Altered Hepatic mRNA Expression of Apoptotic Genes during Dimethylnitrosamine Exposure

Thomas L. Horn*,1, Arindam Bhattacharjee2, Lawrence B. Schook* and Mark S. Rutherford*,3

Department of Veterinary PathoBiology, * Toxicology Graduate Program, University of Minnesota, St. Paul, Minnesota 55108

Received February 24, 2000; accepted April 14, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of TNF{alpha} in regulating apoptotic signaling was investigated during subacute, low-dose (5.0 mg/kg) dimethylnitrosamine (DMN)-induced hepatotoxicity. In TNF{alpha} receptor (TNFR) intact (wild-type, WT) mice following 4 and 7 DMN exposures, hepatic transcripts for TNF{alpha} and TNFR-1 were elevated as compared to vehicle controls. DMN hepatotoxicity in WT and TNFR-1/TNFR-2 double knockout (DKO) mice were then compared over a 7-d exposure period. Liver RNA was isolated to measure hepatic expression of TNF{alpha}/Fas-related genes and the Bcl-2 family of genes that impact apoptosis. Hepatic mRNA levels for Fas, the apoptosis-promoting gene Bax, and the anti-apoptotic gene, Bcl-XL, were up regulated following 4 and 7 DMN exposures in both WT and TNFR DKO mice as compared to vehicle controls. Notably, hepatic transcript levels for Bax were higher in TNFR DKO mice treated with DMN compared to identically treated WT mice. However, we detected approximately equal DMN-induced apoptotic degradation of liver DNA following 1, 4, and 7 exposures in WT and TNFR DKO mice. Taken together, these data show DMN-induced hepatic TNF{alpha} expression and suggest that TNFR-1 signaling may be up regulated following 4 and 7 daily DMN exposures. However, TNF{alpha} is not required for apoptotic signaling at the mRNA transcript level within the liver and instead may actually decrease Bax production.

Key Words: apoptosis; Bax; Bcl-XL; dimethylnitrosamine (DMN); Fas (CD95/APO-1); hepatotoxicity; TNF.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The phenomenon of programmed cell death, termed apoptosis, is central to development and homeostasis. Apoptosis can be triggered by developmental or environmental stimuli that activate diverse cellular events that eventually culminate in cellular death. Although necrosis is a passive process that results in the rupture of cellular membranes and release of cytosolic contents, apoptosis is characterized by DNA fragmentation, chromatin condensation, membrane blebbing, cell shrinkage and disassembly into membrane-enclosed vesicles (Searle et al., 1982Go; Wyllie et al., 1980Go).

Apoptosis has been reported for various hepatotoxicants, including carbon tetrachloride (Shi et al., 1998Go), cadmium (Habeebu et al., 1998Go), cyclohexamide (Ledda-Columbano et al., 1992Go), 1,1-dichloroethylene (Reynolds et al., 1984Go), actinomycin D (Leist et al., 1997Go), and thioacetamide (Ledda-Columbano et al., 1991Go), 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, 1989Go; Ray et al., 1992Go). 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, 1996Go).

DMN hepatotoxicity produces a transient inflammatory response associated with elevated serum cytokines for tumor necrosis factor alpha (TNF{alpha}), interleukin (IL)-1ß, and IL-6 (Schook et al., 1992Go), and increased hepatic mRNA expression of acute-phase reactants (Bhattacharjee et al., 1998Go; Horn et al., 1998Go; Lockwood et al., 1994Go). Increased hepatic TNF{alpha} mRNA expression has been observed in other xenobiotic-induced hepatotoxicity models (Blazka et al., 1995Go; Bruccoleri et al., 1997Go; Czaja et al., 1989Go; Kayama et al., 1995Go; Ksontini et al., 1998Go; Luster et al., 1994Go), suggesting a possible central role for hepatic TNF{alpha} in DMN-induced hepatotoxicity. In support of this, we recently detected increased hepatic transcripts for TNF{alpha} following acute, high-dose DMN exposure (Horn et al., 2000Go).

The biologic activities of TNF{alpha} are mediated by two structurally related but functionally distinct receptors, TNF{alpha} receptor (TNFR)-1 (p55) and TNFR-2 (p75) (Grell et al., 1994Go; Tartaglia and Goeddel, 1992Go; Tartaglia et al., 1993Go). Whereas TNFR-1 signaling is the primary pathway in most cell types that mediate classical TNF{alpha}-related immune or inflammatory responses, TNFR-2 mediates several responses, including apoptosis of activated, mature T lymphocytes (Peschon et al., 1998Go; Sheehan et al., 1995Go; Zheng et al., 1995Go). Recent evidence also links TNFR-2 signaling in mediating the migration of Langerhans' cells (Wang et al., 1996Go, 1997Go). Thus, the importance of TNF{alpha} 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., 1993Go). To date, Fas-Fas ligand (FasL) interactions during xenobiotic exposure have not been extensively examined.

TNF{alpha} has been shown to be a hepatoprotective agent that prevents Fas-mediated apoptosis during liver regeneration (Takehara et al., 1998Go). Because TNF{alpha} is known to modulate apoptotic responses in the liver (Leist et al., 1995aGo, 1995bGo, 1997Go; Rolfe et al., 1997Go), the current study was undertaken to determine how signaling through TNF{alpha} 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{alpha} can regulate, but is not required for, apoptotic gene transcript expression in the liver during DMN hepatotoxicity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mice and dosing regimen.
Specific pathogen-free WT (C57BL/6) mice were obtained from Jackson Laboratory (Bar Harbor, ME). TNFR DKO mice ([C57BL/6 x 129] Peschon et al., 1998) were obtained from Immunex Corporation (Seattle, WA) and were bred in microisolator cages at the College of Veterinary Medicine, University of Minnesota. All breeding pairs were genotyped by PCR analysis of genomic DNA to confirm TNFR genotype (Peschon et al., 1998Go). All mice were housed in microisolator cages on a 12-h light/dark cycle and received tap water and mouse chow ad libitum. DMN (Sigma Chemical Co., St. Louis, MO) was diluted in PBS (pH 7) and stored at 4°C for less than 3 weeks. Solutions were tested for endotoxin by the limulus amebocyte assay (Associates of Cape Cod, Woods Hole, MA) prior to use. All mice (~ 20 g) were between 6 and 8 weeks of age and were rested for 1 week prior to dosing. Mice received 0.2 ml of either vehicle (PBS) or DMN (5.0 mg/kg) intraperitoneally (ip) once daily between 8 AM and 10 AM for up to 7 days. Mice were sacrificed 6 h postexposure on days 1, 4, and 7, and livers were removed. The dosing regimen is consistent with low-dose DMN exposure as previously documented (Bhattacharjee et al., 1998Go; Lockwood et al., 1994Go; Schook et al., 1992Go). Endotoxin (lipopolysaccharide [LPS] 50 µg E. coli 055:B55, Difco Laboratories, Detroit, MI) diluted in PBS was administered ip for 6 h and livers removed. In separate experiments, WT or TNFR DKO mice were exposed ip for 6 or 24 h with DMN (100 mg/kg). Additionally, WT mice were injected ip daily for up to 3 days with 15 or 25 mg/kg DMN and sacrificed 6 h postexposure on days 1 and 3. All animal procedures followed standard protocols approved by the University of Minnesota Animal Welfare Committee.

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{alpha}, 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{alpha}, ACA GAA AGC ATG ATC CGC; 3`-TNF{alpha}, GTA GAC CTG CCC GGA CTC. Amplification conditions were (94°C 15 s, 54°C 1 min, 72°C for 30 s) for 15–35 cycles. The expected amplicon lengths were 148 bp for HPRT, 477 bp for Fas, and 692 bp for TNF{alpha}. 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{alpha} 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., 1997Go). TNF{alpha} and HPRT RT-PCR amplicons were separated on 1.2% agarose gels as described above. Double-stranded linearized or PCR-derived cDNAs for TNF{alpha} and HPRT, respectively, were labeled with [{alpha}-32P]-dATP using RadPrime labeling kit (Life Technologies), and Southern blotting was performed according to standard protocols (Sambrook et al., 1989Go). 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 40–50°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 2.4–4 x 105 cpm/µg and hybridized at 56°C for 16–18 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 [{alpha}-32P]-dCTP and Klenow enzyme at room temperature for 15 min (Rösl, 1992Go). 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 = 3–4). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatic TNF{alpha} and TNFR mRNA Expression in WT Mice
TNF{alpha} is a modulator of apoptosis and liver regeneration in the liver. We have previously reported increased levels of serum TNF{alpha} following DMN exposure (Schook et al., 1992Go). To determine if the liver could serve as an autocrine source of TNF{alpha} production, we measured hepatic TNF{alpha} and TNFR gene expression during a 7-day DMN exposure period in WT mice. Six hours following a single daily exposure, elevated TNF{alpha} transcript levels were detected in livers from both vehicle- and DMN-treated mice. Following 4 daily DMN exposures, hepatic TNF{alpha} mRNA transcripts were up regulated (~ 90% of that induced by a single LPS dose) (Fig. 1AGo). TNF{alpha} mRNA levels following the 7th daily DMN exposure, although elevated compared to vehicle controls, were markedly reduced compared to the 4th daily DMN exposure. When TNFR-1 transcript levels were examined, we observed ~ 36% and ~ 120% up regulation following 1 and 4 daily DMN exposures, respectively, compared to vehicle controls. Following 7 daily DMN exposures, hepatic TNFR-1 transcript levels were similar to vehicle controls (Fig. 1BGo). In contrast, TNFR-2 expression was depressed ~ 40% compared to vehicle treatment during 1 and 7 DMN exposures (Fig. 1BGo). These data demonstrate DMN-induced hepatic TNF{alpha} expression and suggest temporal shifts in TNFR-1/TNFR-2 signaling.



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FIG. 1. Hepatic mRNA levels of TNF{alpha}, TNFR-1, and TNFR-2 in WT mice. Liver RNA was isolated from naive (N) mice, mice exposed ip to a single LPS (L) injection, or exposed to either vehicle (PBS; V) or DMN (5.0 mg/kg; D) for 1, 4, or 7 daily exposures. (A) Liver RNA (2 µg) was subjected to RT-PCR amplification and Southern blotting for the detection of TNF{alpha}. (B) Hepatic mRNA transcript expression for TNFR-1, TNFR-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured by RPA from 10 µg total liver RNA. Phosphorimagery ratios of (A) TNF{alpha}:HPRT or (B) TNFR:GAPDH are indicated below each lane and are representative for two to three different identically treated mice. TNF{alpha} and TNFR expression were normalized to the housekeeping genes HPRT or GAPDH, respectively, by dividing the TNF{alpha} or TNFR phosphorimagery value by the corresponding housekeeping phosphorimagery value and multiplying by 100.

 
Hepatic Fas mRNA Expression in WT and TNFR DKO Mice
To determine the possible involvement of Fas and its regulation by TNF{alpha}, hepatic Fas mRNA expression was measured in WT and TNFR DKO mice. RNA was isolated from liver lobes from three WT mice per treatment group 6 h following 1, 4, or 7 daily DMN or vehicle exposures. For comparison, liver RNAs were isolated from naive mice or mice treated with LPS (50 µg) ip for 6 h. Semiquantitative RT-PCR indicated an up regulation of Fas mRNA in WT mice following 4 and 7 daily DMN exposures and in TNFR DKO mice during 7 daily DMN exposures compared to vehicle controls (Fig. 2Go).



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FIG. 2. Hepatic Fas expression in WT and TNFR DKO mice. Liver RNA (2 µg) was isolated from naive (N) mice, mice exposed ip to a single LPS (L) injection, or exposed to either vehicle (PBS; V) or DMN (5.0 mg/kg; D) for 1, 4, or 7 daily exposures. Hepatic Fas and HPRT transcripts were detected by RT-PCR and ethidium bromide-stained gels were visualized under UV light. Fas:HPRT ratios are indicated below each lane representing three different identically treated mice and were obtained by dividing the Fas value by the HPRT value and multiplying by 100.

 
Fas transcript up regulation was further confirmed by a more quantitative RPA (Fig. 3Go). Following the 4th daily exposure, steady-state Fas transcript levels were elevated 4-fold in WT mice exposed to DMN compared to vehicle controls. Following the 7th daily DMN exposure, Fas mRNA levels were still elevated 1.9-fold compared to vehicle controls, but differences were not statistically significant (p < 0.05) (Figs. 3A and 3CGoGo). When TNFR DKO mice were examined, a 2.6-fold increase in Fas mRNA was observed following the 4th daily DMN exposure, and Fas mRNA levels remained elevated (1.9-fold) through 7 daily DMN exposures compared to vehicle controls (Figs. 3B and 3CGoGo). In addition, Fas transcripts following 7 daily DMN exposures in TNFR DKO mice were 1.6-fold (p < 0.05) higher as compared to WT controls (Fig. 3CGo). Thus, daily DMN exposure increased hepatic Fas transcripts in both WT and TNFR DKO mice.



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FIG. 3. Hepatic mRNA levels for Fas- and TNFR-1-associated proteins. (A) WT and (B) TNFR DKO mice were exposed to either vehicle (PBS) or DMN (5.0 mg/kg) for 1, 4, or 7 daily exposures. Liver RNA (10 µg) was isolated and individually probed with multiple antisense RNA probes for Fas, TNFR-1, FADD, TRADD, RIP, FasL, TRAIL, FAP, FAF, caspase-8, L32 ribosomal protein, and GAPDH (mAPO3 template DNA; PharMingen). L32 and GAPDH (housekeeping genes) serve as internal loading controls. DP, diluted undigested RPA probes (ladders); Y, yeast tRNA (negative control); P, PharMingen control RNA (2 µg); N, naive; L, LPS (50-µg dose for 6 h); V, vehicle; D, DMN. (C) Fas expression in both WT (empty bars) and TNFR DKO (filled bars) mice was normalized to GAPDH for each sample by dividing the Fas densitometry value by the GAPDH densitometry value and multiplying by 100. To equalize for naive expression, Fas ratios were also divided by the naive value (i.e., naive value = 1) and expressed as average ratios ± SE. Results shown are representative for three to four independent identically treated groups. Asterisk (*), statistically significant compared to vehicle-treated mice within the same strain; plus sign (+), TNFR DKO statistically significant compared to identically treated WT mice (p < 0.05).

 
Hepatic Expression of TNFR Superfamily Associated Signaling Molecules
The biological responses mediated by Fas or TNF{alpha} are dependent on receptor-associated signaling complex proteins that interact with the cytoplasmic tail of Fas or TNFR (Allen et al., 1998Go). We observed no obvious alterations in hepatic transcript levels for Fas-associated death domain (FADD), caspase-8 (FADD protein-like IL-1-converting enzyme [FLICE]), Fas-associated factor (FAF), TNF-related apoptosis-inducing ligand (TRAIL or Fas2L), TNFR-associated death domain (TRADD), and receptor-interacting protein (RIP) following DMN exposure in either WT or TNFR DKO mice (Figs. 3A and 3BGoGo). Hepatic transcripts for FasL and Fas-associated phosphatase (FAP) were not detected by this assay in WT or TNFR DKO mice following any treatment (Figs. 3A and 3BGoGo). This shows that DMN exposure does not alter hepatic transcript levels for Fas- and TNFR-associated signaling molecules and that their expression is not TNF{alpha}-dependent. Further, these data suggest that intact Fas and TNFR signaling pathways are maintained during DMN exposure.

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., 1998Go). 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. 4Go), 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{alpha} 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. 4BGo), suggesting a role for TNF{alpha} in down regulating hepatic Bax expression during prolonged DMN exposure.



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FIG. 4. Hepatic mRNA levels for apoptosis machinery proteins. (A) WT and (B) TNFR DKO mice were exposed to either vehicle (PBS) or DMN (5.0 mg/kg) for 1, 4, or 7 daily exposures. Liver RNA (10 µg) was isolated and individually probed with multiple antisense RNA probes for Bcl-2 family members that block (Bcl-2, Blf-1, Bcl-W, and Bcl-XL) and promote (Bax, Bak, Bad, and Bcl-XS) apoptosis (mAPO2 template DNA; PharMingen). DP, diluted undigested RPA probes (ladders); Y, yeast tRNA (negative control); P, PharMingen control RNA (2 µg); N, naive; L, LPS (50-µg dose for 6 h); V, vehicle; D, DMN. (C) Bcl-XL and (D) Bax mRNA expression in WT (empty bars) and TNFR DKO (filled bars) mice were normalized to GAPDH for each sample by dividing the respective densitometry value by the GAPDH densitometry value and multiplying by 100. To equalize for naive expression, Bcl-XL and Bax ratios were also divided by the naive value (i.e., naive value = 1) and are expressed as average ratios ± SE. Results shown are representative for three to four independent identically treated groups. Asterisk (*), statistically significant compared to vehicle-treated mice within the same strain; plus sign (+), TNFR DKO statistically significant compared to identically treated WT mice (p < 0.05).

 
In agreement with previous studies (Hockenbery et al., 1991Go; Horn et al., 2000Go), hepatic Bcl-2 expression was not detected during any treatment in either strain (Fig. 4Go). We previously reported TNF{alpha} regulation for hepatic Bfl-1 expression during acute carbon tetrachloride exposure (Horn et al., 2000Go). However, during low-dose, subacute DMN exposure, TNF{alpha} regulation was not observed, as both WT and TNFR DKO have up regulated hepatic Bfl-1 transcripts following 4 and 7 exposures (Fig. 4Go). In WT and TNFR DKO mice, differences in hepatic expression between vehicle and DMN were not observed for other Bcl-2 family members.

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, 1992Go) 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. 5AGo). 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, 1989Go; Ray et al., 1992Go), 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. 5BGo). Because we have observed differential effects of acute high-dose DMN exposure in WT versus TNFR DKO mice (Horn et al., 2000Go), 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. 5BGo). Taken together, low-dose DMN exposures induce hepatic apoptosis in both WT and TNFR DKO mice.



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FIG. 5. DMN-induced hepatic DNA fragmentation. (A) Wild-type and TNFR DKO mice were injected ip with vehicle (PBS; V) or DMN (5.0 mg/kg; D5) daily for up to 7 exposures and sacrificed 6 h postexposure on days 1, 4, or 7. (B) WT and TNFR DKO mice were exposed ip to high-dose DMN (100 mg/kg; D100) for 6 or 24 h. Additionally, WT mice were exposed to DMN (15 or 25 mg/kg; [D15 or D25, respectively]) for up to three exposures and sacrificed 6 h postexposure on days 1 or 3. Isolated liver DNA (2 µg) was labeled with 32P, separated on 1.8% agarose gels, and exposed to X-ray film. Results shown are representative for two independent identically treated groups (MW, DNA molecular weight marker in kilobases; N, naive).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study examined hepatic transcripts for apoptotic signaling components and machinery during low-dose, subacute DMN exposure, and the role of TNF{alpha} in regulating expression of these transcripts. Previous reports have linked TNF{alpha} to apoptosis within the liver (Leist et al., 1995bGo, 1997Go; Rolfe et al., 1997Go; Takehara et al., 1998Go). We (Horn et al., 2000Go) and others (Pritchard and Butler, 1989Go; Ray et al., 1992Go) have shown that acute, high-dose DMN (> 25 mg/kg) induces necrosis and apoptosis. Results presented here confirm that DNA fragmentation, an indicator of apoptosis (Compton, 1992Go), occurs following exposure to high-dose DMN (15, 25, and 100 mg/kg) in WT mice (Fig. 5BGo). More importantly, low-dose, prolonged DMN exposures and the molecular mechanisms associated with such responses have not been investigated. We now show that DNA ladders are evident following 1, 4, and 7 low-dose DMN (5.0 mg/kg) exposures in both WT and TNFR DKO mice (Fig. 5AGo), suggesting that both apoptosis and necrosis occur following low-dose DMN exposure. That DNA fragmentation was also observed in TNFR DKO mice suggests that TNF{alpha} signaling is not necessary for apoptosis during low-dose DMN exposures.

TNF{alpha} transcripts were detected in the liver during subacute, low-dose DMN exposures (Fig. 1AGo). 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., 1998Go; Leist et al., 1995bGo, 1997Go). We previously demonstrated biologically active TNF{alpha} in the serum through 3 daily DMN (5.0 mg/kg) exposures (Schook et al., 1992Go). Our current studies show that the liver is a potential source for TNF{alpha} during DMN exposure, consistent with other models of xenobiotic-induced hepatotoxicity that have associated TNF{alpha} expression in the liver with liver pathology (Blazka et al., 1995Go; Bruccoleri et al., 1997Go; Czaja et al., 1989Go; Kayama et al., 1995Go). It remains to be determined whether resident Kupffer cells, hepatocytes, vascular endothelial cells, or infiltrating immune cells account for up-regulated expression of TNF{alpha} 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{alpha} 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{alpha} transcripts that subsided following 4 and 7 daily exposures (Fig. 1AGo). 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., 2000Go; Lockwood et al., 1991Go, 1994Go; Schook et al., 1992Go) 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{alpha} 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. 2–4GoGoGo). However, TNFR DKO mice have an increased relative expression of Fas and Bax mRNA compared to WT mice (Figs. 3C and 4DGoGo). These data suggest that TNF{alpha}, directly or indirectly by an unknown mechanism(s), appears to limit hepatic Fas and Bax expression following DMN exposure. Thus, hepatic TNF{alpha} 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. 3Go) 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., 1997Go). If FasL is not present in the hepatic microenvironment, the apoptosis we observed (Fig. 5Go) must be affected by other modulators downstream of Fas ligation or via Fas/FasL-independent mechanisms (Evan and Littlewood, 1998Go; Zheng et al., 1995Go).

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, 1998Go). 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{alpha}-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 3BGoGo).

We examined hepatic mRNA expression of prosurvival or proapoptotic genes during DMN exposure because these genes may modulate Fas- and/or TNF{alpha}-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., 1998Go; Boise and Thompson, 1997Go; Green and Reed, 1998Go; Itoh et al., 1993Go; Kren et al., 1996Go). Although Bcl-XL and Bax have opposing functions, they can form heterodimeric complexes to control caspase activation (Adams and Cory, 1998Go; Sato et al., 1994Go; Sedlak et al., 1995Go). 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., 1998Go). Whereas Bcl-XL mRNA levels in WT mice increase less than 2-fold (Fig. 4CGo), Bax transcripts are induced approximately 3-fold (Fig. 4DGo). Thus, the Bcl-XL:Bax ratio changes toward one that favors apoptosis. DNA fragmentation patterns (Fig. 5AGo) 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., 1993Go), 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{alpha} 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{alpha} 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{alpha}, TNFR-1, and Fas signals that affect the relative balance of Bcl-XL and Bax. This antiapoptotic response mediated by TNF{alpha} may serve to prevent undue death of hepatocytes. Despite greater Bax transcripts in TNFR DKO mice exposed to DMN (Fig. 4DGo), we did not observe greater DNA laddering compared to WT mice (Fig. 5AGo).

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., 2000Go). Genomic DNA prepared from TNFR DKO mice at 24 h postexposure did not show DNA laddering as did WT mice (Fig. 5BGo). 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., 1998Go), TNF{alpha} has the potential to promote development of liver carcinogenesis by suppressing apoptotic removal of damaged hepatocytes. Thus, the impact of TNF{alpha} on individual processes associated with DMN hepatotoxicity must be carefully evaluated at low-dose, multiple exposures as well as high-dose, acute exposures.


    ACKNOWLEDGMENTS
 
We would like to thank Drs. Raymond G. Goodwin and Jacques J. Peschon at Immunex Corporation (Seattle, WA) for the breeding pairs of TNFR DKO mice used to establish our breeding colony. The assistance of Victoria Lappi with RPA and Gail Flickinger with liver DNA isolation were greatly appreciated. Technical advice regarding apoptotic DNA labeling was provided by Drs. Curtis Klaassen and Sultan Habeebu from the University of Kansas Medical Center, Kansas City, Kansas. This work was supported through National Institutes of Health grants ES08395 (M.S.R.) and ES04348 (L.B.S.). T.L.H. was supported in part by a USDA National Needs Graduate Student Fellowship.


    NOTES
 
This research was presented in part as a thesis submitted to the University of Minnesota Graduate School by T. L. Horn in partial fulfillment of the requirements for Ph.D. degree.

1 Current address: VA Medical Center, Research Service (151), 1 Veterans Dr., Minneapolis, MN 55417. Back

2 Current address: Dana-Farber Cancer Institute, Adult Oncology, Mayer 433, 44 Binney St., Boston, MA 02115. Back

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. Back


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