Corn oil rapidly activates nuclear factor-
B in hepatic Kupffer cells by oxidant-dependent mechanisms
Ivan Rusyn1,2,5,
Cynthia A. Bradham3,
Leslie Cohn1,
Robert Schoonhoven4,
James A. Swenberg1,4,
David A. Brenner3 and
Ronald G. Thurman1,2
1 Curriculum in Toxicology,
2 Laboratory of Hepatobiology and Toxicology,
Department of Pharmacology,
3 Department of Medicine and
4 Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599-7365, USA
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Abstract
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N-6 polyunsaturated fatty acids (N-6 PUFAs), major constituents of corn oil and natural ligands for peroxisome proliferator-activated receptors, increase the rate of growth of established tumors. It has been proposed that chemical peroxisome proliferators increase hepatocyte proliferation by mechanisms involving activation of nuclear factor-
B (NF-
B) and production of low levels of tumor necrosis factor
(TNF
) by Kupffer cells; however, how N-6 PUFAs are involved in increased cell proliferation in liver is not well understood. Here, the hypothesis that N-6 PUFAs increase production of mitogens by activation of Kupffer cell NF-
B was tested. A single dose of corn oil (2 ml/kg, i.g.), but not olive oil or medium-chain triglycerides (saturated fat), caused an ~3-fold increase in hepatocyte proliferation. Similarly, when activity of NF-
B in whole rat liver or isolated hepatocytes and Kupffer cells was measured at various time intervals for up to 36 h, only corn oil activated NF-
B. Corn oil increased NF-
B activity ~3-fold 12 h after treatment exclusively in the Kupffer cell fraction. In contrast, increases were small and only occurred after ~8 h in hepatocytes. The activation of NF-
B at 2 h and increases in cell proliferation at 24 h due to corn oil were prevented almost completely when rats were pretreated for 4 days with either dietary glycine (5% w/w), an agent that inactivates Kupffer cells, or the NADPH oxidase inhibitor, diphenyleneiodonium (s.c., 1 mg/kg/day). Furthermore, arachidonic acid (100 µM) activated superoxide production ~4-fold when added to isolated Kupffer cells in vitro. This phenomenon was not observed with oleic or linoleic acids. Interestingly, a single dose of corn oil increased TNF
mRNA nearly 2-fold 8 h after treatment. It is concluded that corn oil rapidly activates NF-
B in Kupffer cells via oxidant-dependent mechanisms. This triggers production of low levels of TNF
which is mitogenic in liver and promotes growth of hepatocytes.
Abbreviations: BrdU, 5-bromo-2'deoxyuridine; DPI, diphenyleneiodonium; MCT, medium chain triglycerides; N-6 PUFAs, N-6 polyunsaturated fatty acids; NF-
B, nuclear factor-
B; PKC, protein kinase C; PPARs, peroxisome proliferator activated receptors.
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Introduction
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The biochemical links between diet and the frequency of certain forms of cancer have been the subject of extensive epidemiological and fundamental research (1). For example, a strong correlation between certain types of lipid in the diet and cancer exists in laboratory animals (14) and humans (5). It was found that dietary fat promotes the growth of initiated cells, thus contributing to the higher risk of many human cancers (6). Furthermore, high-fat diets containing corn oil, soybean oil or safflower oil, which are 5580% linoleic acid (C18:2; N-6), increase the rate of growth of established tumors (7). Importantly, it is known that olive oil and saturated fat are much weaker than corn oil in promotion models (7,8). The principal N-6 polyunsaturated fatty acid (N-6 PUFA) in the diet is linoleic acid, which is metabolized in many tissues and tumor cells to arachidonic acid, a precursor for eicosanoids. It has been suggested that eicosanoids are directly responsible for growth stimulation by N-6 PUFAs (6); however, promotion by cytokines is also possible.
Fatty acids and eicosanoids are ligands of peroxisome proliferator activated receptors (PPARs), and high-fat diets cause peroxisome proliferation in rodent liver (9). Peroxisomes, cytoplasmic organelles in eukaryotic cells, contain enzymes that participate in fatty acid ß-oxidation, the initial reactions of ether lipid synthesis, alcohol oxidation, transaminations and purine polyamine catabolism (10). The peroxisome proliferator response has drawn considerable interest due to the fact that a number of peroxisome proliferators, such as hypolipidemic drugs, phthalate esters and solvents, cause hepatocellular carcinomas in rodents (11). While many hypotheses have been proposed to explain this phenomenon, increased cell proliferation in liver is most likely involved (12).
It is well known that Kupffer cells, the resident hepatic macrophages, are a rich source of mitogens and co-mitogens in liver (13). Since Kupffer cells are the major source of the direct hepatocyte mitogen tumor necrosis factor
(TNF
) (14), it was hypothesized recently that they participate in the mechanism of action of peroxisome proliferators (15). Indeed, hepatocyte proliferation induced by WY-14,643 was prevented by inactivation of Kupffer cell TNF
production with methyl palmitate (16) and dietary glycine (17), or with an antibody to TNF
(18). Since TNF
synthesis is dependent on nuclear factor
B (NF-
B), it was hypothesized that proliferation of hepatocytes is stimulated via early activation of the transcription factor NF-
B in Kupffer cells leading to increased TNF
production which triggers subsequent proliferation in hepatocytes. Indeed, it was discovered recently that activation of NF-
B by WY-14,643 occurs rapidly in Kupffer cells (19). These new data support the idea that Kupffer cell NF-
B is activated by oxidants which trigger cell proliferation in rodent liver following treatment with peroxisome proliferators via TNF
. Here, experiments were designed to test the hypothesis that N-6 PUFAs present in corn oil activate NF-
B in Kupffer cells via oxidant-dependent mechanisms similar to WY-14,643. This, in turn, stimulates production of TNF
and increases cell proliferation in nearby parenchymal cells.
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Materials and methods
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Animals
Female SpragueDawley rats weighing between 175 and 225 g were used in all experiments. Animals were housed two to three per cage with a 12 h day/night cycle and given lab chow and water ad libitum. Animals were acclimated at least 1 week before treatment, and Institutional Animal Care and Use Committee criteria were followed. Corn oil was a generous gift of Novartis Nutrition (Minneapolis, MN). It was supplied in 200 ml aliquots under nitrogen and stored at 4°C to prevent oxidation. Neobee M-5 (medium chain triglycerides), a source of saturated fat, was purchased from Stepan (Maywood, NJ) and olive oil was obtained fresh from local suppliers. Linoleic, arachidonic and oleic acids were purchased from Cayman Chemical (Ann Arbor, MI). Stearic acid was from Sigma (St Louis, MO). Diphenyleneiodonium sulfate was purchased from Toronto Research Chemicals (Toronto, Ontario). All other chemicals and reagents were of the highest available purity from standard commercial sources. Different types of dietary fats were administered to rats by gavage (0.24 ml/kg) as a single dose. Treatments were performed between 09:00 and 12:00 h and rats were killed under pentobarbital anesthesia at different time intervals ranging from 1 to 36 h. In some experiments, rats were fed semi-synthetic control or 5% glycine diets for 4 days as described elsewhere (17).
Cell isolation
Hepatocyte and Kupffer cell fractions were prepared by collagenase digestion and differential centrifugation using Percoll (Pharmacia, Uppsala, Sweden) gradients as described elsewhere with slight modifications (20). To obtain pure Kupffer cells, the non-parenchymal cell fraction was seeded onto Petri dishes and cultured for 1 h at 37°C in Dulbecco's modified Eagle's medium (DMEM-H; 1000 mg/l glucose) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml of penicillin G and 100 µg/ml of streptomycin sulfate). Non-adherent cells were removed by replacing media with fresh DMEM-H culture medium. Viability was determined by trypan blue exclusion and was >90% in all experiments.
Preparation of nuclear extracts
For studies in whole liver, 100 mg of frozen tissue was placed on a 10 cm Petri dish on ice and minced immediately before the isolation procedure. Freshly prepared Kupffer cells were harvested with a cell scraper, washed and centrifuged at 1500 g for 5 min. Hepatocytes were isolated as detailed above and washed with cold Hank's balanced salt solution (HBSS) twice. Nuclear extracts were prepared from freshly isolated cells as described by Dignam et al. (21) with minor modifications (19). Protein concentration was determined using the Bradford Protein Concentration Assay Kit (Bio-Rad, Hercules, CA) by measuring absorbance spectrophotometrically at 595 nm (22).
Electrophoretic mobility shift assays (EMSAs)
The gel mobility shift assay was used in this study to assess the amount of active protein involved in proteinDNA interactions. Binding conditions for NF-
B were characterized and EMSAs performed as described in detail elsewhere (23). Briefly, equal amounts (840 µg) of nuclear extract from cells or liver tissue were pre-incubated 10 min on ice with 1 µg poly(dIdC) and 20 µg BSA (both from Pharmacia Biotech, Piscataway, NJ) and 2 µl of a 32P-labeled DNA probe (10 000 c.p.m./µl, Cerenkov) containing 1 ng of double-stranded oligonucleotide in a total volume of 20 µl. Mixtures were incubated for 20 min on ice and resolved on 5% polyacrylamide (29:1 cross-linking) and 0.4x TBE gels. After electrophoresis, gels were dried and exposed to X-OMAT LS Kodak film. Specificity of NF-
B binding was verified by competition assays and ability of specific antibodies to supershift proteinDNA complexes. In competition assays, 100-fold excess of unlabeled oligonucleotide was added 10 min before addition of the labeled probe. In supershift experiments, 4 µg of rabbit antisera against p50 or p65 protein (Santa Cruz Biotech, Santa Cruz, CA) was added to the reaction mixture after incubation with labeled probe which was further incubated at 25°C for 30 min. Labeled and unlabeled oligonucleotides contained the consensus sequence for NF-
B (24). Data were quantitated by scanning autoradiograms with GelScan XL (Pharmacia LKB, Uppsala, Sweden).
Reverse transcriptionpolymerase chain reaction (RTPCR)
Total liver RNA was extracted by CsCl centrifugation as described elsewhere (25). RNA (1 µg) was subjected to RT in 25 µl TrisEDTA buffer (10 mM TrisHCl, 1 mM EDTA, pH 8.0) using oligo(dT) as a primer and MMLV reverse transcriptase (Gibco BRL, Gaithersburg, MD). TNF
and ß-actin mRNA were amplified using primers, buffer and amplification conditions described elsewhere (26). For semi-quantitation, 1, 2.5 and 5 µl of RT reaction were amplified. The amplified PCR products were separated by electrophoresis through 2.5% agarose gels at 60 V for 90 min. Bands of cDNA were visualized by UV illumination after staining with 0.5 µg/ml ethidium bromide.
Superoxide assay
Kupffer cells were seeded in RPMI 1640 supplemented with fatty-acid-free BSA at a density of 106 cells/well in 24-well plates. Before experiments, medium was removed and Kupffer cells were washed twice with HBSS. Stock solutions of all fatty acids were prepared fresh for each experiment as sodium salts (27) and bound to defatted albumin prior to use (fatty acid:albumin ratio 4:1) (28). Equal amounts of defatted albumin were added in control experiments. Various concentrations of linoleic, arachidonic, oleic or stearic (5150 µM) acids were added to cells and incubated in HBSS. After 30 min, cytochrome c (50 µM) was added to each well and the reaction was allowed to proceed for an additional 30 min. Superoxide generation was measured as reduction of cytochrome c inhibitable by superoxide dismutase as described elsewhere (29).
Cell proliferation
In some experiments, rats were given 5-bromo-2'-deoxyuridine (BrdU) (100 mg/kg, i.p.; Sigma) 1 h before killing to assess rates of cell proliferation in liver. Livers were perfused with HBSS to remove blood and fixed with 4% paraformaldehyde. A section of duodenum, a tissue which proliferates rapidly, was collected as a positive control for BrdU incorporation. Tissue sections were deparaffinized, rehydrated and hydrolyzed in 4 N HCl for 20 min at 37°C. Immunohistochemical staining was performed using the DAKO Envision System Peroxidase Staining Kit (DAKO, Carpinteria, CA) and a primary monoclonal antibody to BrdU (DAKO, clone Bu20a) as described elsewhere (17). Cel1 proliferation was quantitated by determining the percentage of BrdU-positive hepatocytes in 10 random high-power fields/slide (1000 hepatocytes/slide).
Statistics
Results are reported as means ± SEM with n = 45 in each group. Treatment groups were compared using one-way ANOVA followed by StudentNeumanKeuls post-hoc test, or two-way ANOVA using StudentNeumanKeuls post-hoc test, where appropriate. A P-value <0.05 was selected prior to the study to determine statistical differences between groups.
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Results
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To determine if a single dose of dietary oil could increase cell proliferation in liver, rats were gavaged with 2 ml/kg of corn oil, olive oil, or saturated fat [medium-chain triglycerides (MCT)]. Increases in hepatocyte DNA synthesis 24 h after treatment were identified from the incorporation of BrdU in nuclei of proliferating cells. Corn oil increased cell proliferation ~3.5-fold in hepatocytes (P < 0.05 compared with control; Table I
). However, olive oil and MCT had no significant effect on hepatocyte proliferation.
It was shown that chemical peroxisome proliferators rapidly activate the transcription factor NF-
B in whole rat liver (19). Since NF-
B is involved in production of mitogenic TNF
and activation of cell proliferation (30), here the hypothesis that NF-
B is involved in the mechanism of increases in cell proliferation due to corn oil was tested. Specifically, rats were given different oils by gavage (2 ml/kg) and activity of NF-
B in liver was measured 2 h later. Interestingly, corn oil, but not olive oil or saturated fat (MCT), activated NF-
B ~3-fold (Figure 1
). Protein binding in nuclear extracts to labeled oligonucleotide probe was confirmed to be specific for the active p50/p50 and p50/p65 forms of NF-
B by gel shift assay (Figure 2
). In the absence of nuclear proteins, no proteinDNA complex was detected (lane 1). DNA binding with a 32P-labeled probe (lane 2) was markedly reduced in the presence of an excess of unlabeled oligonucleotide containing the NF-
B binding site (lane 3). Furthermore, addition of p50 or p65 antiserum reduced the intensity of the complex and produced supershifted complexes with a higher molecular mass (lanes 4 and 5, respectively).
Activation of NF-
B by corn oil was both time and dose dependent (Figure 3
). A rapid maximal 3-fold increase in NF-
B activity was observed between 2 and 8 h after treatment and was followed by a steady decline to near control levels by 36 h (Figure 3A
). Similarly, activation of NF-
B was dependent on the amount of corn oil given. A maximal response was observed with 2 ml/kg; however, doses as small as 0.5 ml/kg activated NF-
B significantly (Figure 3B
).
Low levels of TNF
are mitogenic in liver (14). To determine whether TNF
mRNA levels are altered by corn oil, changes in TNF
mRNA transcripts were measured using RTPCR. TNF
is expressed in minute amounts in livers of untreated animals (Figure 4A
); however, corn oil increased TNF
mRNA significantly ~2-fold between 8 and 24 h after a single dose (P < 0.05, n = 4).
Since WY-14,643 rapidly activated NF-
B in Kupffer cells, it was hypothesized that the rapid activation of NF-
B in rat liver due to corn oil administration occurs first in Kupffer cells. Accordingly, rats were given corn oil (2 ml/kg) and parenchymal and Kupffer cells were isolated at various time intervals from 0 to 24 h. Indeed, NF-
B activity was elevated ~3-fold 12 h after corn oil treatment (P < 0.05, n = 4) almost exclusively in the Kupffer cell fraction (Figure 4B
, upper panel). In contrast, hepatocytes exhibited significant increases of 34-fold (P < 0.05, n = 4) only after 8 h (Figure 4B
, lower panel).
To determine if fatty acids can activate Kupffer cells in vitro, cells were isolated from untreated rats, cultured and superoxide production was measured. Arachidonic acid, a major metabolite of linoleic acid, the most abundant fatty acid in corn oil, maximally increased superoxide production nearly 4-fold by isolated Kupffer cells (Figure 5
) in a dose-dependent manner with a peak at 100 µM (data not shown). However, linoleic acid had no effect at the same concentration. Interestingly, oleic acid had no appreciable effect (Figure 5
). Furthermore, when Kupffer cells were pretreated with diphenyleneiodonium (DPI; 10 µM), an NADPH oxidase inhibitor, or glycine (1 mM), an agent that inactivates Kupffer cells (31), activation of superoxide production by arachidonic acid was decreased by ~75% (data not shown).

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Fig. 5. Effect of fatty acids on superoxide production by Kupffer cells. Arachidonic, linoleic and oleic acids (AA, LA and OA, respectively; 100 µM) were added to cultured Kupffer cells and superoxide production was measured as described in Materials and methods. Data shown are means ± SEM. *, Statistical difference from control (P < 0.05) by one-way ANOVA with Bonferoni's post-hoc test (n = 45 in each group).
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To determine if antioxidants (e.g. DPI) or agents that inactivate Kupffer cells (e.g. glycine) could prevent rapid activation of NF-
B caused by a single dose of corn oil in vivo, rats were pretreated with either DPI (1 mg/kg, s.c.) or dietary glycine (5% w/w) for 4 days. Control powdered diet or pretreatment with vehicle had no effect (data not shown). However, both DPI and dietary glycine significantly blunted the rapid activation of NF-
B caused by corn oil, by 7580% (Figure 6
). Furthermore, both DPI and glycine completely prevented the increase in hepatocyte proliferation caused by corn oil (Table 1
).
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Discussion
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Different dietary fats have been directly linked to chemically induced hepatocarcinogenesis (32). The liver is not only a major site of storage and bioactivation of many procarcinogens, it is also a primary site of metabolism of many hormones and cytokines which have profound modulatory influences on many site-specific tumors (33). While many in vitro and in vivo studies support the hypothesis that nutritional factors such as fat play an important role in the etiology of cancer, precise cellular and molecular mechanisms are only now beginning to be elucidated. Here, experiments were designed to test the hypothesis that N-6 PUFAs activate NF-
B in rat liver via oxidant-dependent mechanisms.
NF-
B may play a central role in the mechanism of increased cell proliferation by corn oil
The transcription factor NF-
B is ubiquitous and is localized in the cytoplasm of most resting cells bound to inhibitory I
B component. Various inducers (e.g. endotoxin, some viruses, cytokines and reactive oxygen species) trigger the phosphorylation, ubiquitination and proteosomal degradation of I
B, which enables the active dimer of NF-
B to translocate to the nucleus and bind specific DNA sequences (for a review, see ref. 30).
In this study, only the tumor promoter corn oil increased cell proliferation 24 h after treatment and activated nuclear transcription factor
B in rat liver as early as 2 h after a single dose (Table I
; Figure 1
). Furthermore, neither olive oil nor saturated fat (MCT), which do not increase cell proliferation in liver, had a significant effect under similar conditions. Similar to what was observed with the peroxisome proliferator WY-14,643 (19), the activation of NF-
B was time-dependent with a peak at 28 h, followed by a steady decline to basal levels over 36 h (Figure 3A
). Importantly, a dose of corn oil of as little as 0.5 ml/kg, which is in the range of average human consumption, activated NF-
B significantly (Figure 3B
). Since pretreatment with the NADPH oxidase inhibitor DPI blocked the effect of corn oil on both increases in cell proliferation (Table I
) and activation of NF-
B (Figure 6
), it is concluded that oxidants from Kupffer cell are involved in activation of NF-
B due to corn oil treatment.
Kupffer cells are responsible for increased cell proliferation in liver caused by corn oil
Recently, it has been reported that Kupffer cells play a pivotal role in the activation of NF-
B and expression of TNF
in rodent liver by the lipophilic peroxisome proliferator WY-14,643, a potent rodent carcinogen (16,19). Since fatty acids and products of their metabolism (e.g. leukotrienes) also cause proliferation of peroxisomes in rodent liver (34), it was hypothesized that Kupffer cells are involved in the mechanism of promotion by N-6 PUFAs. Moreover, it was reported that arachidonic acid rapidly activated NF-
B in J774 macrophages in vitro (35). The present findings showed that after treatment with corn oil in vivo, the active form of NF-
B was localized predominantly in Kupffer cells where it peaked 1 h after treatment, whereas hepatocytes exhibited activation only after 8 h (Figure 4B
). Furthermore, arachidonic acid, a major metabolite of linoleic acid, activated superoxide production by Kupffer cells in vitro (Figure 5
). In addition, inactivation of Kupffer cells in vivo via pretreatment with dietary glycine (17) prevented both increased cell proliferation and activation of NF-
B in rat liver caused by corn oil (Table I
; Figure 6
). Since hepatocytes are the dominant cell type in the liver in terms of size and number, later activation of NF-
B in hepatocytes, observed here (Figure 4B
), occurs most likely as a consequence of TNF
production following activation of Kupffer cells by corn oil since binding of TNF
to its receptor and induction of a chain of intracellular events can activate NF-
B (36) and stimulate cell proliferation (37). Indeed, a single dose of corn oil (2 ml/kg) upregulated expression of TNF
in rat liver ~2-fold (Figure 4A
). Taken together, these data strongly support the hypothesis that Kupffer cell NF-
B acts as a trigger of increased cell proliferation in rodent liver due to the tumor promoter corn oil.
Chemical and dietary peroxisome proliferators could increase proliferation of hepatocytes via similar mechanisms
Several mechanisms have been proposed to explain how fatty acids and chemical peroxisome proliferators cause tumors in liver (32,38). The induction of oxidative stress and lipid peroxidation in target tissues have been proposed as possible mechanisms of action for non-genotoxic carcinogens (39). It has been hypothesized that H2O2 from acyl-CoA oxidase causes oxidative stress leading to DNA damage (40). A similar mechanism has been proposed to explain fat-induced rodent hepatocarcinogenesis (32). However, with the exception of a few reports (41), there has not been overwhelming experimental support for this idea. Furthermore, sustained stimulation of cell proliferation is most likely a key step in the ultimate development of cancer in liver (12) and oxidants may act by triggering increases in cell proliferation due to peroxisome proliferators via activation of transcription factors and production of mitogens (19).
Here, we hypothesize that polyunsaturated dietary lipids such as those in corn oil activate Kupffer cells. This triggers production of oxidants and activation of NF-
B leading to synthesis of mitogenic cytokines such as TNF
which increases cell proliferation in parenchymal cells, causing promotion of previously initiated cells leading to tumors. Fatty acids are known activators of protein kinase C (PKC), an enzyme associated with tumor promotion. The question then arises, how do fatty acids activate NF-
B? Indeed, hepatic PKC is activated by phorbol esters, which act not only on the skin where they have been studied extensively, but also in liver (42); therefore, it is possible that fatty acids act via PKC which may be involved in activation of NF-
B via two separate mechanisms. It is known that PKC can indirectly increase production of oxidants (e.g. superoxide anion), which are redox modulators of NF-
B activity, by phosphorylation of NADPH oxidase (reviewed in ref. 43). Alternatively, the
isoform of PKC can directly phosphorylate I
B (44).
Fatty acids bind and activate PPARs (e.g. PPAR
) (45). PPAR
plays a role in increases in cell proliferation and carcinogenesis in liver (46). For instance, it is possible that N-6 PUFAs and their products activate PPAR
leading to enhanced activity of many enzymes, including protein kinases (47); still, the link between TNF
from Kupffer cells and the PPAR
remains unknown. Moreover, it has been reported recently that macrophages do not contain PPAR
(48) which raises the possibility of involvement of other PPAR isoforms in this mechanism.
In conclusion, the data presented here support the hypothesis that corn oil activates NF-
B in Kupffer cells via oxidant-dependent mechanisms. This is probably a key early event resulting in subsequent increases in TNF
leading to increased cell proliferation in the liver.
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Acknowledgments
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This work was supported, in part, by a grant from NIEHS (ES-04325).
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Notes
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5 To whom correspondence should be addressed Email: iir{at}med.unc.edu 
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Received June 7, 1999;
revised July 19, 1999;
accepted July 23, 1999.