* Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721; and
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
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ABSTRACT |
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Key Words: Kupffer cell; Fischer-344 rat; Sprague-Dawley rat; 1,2-dichlorobenzine.
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INTRODUCTION |
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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, 1990; Kuester et al., 2002
; Laskin and Pedino, 1995
; Laskin et al., 1986
; Sipes et al., 1989
). 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-
) 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., 1998
; Horbach et al., 1997
; Spitzer et al., 1996
; Yamada et al., 1999
). 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-
and CINC following exposure to hydrogen peroxide (Horbach et al., 1997
), heavy metals (Dong et al., 1998
), and during conditions of ischemia (Kataoka et al., 2002
). The processes that mediate cytokine/chemokine production and release by hepatocytes are unclear but may involve oxidative stress (Horbach et al., 1997
).
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., 2000). 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-
B), activator protein1 (AP-1) and electrophile responsive element (EpRE) transcription factor-binding activity and the production of TNF-
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.
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MATERIALS AND METHODS |
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Animals.
Male F-344 (161190 g, 89 weeks old) and SD (175199 g, 89 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). Briefly, animals were anesthetized with pentobarbital (50 mg/kg, ip) and the portal vein was exposed. Hanks 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., 2000). 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). 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-
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-
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). Briefly, 50 ml of internal standard,
-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).
Determination of nuclear transcription factor-binding activity.
NF-B- AP-1-, and EpRE-bnding activity were determined using the electrophoretic mobility shift analysis as described by Dignam et al. (1983)
. Briefly, hepatocytes (2 x 106 cells) or KC (1012 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 (2.55 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-
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- and CINC.
CINC and TNF- 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 SDSPAGE followed by transfer to a nitrocellulose membrane (Stamer et al., 1994
). The membrane was incubated with rabbit monoclonal antibody against rat CINC-1 protein (Peptide International, Louisville, KY) or rabbit polyclonal antibody against mouse TNF-
(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). 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 (179) 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). 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 (115 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 Bonferronis 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).
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RESULTS |
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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. 6). The background labeling of the secondary antibodies was minimal.
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DISCUSSION |
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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., 1993; Hoglen et al., 1998
; Kulkarni et al., 1997
, 1999
). In the SD rat, maximal liver injury occurs later (48 h post dose) and is less pronounced. Figure 8
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., 2000
). 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)
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., 1993
; Hoglen et al., 1998
). However, the role of hepatocellular oxidative stress in 1,2-DCB-induced liver damage is less clear.
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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-B and AP-1 transcription-factor activation (Bowie and ONeill, 2000
; Karin et al., 2001
). Indeed, the results of this study demonstrate enhanced DNA-binding activity of AP-1 and NF-
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-
B was approximately 2-fold greater in F-344 than in SD rat hepatocytes. The enhanced binding activity of NF-
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., 2000
). 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., 2000
).
NF-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-
B upregulates the transcription of cytokines and chemokines such as TNF-
and CINC (Horbach et al., 1997
), which function, at least in part, to activate inflammatory cells. TNF-
can prime KC for activation, and CINC enhances the migration of neutrophils and their production of reactive oxygen species (Beyaert and Fiers, 1994
; Thornton et al., 1992
). The expression of CINC, TNF-
or other cytokines have been observed in hepatocytes treated with the hepatotoxicants ethanol (Spitzer et al., 1996
; Yamada et al., 1999
), acetaminophen (Dambach et al., 2002
; Horbach et al., 1997
), cadmium, and hydrogen peroxide (Dong et al., 1998
). 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-
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-
B, as this redox-sensitive dimeric protein promotes the transcription of the CINC gene (Lakshminarayanan et al., 1998
; Ohtsuka et al., 1996
; Ren et al., 2002
). Therefore, the increase in NF-
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-B and produce increased levels of nitric oxide. Classic activators of KC function such as LPS (Okada et al., 2000
), ethanol (Tran-Thi et al., 1995
; Tsukamoto et al., 1999
) and TNF-
(Tran-Thi et al., 1995
) also trigger increased NF-
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., 1999
). While the expression of CXCR1 is exclusive to neutrophils, CXCR2 is also expressed by monocytes (Saccani et al., 2000
). 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., 1998
). 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, 1999
). 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-
B activity and IL-6 production by KC (Luckey and Petersen, 2002
). 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., 1998
).
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, 1997). 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., 1996
). 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., 1995
; Monks and Jones, 2002
) 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., 1997, 1999
; Soni et al., 1999
) 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., 2001
). It is likely that multiple pathways influence the susceptibility of these rat strains to 1,2-DCB and individuals to hepatotoxicants as a whole.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bone-Larson, C. L., Hogaboam, C. M, Evanhoff, H., Strieterm R. M., and Kunkel, S. L. (2001). IFN-gamma-inducible protein-10 (CXCL10) is hepato-protective during acute liver injury through the induction of CXCR2 on hepatocytes. J. Immunol. 167, 70777783.
Bowie, A., and ONeill, L. A. (2000). Oxidative stress and nuclear factor-kappaB activation: A reassessment of the evidence in the light of recent discoveries. Biochem. Pharmacol. 59, 1323.[CrossRef][ISI][Medline]
Dambach, D. M., Watson, L. M., Gray, K. R., Durham, S. K., and Laskin, D. L. (2002). Role of CCR2 in macrophage migration into the liver during acetaminophen-induced hepatotoxicity in the mouse. Hepatology 35, :10931103.[CrossRef][ISI][Medline]
Decker, K. (1990). Biologically active products of stimulated liver macrophages (Kupffer cells). Eur. J. Biochem. 192, 245261.[ISI][Medline]
Dignam, J. D., Martin, P. L., Shastry, B. J., and Roeder, R. G. (1983). Eukaryotic gene transcription with purified components. Methods Enzymol. 101, 582598.[ISI][Medline]
Dong, W., Simeonova, P., Gallucci, R., Matheson, J., Flood, L., Wang, S., Hubbs, A., and Luster, M. I. (1998). Toxic metals stimulate inflammatory cytokines in hepatocytes through oxidative stress mechanisms. Toxicol. Appl. Pharmacol. 151, 359366.[CrossRef][ISI][Medline]
Fariss, M. W., and Reed, D. J. (1987). High-performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives. Methods Enzymol. 143, 101109.[ISI][Medline]
Fujino, H., Pierce, K. L., Srinivasan, D., Protzman, C. E., Krauss, A. H., Woodward, D. F., and Regan, J. W. (2000). Delayed reversal of shape change in cells expressing FP(B) prostanoid receptors: Possible role of receptor resensitization. J. Biol. Chem. 275, 2990729914.
Green, L., Wagner, D., Glowgoski, J., Skipper, P., Wishnok, J., and Tannenbaum, S. (1992). Analysis of nitrate, nitrite, and [15N]nitrite in biological fluids. Anal. Biochem. 126, 131138.
Gunawardhana, L., Mobley, S. A., and Sipes, I. G. (1993). Modulation of 1,2-dichlorobenzene hepatotoxicity in the Fischer-344 rat by a scavenger of superoxide anions and an inhibitor of Kupffer cells. Toxicol. Appl. Pharmacol. 119, 205213.[CrossRef][ISI][Medline]
Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. (1993). Identification of an oncoprotein and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev. 7, 21352148.[Abstract]
Hissink, A., Oudshoorn, M., Van Ommen, B., Guido, R., Haenen, M., and Van Bladeren, P. (1996). Differences in cytochrome P450-mediated biotransformation of 1,2-dichlorobenzene by rat and man: Implications for human risk assessment. Chem. Res. Toxicol. 9, 12491256.[CrossRef][ISI][Medline]
Hoglen, N. C., Younis, H. S., Hartley, D. P., Gunawardhana, L., and Sipes, I. G. (1998).1,2-Dichlorobenzene-induced lipid peroxidation in male Fischer-344 rats is Kupffer-cell dependent. Toxicol. Sci. 46, 376385.[Abstract]
Horbach, M., Gerer, E., and Kahl, R. (1997). Influence of acetaminophen treatment and hydrogen peroxide treatment on the release of a CINC-related protein and TNF- from rat hepatocyte cultures. Toxicology 121, 117126.[CrossRef][ISI][Medline]
Jaeschke, H., and Benzick, A. E. (1992). Pathological consequences of enhanced intracellular superoxide formation in isolated perfused rat liver. Chem.-Biol. Interact. 84, 5568.[CrossRef][ISI][Medline]
Karin, M., Takahashi, T., Kapahi, P., Delhase, M., Chen, Y., Makris, C., Rothwarf, D., Baud, V., Natoli, G., Guido, F., and Li, N. (2001) Oxidative stress and gene expression: The AP-1 and NF-B connections. Biofactors 15, 8789.[ISI][Medline]
Kataoka, M., Shimizu, H., Mitsuhashi, N., Ohtsuka, M., Wakabayashi, Y., Ito, H., Kimura, F., Nakagawa, K., Yoshidome, H., Shimizu, Y., et al. (2002). Effect of cold-ischemia time on C-X-C chemokine expression and neutrophil accumulation in the graft liver after orthotopic liver transplantation in rats. Transplantation 73, 17301735.[ISI][Medline]
Kuester, R. K., Waalkes, M. P., Goering, P. L., Fisher, B. L., McCuskey, R. S., and Sipes, I. G. (2002). Differential hepatotoxicity induced by cadmium in Fischer 344 and Sprague-Dawley rats. Toxicol. Sci. 65, 151159.
Kulkarni, S. G., Harris, A. J., Casciano, D. A., and Mehendale, H. M. (1999). Differential protooncogene expression in Sprague-Dawley and Fischer-344 rats during 1,2-dichlorobenzene-induced hepatocellular regeneration. Toxicology 139, 119127.[CrossRef][ISI][Medline]
Kulkarni, S. G., Warbritton, A., Bucci, T. J., and Mehendale, H. M. (1997). Antimitotic intervention with colchicine alters the outcome of o-DCB-induced hepatotoxicity in Fischer 344 rats. Toxicology 120, 7988.[CrossRef][ISI][Medline]
Lakshminarayanan, V., Weiss, E., and Roebuck, K. (1998). H2O2 and tumor necrosis factor- induce differential binding of the redox-responsive transcription factors AP-1 and NF-
B to the interleukin-8 promoter in endothelial and epithelial cells. J. Biol. Chem. 273, 3267032678.
Laskin, D. L., and Pedino, K. J. (1995). Macrophages and inflammatory mediators in tissue injury. Annu. Rev. Pharmacol. Toxicol. 35, 655677.[CrossRef][ISI][Medline]
Laskin, D. L., Pilaro, A. M., and Ji, S. (1986). Potential role of activated macrophages in acetaminophen hepatotoxicity. Toxicol. Appl. Pharmacol. 86, 216226.[ISI][Medline]
Luckey, S. W., and Petersen, D. R. (2002). Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats. Exp. Mol. Pathol. 71, 226240.[CrossRef][ISI]
Maher, J. J., Lozier, J. S., and Scott, M. K. (1998). Rat hepatic stellate cells produce cytokine-induced neutrophil chemoattractant in culture and in vivo. Am. J. Physiol. 275, G847853.[ISI][Medline]
McQueen, C. A., Rosado, R. R., and Williams, G. M. (1989). Effect of nalidixic acid on DNA repair in rat hepatocytes. Cell. Biol. Toxicol. 5, 201206.[ISI][Medline]
Mertens, J. J., Gibson, N. W., Lau, S. S., and Monks, T. J. (1995). Reactive oxygen species and DNA damage in 2-bromo-(glutathion-S-yl) hydroquinone-mediated cytotoxicity. Arch. Biochem. Biophys. 320, 5158.[CrossRef][ISI][Medline]
Monks, T. J., and Jones, D. C. (2002). The metabolism and toxicity of quinones, quinonimines, quinone methides, and quinone-thioethers. Curr. Drug. Metab. 3,:425438.[Medline]
NTP (National Toxicology Program) (1985). Technical Report Series No. 255. Toxicology and carcinogenesis studies of 1,2-dichlorobenzene (CAS No. 95501) in F-344/N and B6C3F1 mice (gavage studies). U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health. NIH Publication No. 86, 2511. Research Triangle Park, NC.
Ohtsuka, T., Kubota, A., Hirano, T., Watanabe, K., Yoshida, H., Tsurufuji, M., Lizuka, Y., Konishi, K., and Tsurufuji, S. (1996). Glucocorticoid-mediated gene suppression of rat cytokine-induced neutrophil chemoattractanct CINC/gro, a member of the interleukin-8 family, through impairment of NF-B activation. J. Biol. Chem. 71, 16511659.[CrossRef]
Okada, K., Marubayashi, S., Fukuma, K., Yamada, K., and Dohi, K. (2000). Effect of the 21-aminosteroid on nuclear factor-kappa B activation of Kupffer cells in endotoxin shock. Surgery. 127, 7986.[CrossRef][ISI][Medline]
Petering, H., Gotze, O., Kimmig, D., Smolarski, R., Kapp, A., and Elsner, J. (1999). The biologic role of interleukin-8: Functional analysis and expression of CXCR1 and CXCR2 on human eosinophils. Blood 93, 694702.
Ren, X., Kennedy, A., and Colletti, L. M. (2002). Cxc chemokine expression after stimulation with interferon-gamma in primary rat hepatocytes in culture. Shock. 6,:513520.
Saccani, A., Saccani, S., Orlando, S., Sironi, M., Bernasconi, S., Ghezzi, P., Mantovani, A., and Sica, A. (2000). Redox regulation of chemokine receptor expression. PNAS 97, 27612766.
Sauer, J. M., Waalkes, M. P., Hooser, S. B., Kuester, R. K., McQueen, C. A., and Sipes, I. G. (1997). Suppression of Kupffer-cell function prevents cadmium-induced hepatocellular necrosis in the male Sprague-Dawley rat. Toxicology 121, 155164.[CrossRef][ISI][Medline]
Sipes, I. G., Elsisi, A. E., Sim, W., Mobley, S., and Earnest, D. (1989). Role of reactive oxygen species secreted by activated Kupffer cells in the potentiation of carbon tetrachloride hepatotoxicity by hypervitaminosis A. In Cells of the Hepatic Sinusoid (E. Wisse, D. Knook, and K. Decker, Eds.), pp. 2, 376379. Elsevier Biomedical Press, New York.
Soni, M. G., Ramaiah, S. K., Mumtaz, M. M., Clewell, H., and Mehendale, H. M. (1999). Toxicant-inflicted injury and stimulated tissue repair are opposing toxicodynamic forces in predictive toxicology. Regul. Toxicol. Pharmacol. 29(2, Pt 1), 165174.[CrossRef][ISI][Medline]
Spitzer, J. A., and Zhang, P. (1996). Gender differences in phagocytic response in the blood and liver, and the generation of cytokine-induced neutrophil chemoattractant in the liver of acutely ethanol-intoxicated rats. Alcohol. Clin. Exp. Res. 20, 914920.[ISI][Medline]
Stamer, W., Snyder, R., Smith, B., Agre, P., and Regan, J. (1994). Localization of aquaporin chip in the human eye: Implications in the pathogenesis of glaucoma and other disorders of ocular fluid balance. Investig. Ophthalmol. Vis. Sci. 35, 38673872.[Abstract]
Stine, E., Gunawardhana, L., and Sipes, I. G. (1991). The acute hepatotoxicity of the isomers of dichlorobenzene in Fisher-344 and Sprague-Dawley rats: Isomer-specific and strain-specific differential toxicity. Toxicol. Appl. Pharmacol. 109, 472481.[ISI][Medline]
Swope, M. D., and Lolis, E. (1999). Macrophage migration inhibitory factor: Cytokine, hormone, or enzyme? Rev. Physiol. Biochem. Pharmacol. 139, 132.[ISI][Medline]
Thornton, A. J., Ham, J., and Kunkel, S. L. (1992). Kupffer cell-derived cytokines induce the synthesis of a leukocyte chemotactic peptide, interleukin-8, in human hepatoma and primary hepatocyte cultures. Hepatology 15, 11121122.[ISI][Medline]
Tran-Thi, T. A., Decker, K., and Baeuerle, P. A. (1995). Differential activation of transcription factors NF-B and AP-1 in rat liver macrophages. Hepatology 22, 613619.[Medline]
Tsukamoto, H., Lin, M., Ohata, M., Giulivi, C., French, S. W., and Brittenham, G. (1999). Iron primes hepatic macrophages for NF-B activation in alcoholic liver injury. Am. J. Physiol. 277(6, Pt.1), G12401250.[ISI][Medline]
Wilkinson, J., and Clapper, M. L. (1997). Detoxification enzymes and chemoprevention. Proc. Soc. Exp. Bio. Med. 216, 192200.[Abstract]
Yamada, S., Matsuoka, H., Harada, Y., Momosaka, Y., Isumi, H., Kohno, K., Yamaguchi, Y., and Eto, S. (1999). Effect of long-term ethanol consumption on ability to produce cytokine-induced neutrophil chemattractant-1 in the rat liver and its gender difference. Alcohol. Clin. Exp. Res. 23(Suppl. 4), 6166S.
Younis, H. S., Hoglen, N. C., Kuester, R. K., Gunawardhana, L., and Sipes, I. G. (2000).1,2-Dichlorobenzene-mediated hepatocellular oxidative stress in Fischer-344 and Sprague-Dawley rats. Toxicol. Appl. Pharmacol. 163, 141148.[CrossRef][ISI][Medline]
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