Department of Veterinary PathoBiology, * Toxicology Graduate Program, University of Minnesota, St. Paul, Minnesota 55108
Received February 24, 2000; accepted April 14, 2000
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ABSTRACT |
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Key Words: apoptosis; Bax; Bcl-XL; dimethylnitrosamine (DMN); Fas (CD95/APO-1); hepatotoxicity; TNF.
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INTRODUCTION |
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Apoptosis has been reported for various hepatotoxicants, including carbon tetrachloride (Shi et al., 1998), cadmium (Habeebu et al., 1998
), cyclohexamide (Ledda-Columbano et al., 1992
), 1,1-dichloroethylene (Reynolds et al., 1984
), actinomycin D (Leist et al., 1997
), and thioacetamide (Ledda-Columbano et al., 1991
), yet the molecular mechanism(s) of xenobiotic-induced toxicity remain poorly understood. Dimethylnitrosamine (DMN), a common environmental contaminant, induces hepatotoxicity and has carcinogenic potential in humans and animals. Liver-specific DMN metabolism leads to hepatic hemorrhaging and centrilobular necrosis (reviewed in Myers and Schook, 1996). Evidence also indicates that acute high-dose DMN exposure (> 25 mg/kg) induces an apoptotic mechanism that may contribute to toxicity (Pritchard and Butler, 1989
; Ray et al., 1992
). However, the extent of apoptosis following multiple, low-dose DMN exposure as well as the hepatic apoptotic genes involved have not been examined, despite accumulating liver injury observed under such treatment regimens (Myers and Schook, 1996
).
DMN hepatotoxicity produces a transient inflammatory response associated with elevated serum cytokines for tumor necrosis factor alpha (TNF), interleukin (IL)-1ß, and IL-6 (Schook et al., 1992
), and increased hepatic mRNA expression of acute-phase reactants (Bhattacharjee et al., 1998
; Horn et al., 1998
; Lockwood et al., 1994
). Increased hepatic TNF
mRNA expression has been observed in other xenobiotic-induced hepatotoxicity models (Blazka et al., 1995
; Bruccoleri et al., 1997
; Czaja et al., 1989
; Kayama et al., 1995
; Ksontini et al., 1998
; Luster et al., 1994
), suggesting a possible central role for hepatic TNF
in DMN-induced hepatotoxicity. In support of this, we recently detected increased hepatic transcripts for TNF
following acute, high-dose DMN exposure (Horn et al., 2000
).
The biologic activities of TNF are mediated by two structurally related but functionally distinct receptors, TNF
receptor (TNFR)-1 (p55) and TNFR-2 (p75) (Grell et al., 1994
; Tartaglia and Goeddel, 1992
; Tartaglia et al., 1993
). Whereas TNFR-1 signaling is the primary pathway in most cell types that mediate classical TNF
-related immune or inflammatory responses, TNFR-2 mediates several responses, including apoptosis of activated, mature T lymphocytes (Peschon et al., 1998
; Sheehan et al., 1995
; Zheng et al., 1995
). Recent evidence also links TNFR-2 signaling in mediating the migration of Langerhans' cells (Wang et al., 1996
, 1997
). Thus, the importance of TNF
in the pathophysiology of DMN-induced toxicity is of interest for dissecting the molecular pathways that impact hepatocellular injury.
Related proteins belonging to the Bcl-2 family regulate the apoptotic machinery. These include proteins that inhibit apoptosis, such as Bcl-2 and Bcl-XL, and others that promote apoptosis, including Bax, Bak, and Bad. The ratio of such Bcl-2 family dimers determines the fate of the cell (reviewed in Allen et al., 1998). Activation of the apoptotic machinery can be mediated by Fas (CD95/APO-1), a type I membrane protein belonging to the TNFR superfamily (reviewed in Nagata and Golstein, 1995) that has been implicated in massive liver apoptosis and fulminant hepatic failure (Ogasawara et al., 1993). To date, Fas-Fas ligand (FasL) interactions during xenobiotic exposure have not been extensively examined.
TNF has been shown to be a hepatoprotective agent that prevents Fas-mediated apoptosis during liver regeneration (Takehara et al., 1998
). Because TNF
is known to modulate apoptotic responses in the liver (Leist et al., 1995a
, 1995b
, 1997
; Rolfe et al., 1997
), the current study was undertaken to determine how signaling through TNF
influences the hepatic mRNA expression of apoptotic modulators during subacute, low-dose DMN exposure. Hepatic expression of TNFR, Fas, Fas signaling molecules, and the Bcl-2 family of apoptotic genes was measured in wild-type (WT) and TNFR-1/TNFR-2 (p55/p75/) double knockout (DKO) mice following subacute DMN (5.0 mg/kg) exposure. Results showed that hepatic transcripts for TNFR-1, Fas, Bcl-XL, and Bax are up regulated following DMN exposure. However, TNF
can regulate, but is not required for, apoptotic gene transcript expression in the liver during DMN hepatotoxicity.
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MATERIALS AND METHODS |
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RNA isolation.
Liver lobes were thawed and homogenized in 4 ml Trizol (Life Technologies, Grand Island, NY) using baked, frosted-end slides. Total cellular RNA was extracted according to manufacturer's directions. RNA was resuspended in RNase-free water, quantitated using UV spectrophotometry, and stored at 80°C.
Reverse transcription-polymerase chain reaction (RT-PCR).
Chromosomal DNA contamination in total liver RNA was removed by DNase I digestion (Promega, Madison, WI). RT was performed in a 20-µl volume that contained 5 µM oligo dT12-18, 2 µg total liver RNA, 200 U SuperscriptTM II reverse transcriptase (Life Technologies) at 24°C for 10 min followed by 42°C for 1 h. In a total volume of 20 µl, the PCR mixture contained 150 µM dNTPs, 1 µM antisense and sense primers for either Fas, hypoxanthine phosphoribosyl transferase (HPRT), or TNF, 1 µl reverse-transcribed cDNA, and 2 U Taq polymerase (PE Applied Biosystems, Foster City, CA). The sequences of oligonucleotide primers were: 5`-Fas, CGC CTA TGG TTG TTG ACC; 3`-Fas, CTC CAG ACA TTG TCC TTC; 5`-HPRT, CAG TAC AGC CCC AAA ATG; 3`-HPRT, ACT TGC GCT CAT CTT AGG; 5`-TNF
, ACA GAA AGC ATG ATC CGC; 3`-TNF
, GTA GAC CTG CCC GGA CTC. Amplification conditions were (94°C 15 s, 54°C 1 min, 72°C for 30 s) for 1535 cycles. The expected amplicon lengths were 148 bp for HPRT, 477 bp for Fas, and 692 bp for TNF
. An aliquot of the RT-PCR reactants (10 µl) was separated on a 1.2% agarose gel containing ethidium bromide, visualized under UV light, and analyzed using NIH Image software (http://rsb.info.nih.gov/nih-image/).
RT-PCR Southern blotting.
TNF cDNA clone was obtained from American Type Culture Collection (Rockville, MD). Reverse-transcribed HPRT transcripts were amplified by gene-specific oligonucleotide primers and cloned into EcoRV site of pBluescript SK+ by a modified TA cloning method (Bhattacharjee et al., 1997
). TNF
and HPRT RT-PCR amplicons were separated on 1.2% agarose gels as described above. Double-stranded linearized or PCR-derived cDNAs for TNF
and HPRT, respectively, were labeled with [
-32P]-dATP using RadPrime labeling kit (Life Technologies), and Southern blotting was performed according to standard protocols (Sambrook et al., 1989
). Hybridization was conducted at 42°C for 1 h, followed by two 15-min washes at 24°C in 1x SSC, 0.1% SDS, followed by a final wash at 4050°C in 0.25x SSC, 0.1% SDS. Blots were analyzed using phosphorimagery (Molecular Dynamics, Sunnyvale, CA).
Multi-probe RNase protection assay (RPA).
The DNA templates (mCR4, mAPO2, and mAPO3), in vitro transcription and RPA kits were purchased from PharMingen (San Diego, CA). Briefly, RNA probes were generated using 1 µg of template DNA and in vitro transcribed using T7 RNA polymerase. Yeast tRNA (10 µg) and PharMingen-supplied mouse control RNA (2 µg) were used as negative and positive controls, respectively. Total liver RNA (10 µg) was dried and resuspended in 2.44 x 105 cpm/µg and hybridized at 56°C for 1618 h. Following hybridization, RNA samples were RNase A/T1 digested for 45 min at 30°C, followed by incubation with Proteinase K for 15 min at 37°C, then ethanol precipitated. Protected RNAs were separated on a 6% polyacrylamide/7 M urea denaturing gel. Following electrophoresis, the gels were dried and exposed to phosphorimager screens or X-ray film, and bands were quantitated using phosphorimagery or densitometry, respectively (Molecular Dynamics).
DNA fragmentation.
DNA isolation for fragmentation assay was modified from the protocol described by Habeebu et al. (1998). Briefly, liver lobes (100 mg) were minced and homogenized in 500 µl lysis buffer. Proteinase K ([250 µg/ml] Sigma) was added to the homogenate and incubated for 16 h at 55°C. Thereafter, 100 µg/ml RNase A (Sigma) was added and the reaction was incubated at 37°C for 1 h. DNA was phenol, phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform/isoamyl alcohol (24:1) extracted, ethanol precipitated, and resuspended in TE buffer. DNA samples (2 µg) were radiolabeled by incubation with [-32P]-dCTP and Klenow enzyme at room temperature for 15 min (Rösl, 1992
). Equal amounts of labeled DNA were separated on 1.8% agarose gels. One microgram of an EcoRI/HindIII digest of lambda DNA (Sigma) served as a molecular weight marker. Following electrophoresis, gels were dried and exposed to X-ray film.
Statistics.
All hepatic mRNA expression data are presented as the mean ± SE (n = 34). Relative concentration of mRNA transcripts was analyzed by two-sample Student's t test, and a 95% confidence level was used to define statistical significance.
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RESULTS |
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Hepatic Expression of the Bcl-2 Family of Apoptosis Regulatory Genes
Because hepatic TNFR and Fas and their associated signaling pathways appear intact during subacute DMN exposures, we measured expression of the Bcl-2 family members Bcl-2, Bcl-XL, Blf-1, and Bcl-W that are implicated in preventing apoptosis. In addition, we examined the expression of Bax, Bad, Bak, and Bcl-XS that are known to promote apoptosis (Allen et al., 1998). Results indicated that during DMN exposure, hepatic transcripts for Bcl-XL and Bax were maximally induced in both WT and TNFR DKO mice following 4 daily DMN exposures (Fig. 4
), and remained elevated following 7 daily exposures in both WT and TNFR DKO mice compared to vehicle-exposed mice. Interestingly, following a single DMN exposure, significant increases in Bcl-XL and Bax transcripts were observed only in TNFR DKO mice. Thus, hepatic expression of Bcl-XL and Bax, two opposing members of the Bcl-2 family, are up regulated during DMN exposure, but TNF
signals are not required for this induction. However, following 4 and 7 daily DMN exposures, the relative mRNA abundance for Bax from TNFR DKO mice was statistically higher (p < 0.05) compared to WT mice (Fig. 4B
), suggesting a role for TNF
in down regulating hepatic Bax expression during prolonged DMN exposure.
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DMN-Induced DNA Fragmentation
In order to determine whether the observed changes in apoptotic gene expression are reflected by hepatocellular death, we examined the extent of DNA fragmentation as an indicator of apoptosis (Compton, 1992) via separation of 32P-labeled total genomic liver DNA. Faint DNA ladders in WT and TNFR DKO mice were detected in naive mice or mice exposed to vehicle and are indicative of normal liver homeostasis. In contrast, WT and TNFR DKO mice receiving 1, 4, or 7 DMN (5.0 mg/kg) exposures showed considerably more DNA laddering, consistent with apoptotic DNA degradation (Fig. 5A
). However, DMN-induced DNA laddering was nearly equivalent in WT and TNFR DKO mice at all times examined. Because apoptosis has been implicated following high-dose (> 25 mg/kg) DMN exposure (Pritchard and Butler, 1989
; Ray et al., 1992
), we examined DNA fragmentation in WT and TNFR DKO mice. WT mice were exposed to DMN (15 or 25 mg/kg) for up to 3 days, and were sacrificed 6 h postexposure on days 1 and 3 as additional positive controls for apoptotic DNA fragmentation. Following 3 daily exposures to DMN (15 or 25 mg/kg), WT mice showed extensive DNA laddering (Fig. 5B
). Because we have observed differential effects of acute high-dose DMN exposure in WT versus TNFR DKO mice (Horn et al., 2000
), mice were treated with 100 mg/kg of DMN. Whereas DNA smearing was primarily observed at 6 h postexposure in both WT and TNFR DKO mice, at 24 h DNA fragmentation was evident in WT mice. In contrast, TNFR DKO mice still showed smearing of DNA at 24 h postexposure, suggesting necrotic DNA degradation (Fig. 5B
). Taken together, low-dose DMN exposures induce hepatic apoptosis in both WT and TNFR DKO mice.
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DISCUSSION |
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TNF transcripts were detected in the liver during subacute, low-dose DMN exposures (Fig. 1A
). This pleiotropic cytokine has been implicated in multiple forms of experimental liver injury, including hepatitis induced by D-galactosamine/endotoxin exposure or concanavalin A treatment after transcriptional block (Ksontini et al., 1998
; Leist et al., 1995b
, 1997
). We previously demonstrated biologically active TNF
in the serum through 3 daily DMN (5.0 mg/kg) exposures (Schook et al., 1992
). Our current studies show that the liver is a potential source for TNF
during DMN exposure, consistent with other models of xenobiotic-induced hepatotoxicity that have associated TNF
expression in the liver with liver pathology (Blazka et al., 1995
; Bruccoleri et al., 1997
; Czaja et al., 1989
; Kayama et al., 1995
). It remains to be determined whether resident Kupffer cells, hepatocytes, vascular endothelial cells, or infiltrating immune cells account for up-regulated expression of TNF
during DMN exposure. We also observed time-dependent up-regulated transcript levels for TNFR-1, and down-regulated TNFR-2 transcript levels during DMN treatment, suggesting that the quality of the TNF
signal changes throughout the course of exposure.
Following a single daily exposure, a vehicle-induced response was observed, as evidenced by up regulation of TNF transcripts that subsided following 4 and 7 daily exposures (Fig. 1A
). This is consistent with our previous findings showing a transient increase in inflammatory and acute-phase gene expression following a single PBS administration (Horn et al., 2000
; Lockwood et al., 1991
, 1994
; Schook et al., 1992
) and illustrates the importance of comparing naive and vehicle controls when considering molecular data.
Biological responses mediated by Fas and TNFR are dependent on receptor-associated signaling complex proteins. We report here that during DMN hepatotoxicity, stimulation by TNF is not necessary to increase Fas, Bax, and Bcl-XL transcripts within the liver, as evidenced by similar hepatic mRNA expression profiles in TNFR DKO mice as compared to WT mice (Figs. 24
). However, TNFR DKO mice have an increased relative expression of Fas and Bax mRNA compared to WT mice (Figs. 3C and 4D
). These data suggest that TNF
, directly or indirectly by an unknown mechanism(s), appears to limit hepatic Fas and Bax expression following DMN exposure. Thus, hepatic TNF
signals during low-dose, subacute DMN exposure impact the balance of receptors and proteins that regulate apoptosis/cell survival.
Fas-FasL interactions are well documented to induce apoptosis in a variety of cells. Susceptibility toward Fas-mediated apoptosis depends on expression of the Fas receptor, intact Fas-signaling pathways, and the absence of apoptosis-inhibiting molecules that interfere with Fas signal transduction pathways. During DMN-induced liver injury, damaged hepatocytes may be eliminated by up regulating their constitutive expression of Fas. The lethal signal to remove injured cells is potentially delivered by infiltrating or resident immune cells that bear FasL. In support of this, we have observed that following low-dose, subacute DMN exposure, infiltration of immune cells into the liver is significantly increased in WT mice as compared to TNFR DKO mice following day 7 (manuscript in preparation). Therefore, TNFR DKO mice exposed to DMN have fewer infiltrating cells that potentially express FasL, and removal of Fas-positive hepatocytes may be impaired. This may explain the observation that TNFR DKO mice express higher hepatic Fas transcripts. However, even though DMN-induced up regulation of Fas in centrilobular hepatocytes can render hepatocytes more susceptible to apoptosis, our results suggest that FasL may not be involved in initiating apoptotic responses under the DMN exposure tested. We were unable to detect hepatic FasL transcripts by RPA (Fig. 3) or RT-PCR (data not shown), even in WT mice. It may be that the numbers of FasL-positive infiltrating cells are too low for our detection capabilities. Similar to our results for low-dose, subacute DMN exposures, a recent study using gld-lpr and lprcg mutant mice that do not express FasL/Fas or express a dysfunctional Fas receptor, respectively, suggested that hepatic apoptosis induced by acute, high-dose (30 mg/kg) DMN is not mediated by Fas-FasL signals (Oyaizu et al., 1997
). If FasL is not present in the hepatic microenvironment, the apoptosis we observed (Fig. 5
) must be affected by other modulators downstream of Fas ligation or via Fas/FasL-independent mechanisms (Evan and Littlewood, 1998
; Zheng et al., 1995
).
The best described apoptotic-signaling pathway triggered after Fas-FasL or TNFR ligation involves the activation of caspase-8 via the adapter protein FADD. Other pathways such as c-Jun kinase and ceramide have also been proposed (Ashkenazi and Dixit, 1998). Any one of these pathways is sufficient to activate downstream caspase cascades, including caspase-3, which can mediate rapid cellular death. Our observations suggest that components of Fas- and TNF
-mediated signal transduction pathways (FADD, TRADD, and caspase-8) are intact and unaltered at the steady-state mRNA level in both WT and TNFR DKO mice following DMN exposure. In addition, these pathways do not appear to be TNF induced during DMN hepatotoxicity, as their transcript levels in TNFR DKO mice are identical to those found in WT mice (Figs. 3A and 3B
).
We examined hepatic mRNA expression of prosurvival or proapoptotic genes during DMN exposure because these genes may modulate Fas- and/or TNF-mediated hepatocellular disruption. We observed DMN-induced up regulation of Bcl-XL mRNA that peaked following 4 daily DMN exposures in WT and TNFR DKO mice. Hepatic mRNA levels for Bax, a protein that promotes apoptosis, were induced in both WT and TNFR DKO mice across all DMN exposures. The prosurvival proteins Bcl-2 and Bcl-XL that act downstream of caspase-3 activation or Fas activation have been shown to prevent apoptosis (Allen et al., 1998
; Boise and Thompson, 1997
; Green and Reed, 1998
; Itoh et al., 1993
; Kren et al., 1996
). Although Bcl-XL and Bax have opposing functions, they can form heterodimeric complexes to control caspase activation (Adams and Cory, 1998
; Sato et al., 1994
; Sedlak et al., 1995
). Overexpression of Bax can prevent binding of Bcl-XL to members of the caspase pathway and thereby activate caspases to promote apoptosis. Recent evidence suggests that when Bax is not complexed with Bcl-XL, Bax homodimers form pores in mitochondria to disrupt mitochondrial membrane transition potential and release cytochrome c, thereby indirectly activating caspases (Pan et al., 1998
). Whereas Bcl-XL mRNA levels in WT mice increase less than 2-fold (Fig. 4C
), Bax transcripts are induced approximately 3-fold (Fig. 4D
). Thus, the Bcl-XL:Bax ratio changes toward one that favors apoptosis. DNA fragmentation patterns (Fig. 5A
) support that apoptosis is pronounced by day 4 of low-dose DMN exposure. Furthermore, the liver contains relatively small concentrations of the Bcl-XL gene as compared to other tissues such as the brain or bone marrow (Boise et al., 1993
), therefore, a high hepatic Bcl-XL:Bax ratio is unlikely, further favoring apoptosis. Taken together, liver damage following DMN exposure is potentially mediated by the balance of hepatic expression of proapoptotic and antiapoptotic pathways that include Bax.
DMN-induced up regulation of Fas, Bcl-XL, and Bax hepatic mRNAs was observed in TNFR DKO mice, indicating that TNF is not necessary for their induction during DMN liver injury. However, hepatic Bax mRNA expression following 4 and 7 daily DMN exposures was greater (p < 0.05) in TNFR DKO mice as compared to WT mice. This suggests that TNF
production may actually work to counter hepatocellular disruption by down regulating hepatic Fas and Bax expression. This is consistent with a model in which hepatic apoptosis during low-dose, subacute DMN exposure is in part regulated by TNF
, TNFR-1, and Fas signals that affect the relative balance of Bcl-XL and Bax. This antiapoptotic response mediated by TNF
may serve to prevent undue death of hepatocytes. Despite greater Bax transcripts in TNFR DKO mice exposed to DMN (Fig. 4D
), we did not observe greater DNA laddering compared to WT mice (Fig. 5A
).
Previous studies revealed that WT mice have a much higher ratio of Bcl-XL:Bax transcripts than TNFR DKO mice 24 h following high-dose DMN (100 mg/kg) exposure (Horn et al., 2000). Genomic DNA prepared from TNFR DKO mice at 24 h postexposure did not show DNA laddering as did WT mice (Fig. 5B
). This may be the result of continued liver necrosis or because necrotic cells are not removed from the liver between 6 and 24 h, as is likely occurring in WT mice. It is unclear if Bcl-XL, Bax, or Fas expression are related to this observation. It is also interesting to consider that critically injured cells that potentially harbor DMN-induced DNA mutations may be protected from removal by antiapoptotic effects of Bcl-XL expression. In that DMN causes liver carcinogenesis and the maximal mutation frequency is observed following the 7th daily DMN (5.0 mg/kg) exposure (Souliotis et al., 1998
), TNF
has the potential to promote development of liver carcinogenesis by suppressing apoptotic removal of damaged hepatocytes. Thus, the impact of TNF
on individual processes associated with DMN hepatotoxicity must be carefully evaluated at low-dose, multiple exposures as well as high-dose, acute exposures.
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ACKNOWLEDGMENTS |
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NOTES |
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1 Current address: VA Medical Center, Research Service (151), 1 Veterans Dr., Minneapolis, MN 55417.
2 Current address: Dana-Farber Cancer Institute, Adult Oncology, Mayer 433, 44 Binney St., Boston, MA 02115.
3 To whom correspondence should be addressed at Department of Veterinary PathoBiology, University of Minnesota, 295e Animal Science/Veterinary Medicine, 1988 Fitch Ave., St. Paul, MN 55108. Fax: (612) 625-0204. E-mail: ruthe003{at}tc.umn.edu.
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