hepatocytes. The band representing NF-{kappa}B was competitively inhibited with addition of excess (25-fold) unlabeled NF-{kappa}B oligonucleotide, suggesting that this activity was specific for NF-{kappa}B. The immunoprecipitation of CINC from the conditioned media of 1,2-DCB-treated hepatocytes reduced the NF-{kappa}B-binding activity in KC (Fig. 7BGo). This suggests that the presence of CINC in the hepatocyte-conditioned media contributed to the activation of KC.




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FIG. 7. Activation of KC following incubation with conditioned media from 1,2-DCB-treated hepatocytes: Hepatocytes isolated from naive F-344 rats were maintained in monolayer cultures for 2 h and then incubated with DMSO or 1,2-DCB (3.6 µmol) for 24 h. Conditioned media from these hepatocytes was harvested and immediately incubated with KC for 24 h. (A) Representative autoradiograph of NF-{kappa}B-binding activity in KC using EMSA: 1, free probe; 2, no treatment; 3, DMSO; 4, DMSO/LPS; 5, 1,2-DCB; 6, 1,2-DCB/LPS; 7, DMSO/LPS; 8, 1,2-DCB; 9, 1,2-DCB/LPS. Samples of lanes 7–9 were incubated with excess cold NF-{kappa}B oligonucleotide for competition of radiolabeled probe. KC incubated with conditioned media from 1,2-DCB-treated hepatocytes had significantly 4.6 ± 1.1-fold greater NF-{kappa}B-binding activity than KC incubated with conditioned media from DMSO-treated hepatocytes; n = 4. (B) NF-{kappa}B-binding activity by KC incubated with conditioned media from 1,2-DCB-treated hepatocytes (lane 1) was reduced by immunoprecipitation of CINC (lane 2). (C) Nitrite, a marker of nitric oxide, was assayed in conditioned media prior to and following incubation with KC, spectrophotometrically. The data represent percent increase (mean ± SEM) of nitrite concentration following incubation with KC; n = 3.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An inflammatory response is known to contribute to the pathogenesis of liver injury and disease. Several models of hepatocellular injury demonstrate that activation of KC, the resident inflammatory cells of the liver, is crucial for the development of hepatocellular necrosis. Evidence for this is based on studies that show reductions in hepatocellular damage following pretreatment of rodents with chemical inhibitors of KC activity and function. For example, liver injury caused by ischemia/reperfusion (Jaeschke and Benzick, 1992Go), bile duct ligation (Maher et al., 1998Go), and xenobiotic exposure (Laskin and Pedino, 1995Go) is dramatically decreased by GdCl3 or MP, two known inhibitors of KC activity. Although KC produce a number of cytotoxic molecules that include reactive oxygen and nitrogen species and cytotoxic proteins such as TNF-{alpha} to damage the hepatocyte, fulminant liver injury often requires the recruitment of other inflammatory cells (Laskin and Pedino, 1995Go). The infiltration of neutrophils, in particular, is observed in necrotic regions of the liver during chemical-induced hepatotoxicity. The cooperative activities of KC and neutrophils lead to a cascade of events, which produce an inflammatory response in the liver that is responsible for hepatotoxicity.

The studies reported here utilized 1,2-DCB as a model hepatotoxicant to study the mechanisms of KC activation. The results of this work support the hypothesis that mediators released from hepatocytes contribute to the activation of KC during 1,2-DCB-induced liver injury. Evidence of liver damage (serum ALT activity and microscopic hepatocyte necrosis) in F-344 rats is observed by 16 h of 1,2-DCB treatment and is maximal at 24 h (Gunawardhana et al., 1993Go; Hoglen et al., 1998Go; Kulkarni et al., 1997Go, 1999Go). In the SD rat, maximal liver injury occurs later (48 h post dose) and is less pronounced. Figure 8Go illustrates a model of the proposed mechanisms of 1,2-DCB-induced hepatotoxicity that involves two oxidative events. The first oxidative event occurs within the hepatocyte and is independent of KC function (Younis et al., 2000Go). Studies in vivo showed that hepatocellular oxidative stress was observed by 1 h of 1,2-DCB treatment and is due to the bioactivation of 1,2-DCB to reactive intermediates. The in vitro results presented herein reconfirm that 1,2-DCB does generate an oxidative stress in F-344 rat hepatocytes. The increased levels of reactive oxygen species in hepatocytes following 1,2-DCB exposure were averted by pretreatment with catalase, a scavenger of hydrogen peroxide, providing further evidence of the presence of oxidative stress. The activation of KC follows the hepatocellular oxidative-stress phase. Gunawardhana et al.(1993)Go reported that KC produced exaggerated amounts of reactive oxygen species by 16 h of 1,2-DCB treatment that resulted in the development of an extracellular oxidative stress. The consequences of KC-derived extracellular oxidative stress appear to directly contribute to hepatocellular necrosis caused by 1,2-DCB (Gunawardhana et al., 1993Go; Hoglen et al., 1998Go). However, the role of hepatocellular oxidative stress in 1,2-DCB-induced liver damage is less clear.



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FIG. 8. Proposed model of 1,2-dichlorobenzene-induced hepatotoxicity: The results presented here, as well as by others, suggest that the bioactivation of 1,2-DCB by the cytochrome P-450 enzymes of the liver lead to hepatocellular oxidative stress. This event contributes to the activation of KC via hepatocyte-derived cytokines/chemokines. KC activation, and subsequent recruitment of other inflammatory cells to the liver, is responsible for the progression of 1,2-DCB-mediated hepatocellular necrosis, at least in part by release of reactive oxygen species (ROS). SEC, sinusoidal endothelial cells.

 
A hepatocellular redox imbalance may cause a number of cellular and molecular effects to the hepatocyte, the simplest of which is overt cytotoxicity. We found that the in vitro incubation of 1,2-DCB with rat hepatocytes produced a time- and dose-dependent increase in cytotoxicity. Interestingly, the cytotoxic profiles for F-344 and SD cells were similar despite the presence of hepatocellular oxidative stress in F-344 rat cells. This suggests that hepatocellular oxidative stress caused by 1,2-DCB does not contribute to the cytotoxicity of this halobenzene in hepatocytes. However, the absence of any differences in cytotoxicity between F-344 and SD rat hepatocytes following in vitro incubation with 1,2-DCB does not correlate with in vivo studies that demonstrate that F-344 rats are more susceptible than SD rats to 1,2-DCB-mediated hepatocellular necrosis. This paradox is explained by the fact that KC, which contribute to the liver necrosis caused by 1,2-DCB in vivo, are not present in the in vitro model. This phenomenon has been described for other hepatotoxicants such as carbon tetrachloride and cadmium chloride, where KC activation is implicated in liver injury and differential sensitivities exist between F-344 and SD rats in vivo, but not in isolated hepatocytes in vitro (Kuester et al., 2002Go; Sauer et al., 1997Go). The lack of correlation in hepatotoxicity among in vitro and in vivo studies does not undermine the utility of primary cultured hepatocytes for mechanistic studies of 1,2-DCB, because the molecular and cellular changes that occur in in vivo studies such as hepatocellular oxidative stress and cytokine production also occur in primary cultured hepatocytes. Measurement of such endpoints will improve the extrapolation of in vitro findings to the more complex in vivo situation.

Given that hepatocellular oxidative stress is not directly contributing to hepatocyte cell death, other consequences of this redox imbalance may include gene transcription and translation. For example, oxidative stress stimulates signal transduction pathways that enhance NF-{kappa}B and AP-1 transcription-factor activation (Bowie and O’Neill, 2000Go; Karin et al., 2001Go). Indeed, the results of this study demonstrate enhanced DNA-binding activity of AP-1 and NF-{kappa}B in both F-344 and SD rat hepatocytes following incubation with subtoxic concentrations of 1,2-DCB. AP-1-binding activity and JNK phosphorylation were increased to a similar extent in F-344 and SD rat hepatocytes incubated with 1,2-DCB. However, the binding activity of NF-{kappa}B was approximately 2-fold greater in F-344 than in SD rat hepatocytes. The enhanced binding activity of NF-{kappa}B in the F-344 rat as compared to the SD rat correlates with the increased level of hepatocellular oxidative stress produced in this rat strain as demonstrated by enhanced GSSG production. The elevations in AP-1 and JNK phosphorylation may be independent of oxidative stress in this model, or a sufficient threshold of oxidative stress is achieved in the SD rat to produce this effect (Younis et al., 2000Go). Nonetheless, the extent of 1,2-DCB-mediated hepatocellular oxidative stress correlates with the sensitivity of F-344 and SD rats to 1,2-DCB (Younis et al., 2000Go).

NF-{kappa}B may play an important factor in 1,2-DCB-induced hepatotoxicity, given its role to upregulate genes involved in immune stimulation and pro-inflammatory responses. For example, during oxidative stress NF-{kappa}B upregulates the transcription of cytokines and chemokines such as TNF-{alpha} and CINC (Horbach et al., 1997Go), which function, at least in part, to activate inflammatory cells. TNF-{alpha} can prime KC for activation, and CINC enhances the migration of neutrophils and their production of reactive oxygen species (Beyaert and Fiers, 1994Go; Thornton et al., 1992Go). The expression of CINC, TNF-{alpha} or other cytokines have been observed in hepatocytes treated with the hepatotoxicants ethanol (Spitzer et al., 1996Go; Yamada et al., 1999Go), acetaminophen (Dambach et al., 2002Go; Horbach et al., 1997Go), cadmium, and hydrogen peroxide (Dong et al., 1998Go). We found that 1,2-DCB enhanced the production of CINC by F-344 rat hepatocytes and its release into media, but no changes in TNF-{alpha} protein expression were observed. The release of CINC from hepatocytes of F-344 rats incubated with 1,2-DCB was greater than in SD rat hepatocytes. Moreover, hepatocytes from F-344 rats treated with 1,2-DCB in vivo also have enhanced production and release CINC. The production and release of CINC was not observed with 1,4-DCB (a nonhepatotoxic isomer of dichlorobenzene) or with BSO (a glutathione depleter), suggesting that CINC production is not due to depletion of glutathione or solvent effects. The enhanced production and release of CINC by hepatocytes may be linked to NF-{kappa}B, as this redox-sensitive dimeric protein promotes the transcription of the CINC gene (Lakshminarayanan et al., 1998Go; Ohtsuka et al., 1996Go; Ren et al., 2002Go). Therefore, the increase in NF-{kappa}B-binding activity that occurs in F-344 as compared to SD rat hepatocytes within 2 to 6 h of incubation with 1,2-DCB correlates with the enhanced protein expression of CINC by 12 and 24 h. This cascade of molecular events in F-344 rat hepatocytes is likely linked to the initial intracellular oxidative stress as it occurs chronologically after the redox event.

Data presented in this paper demonstrate that factors released from hepatocytes following exposure to 1,2-DCB can activate KC. We found that conditioned media from 1,2-DCB-treated hepatocytes activated KC to enhance the nuclear translocation of NF-{kappa}B and produce increased levels of nitric oxide. Classic activators of KC function such as LPS (Okada et al., 2000Go), ethanol (Tran-Thi et al., 1995Go; Tsukamoto et al., 1999Go) and TNF-{alpha} (Tran-Thi et al., 1995Go) also trigger increased NF-{kappa}B DNA-binding activity and production of nitric oxide in these macrophages. A factor in the hepatocyte-conditioned media that may contribute to the activation of KC is CINC. The ability for CINC to activate KC is unknown. However, the results described herein demonstrate the expression of CXC receptor 2 (CXCR2) protein by KC. CXCR2 is one of six receptors in the CXC receptor family whose ligands include chemokines that contain the CXC motif (Petering et al., 1999Go). While the expression of CXCR1 is exclusive to neutrophils, CXCR2 is also expressed by monocytes (Saccani et al., 2000Go). The expression of CXCR2 by KC suggests that CINC and/or other proteins in its class modulate KC function. The fenestrated endothelium of the liver facilitates the flow of CINC or other factors released from hepatocytes to access KC. Given the proximity of KC and hepatocytes in the liver, release of low levels of CINC protein by hepatocytes may be sufficient to signal KC activation. Indeed, preliminary evidence suggests that removal of CINC from the hepatocyte-conditioned media by immunoprecipitation causes a reduction in KC activity. Given that the release of CINC by hepatocytes is moderate, the activation of KC may require other cytokines and chemokines. In fact, macrophage activation appears to result from a combination of cytokines and chemokines (Maher et al., 1998Go). We have also found that the mRNA expression of the cytokine macrophage migration inhibitory factor (MIF) is elevated in F-344 rat hepatocytes following 1,2-DCB treatment but not in SD cells (data not published). MIF has several functions in inflammation that include neutrophil priming, enhanced macrophage phagocytosis, and killing of parasites and counter-regulation of glucocorticoid-induced cytokine suppression (Swope and Lolis, 1999Go). Therefore, hepatocyte-derived MIF and CINC may also function to recruit migrating neutrophils to the liver. In addition to cytokines and chemokines, products of lipid peroxidation have recently been shown to activate KC. For example, 4-hydroxynonenal (4-HNE) has been shown to stimulate NF-{kappa}B activity and IL-6 production by KC (Luckey and Petersen, 2002Go). The production of 4-HNE is markedly elevated with 1,2-DCB treatment and is much greater in the F-344 rat (Hoglen et al., 1998Go).

An interesting finding in this study was the enhanced binding activity of EpRE to the promoter region of the RNQO1 gene. EpRE promotes the transcription of enzymes involved in the detoxification of electrophilic xenobiotics (Wilkinson and Clapper, 1997Go). The enhanced binding activity of EpRE to the RNQO1 gene in 1,2-DCB treated rat hepatocytes draws attention to the bioactivation of 1,2-DCB by the cytochrome P-450 enzymes and subsequent formation of reactive intermediates, especially quinones. Quinones may be formed as secondary metabolites of 1,2-DCB, but have not been detected as biotransformation products (Hissink et al., 1996Go). However, the upregulation of the transcription of the RNQO1 gene may be considered an indirect measure of their formation. The production of quinones with redox-cycling potential has been observed for other hepatotoxic halobenzenes (Mertens et al., 1995Go; Monks and Jones, 2002Go) and also may account for the hepatocellular oxidative stress associated with 1,2-DCB. Again, differences in oxidative stress and EpRE-binding activity between F-344 and SD rats support these findings.

In summary, this work demonstrates that the release of cytokines and chemokines by compromised hepatocytes may trigger KC activity and the subsequent cascade of inflammatory events that leads to the progression of hepatocellular injury. This suggests that hepatocytes can initiate or upregulate inflammatory cell activity that may ultimately result in their demise. We have identified CINC as one factor that may contribute to KC activation and other similar molecules likely exist. Additional work should focus on the identification of the factors released from hepatocytes that are involved in KC activation and the recruitment of other inflammatory cells to the liver. There is likely frequent communication between hepatocytes and KC during chemical-induced liver damage. This communication is highly complex and involves multiple factors that collectively mediate responses such as activation of KC, inflammatory cell recruitment, and death of hepatocytes. Therefore, the differences in susceptibility between F-344 and SD rats to 1,2-DCB may be due to any of these processes, but may also include differences in bioactivation and repair. For example, Mehendele and coworkers (Kulkarni et al., 1997Go, 1999Go; Soni et al., 1999Go) have demonstrated that enhanced repair processes in the SD rat may explain the differences in sensitivity between F-344 and SD rats to 1,2-DCB. The production of cytokines and chemokines by hepatocytes and/or KC may also contribute to tissue repair processes in the liver (Bone-Larson et al., 2001Go). It is likely that multiple pathways influence the susceptibility of these rat strains to 1,2-DCB and individuals to hepatotoxicants as a whole.


    ACKNOWLEDGMENTS
 
This research was supported, in part, by the Coca-Cola Foundation and Southwest Environmental Health Sciences Center Grant ES 06694. H.S.Y. was supported, in part, by an American Foundation for Pharmaceutical Education Fellowship and a Proctor and Gamble Fellowship.


    NOTES
 
1 To whom correspondence should be addressed at Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, campus P.O. 210207, Tucson, Arizona 85721. Fax: (520) 626-2466. E-mail: sipes{at}email.arizona.edu. Back


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