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B Activation in Rats Administered the Hepatic Tumor Promoter, Phenobarbital1
,**,
2
*
Graduate Center for Nutritional Sciences,
Graduate Center for Toxicology,
**
Department of Pathology and Laboratory Medicine, and
Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, KY 40506
2To whom correspondence should be addressed. E-mail: hglauert{at}uky.edu.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B (NF-B). In this study, we hypothesized that PB-induced NF-B activation can be decreased by dietary vitamin E in rats. Male Sprague-Dawley rats (n = 39) were fed a purified diet with varying levels of dietary vitamin E (10, 50 or 250 mg/kg of dl-
-tocopherol acetate) for 28 d, at which time 8 rats per level of dietary vitamin E were fed the same diet with 500 mg/kg PB for 10 d. In the rats fed the low vitamin E diet, PB increased NF-B DNA binding, but it did not affect NF-B activation in rats fed higher levels of vitamin E (50 and 250 mg/kg). Vitamin E may decrease the oxidative stress created by PB by also enhancing other antioxidants; therefore, we also measured hepatic glutathione S-transferase, glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase and NAD(P)H:quinone reductase (DT-diaphorase) activities and glutathione and ascorbic acid concentrations. Increased dietary -tocopherol did not affect the antioxidants and antioxidant enzymes altered by PB treatment. Thus, the effect of -tocopherol acetate on NF-B activation does not appear to be mediated by alterations in the antioxidant system. These results demonstrate that the activation of NF-B, a transcription factor that affects cell proliferation– and apoptosis-related gene expression, can be inhibited by dietary vitamin E.
KEY WORDS: • vitamin E • phenobarbital • NF-B • antioxidant • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Phenobarbital is nongenotoxic but with chronic administration produces hepatocellular adenomas in rats and hepatocellular adenomas and carcinomas in mice (2
). PB administration in rodents leads to hepatomegaly, resulting from hyperplasia and hypertrophy (3
). The increased cell division returns to control levels within 2 wk of administration, but the hypertrophy is maintained as long as PB is administered (4
). PB administration also inhibits apoptosis in normal liver and in preneoplastic foci (5
) and in primary cultures of rat hepatocytes (6
). Rodents chronically exposed to PB have regression of altered foci when PB administration is removed (3
).
PB may act as a tumor promoter by suppressing apoptosis and/or increasing cell proliferation (4
). It has also been proposed that PB mediates tumor promotion by increasing oxidative stress, most likely from the chronic activation of cytochrome P450 (CYP) and NADPH-CYP reductase (7
–9
). PB induces CYP2B1/2B2 in rat liver. Microsomal mRNA and protein levels of CYP2B1 can be induced as much as 50- to 100-fold and 2B2 can be induced 20-fold by PB treatment (10
). Superoxide and H2O2 are released as by-products of CYP (11
). Inducers of the CYP2B family have hepatic tumor–promoting activity when they are administered chronically to rodents, whereas structurally similar noninducers do not (4
). PB treatment increases hepatic cell replication only transiently, but PB-responsive enzymes, including CYP, can be induced for at least 90 wk as seen in C3H mice treated with 85 mg PB/(kg body · d) (7
). A minimum sequence of 51 bp of DNA has been shown to be PB responsive (PBREM or PB-responsive enhancer module) in rat CYP2B1 and CYP2B2, mouse CYB2B10 and human CYP2B6 genes (12
). In livers exposed to PB, the nuclear receptor, constitutive active receptor, translocates to the nucleus to heterodimerize with the retinoid X receptor to activate PBREM (12
).
PB has been shown to activate the oxidative stress–responsive transcription factor, nuclear factor-B (NF-B) in rat liver (13
). This transcription factor can be activated by multiple stimuli, including oxidative stress (14
). Moreover, NF-B has been shown to be involved in the regulation of cell proliferation and apoptosis (15
,16
). NF-B is a dimer that can consist of several subunits (p50, p52, p65, c-rel, RelB), but the p65:p50 heterodimer is the most common (14
–16
). NF-B is restricted to the cytosol by its inhibitory subunit IB, which masks the nuclear localization signal of the NF-B dimer (14
). Phosphorylation and subsequent degradation of IB allows the NF-B complex to translocate to the nucleus where it can increase the transcription of target genes (14
). Because NF-B regulates genes that affect apoptosis and cell proliferation, NF-B activation may be responsible in part for the metabolic alterations that occur after treatment with PB in rodents.
Many antioxidant variables are altered by PB treatment. Some studies have demonstrated reduced liver vitamin E levels, decreased Mn- and CuZn-superoxide dismutase (SOD) levels, and decreased glutathione peroxidase (GSH Px) activity (6
,17
–19
). Several antioxidant enzymes, including glutathione S-transferase (GST), glutathione reductase (GR) and NAD(P)H:quinone reductase (DT-diaphorase), are increased transcriptionally by PB treatment (11
,19
–21
). Dietary vitamin E can also influence antioxidant status. Several glutathione-related enzymes, including GST, GR and GSH Px in rats, can be elevated by feeding adequate or high levels of dietary -tocopherol, compared with feeding deficient levels (22
) Therefore, vitamin E may be influencing other antioxidants and detoxification enzymes.
In this study, we hypothesized that PB-induced hepatic NF-B activation could be inhibited with increased dietary vitamin E in rats. We chose vitamin E for this study because it is a hydrophobic antioxidant that prevents free radical damage of lipid membranes and tissues (23
), and because PB treatment has been shown to decrease hepatic vitamin E levels (17
, 21
). Dietary vitamin E may decrease the oxidative environment created by PB by increasing vitamin E concentrations in the liver but also by also enhancing other antioxidants. To address these questions, we measured the hepatic DNA binding activity of NF-B as well as the concentrations of hepatic antioxidants and the activities of antioxidant enzymes in Sprague-Dawley rats that were administered 500 mg PB/kg diet for 10 d and fed one of 3 levels of dietary -tocopherol acetate (10, 50 or 250 mg/kg). The results indicated that NF-B DNA binding is sensitive to dietary -tocopherol levels.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Tocopherol-stripped corn oil was obtained from Acros Organics (Morris Plains, NJ). All other constituents of the purified diet were from Teklad Test Diets (Madison, WI). dl--Tocopherol acetate and PB were purchased from Sigma Chemical (St. Louis, MO) as were all other reagents, unless specifically stated otherwise.
Experimental design.
Male Sprague-Dawley rats (n = 39, 100–124 g) were obtained from Harlan Sprague Dawley (Indianapolis, IN) and housed three per cage covered with a microisolator in a temperature- and light-controlled room. After 1 wk of acclimatization, the rats were divided into 3 groups of 13 and fed a purified diet (Table 1
) containing 10, 50 or 250 mg vitamin E/kg diet (as dl--tocopherol acetate). These levels represented low (LVE; deficient), adequate (MVE) and high (HVE) dietary levels of vitamin E, respectively. The adequate level used (50 mg/kg) was slightly lower that used in the AIN-93M diet (75 mg/kg) (24
), but is the same as the NAS/NRC recommendation (25
). The rats consumed the food ad libitum for 28 d, then 8 rats per dietary group were fed the same purified diet with 500 mg/kg phenobarbital for 10 d. The rats were weighed once each week and at the end of the study. Two rats in the high vitamin E group were excluded from the study on wk 4 due to low body weight. Ten days after starting the PB treatment, the rats were euthanized by overexposure to CO2 and their livers were immediately removed and weighed. The livers were frozen in liquid nitrogen and stored at -80°C until time of assay.
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Liver pieces were homogenized in 11.5 g/L KCl with 100 µmol/L EDTA, pH 7.4, using an Ultra-Turrax homogenizer (Tekmar, Cincinnati, OH). One half of the homogenate was immediately divided into aliquots and stored at -80°C to be used for vitamin E, vitamin C, catalase, glutathione and SOD analyses; the other half was used to isolate cytosol and microsomes.
Cytosolic and microsomal isolation.
The cytosol and microsomes for each rat liver were isolated using the method described by Schramm et al. (26
).
Protein assay.
Protein concentrations of the WLH, cytosol and microsomes were determined using the bicinchoninic acid method (Pierce Chemical, Rockford, IL) with bovine 
-globulin as a standard (Bio-Rad, Hercules, CA) at 562 nm.
Alkoxyresorufin O-dealkylation assay.
Benzyloxyresorufin-O-dealkylase (BROD) and pentoxyresorufin-O-dealkylase (PROD) were used as specific substrates for the CYP2B1/2B2 isozymes as described by Burke et al. (27
). With the benzyloxyresorufin substrate, 100 µg of total microsomal protein was used for the PB induced and 250 µg for the uninduced. For the pentoxyresorufin substrate, 400 µg of total protein was added to each reaction for PB induced and uninduced. The absorbance of resorufin was detected with a fluorescence spectrophotometer at an excitation wavelength of 556 nm and an emission wavelength of 589 nm.
Isolation of nuclear extracts.
Hepatic nuclear protein extracts were isolated using a method similar to that of Deryckere and Gannon (28
). Briefly, random pieces of frozen liver were homogenized in ice-cold buffer A [6 mL/L IGEPAL CA-630, 150 mmol/L NaCl, 10 mmol/L HEPES, pH 7.9, 1 mmol/L EDTA, 500 µmol/L phenylmethylsulfonyl fluoride (PMSF)] using a Dounce tissue homogenizer. The homogenate was centrifuged at 270 x g for 30 s to eliminate unbroken tissue. The supernatant was transferred to a 15-mL tube, kept on ice for at least 5 min and centrifuged at 2980 x g for 20 min. The supernatant was discarded. The pellet was resuspended in 1 mL of buffer A, transferred to a microcentrifuge tube and centrifuged at 4000 x g for 3 min. The supernatant was discarded, and the pellet was resuspended with buffer C [250 mL/L glycerol, 20 mmol/L HEPES, pH 7.9, 420 mmol/L NaCl, 1.2 mmol/L MgCl2, 200 µmol/L EDTA, 500 µmol/L PMSF, 500 µmol/L dithiothreitol (DTT), 2 mmol/L Benzamidine, 5 mg/L aprotinin, 5 mg/L leupeptin, 5 mg/L pepstatin A] and incubated on ice for 1 h. The tubes were centrifuged at maximum speed for 10 min in a microcentrifuge. The supernatant (nuclear extract) was removed, divided into aliquots, and stored at -80°C.
Electrophoretic mobility shift assay (EMSA).
The NF-B oligonucleotide (Promega, Madison, WI) was radiolabeled with [-32P] ATP using T4 polynucleotide kinase (New England Biolabs, Beverly, MA). The EMSA reaction included 6 µg nuclear extracts, 1X binding buffer (10 mmol/L HEPES, pH 7.9, 50 mmol/L KCl, 0.2 mmol/L EDTA, 2.5 mmol/L DTT, 100 mL/L glycerol, 0.5 mL/L NP-40), 0.1 µg poly (dI-dC), 100,000 cpm radiolabeled NF-B probe in a total volume of 20 µL. The reaction was incubated for 20 min at room temperature and then electrophoresed on a 7% nondenaturing polyacrylamide gel for 2 h at 150 V using 0.5X Tris-borate-EDTA as the running buffer. To confirm the binding specificity of NF-B, 1 µg of anti-p50, anti-p65 or nonspecific preimmune serum [immunoglobulin (Ig) G] (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the reaction. A positive control was included with each gel (HeLa cell extract, Santa Cruz Biotechnology, Santa Cruz, CA). The radioactivity in the bands was counted with a radioanalytic imaging system (Ambis, San Diego, CA). Net radioactive counts/min (cpm) were determined for NF-B by subtracting background counts from the total counts in the NF-B band.
Western blotting.
Frozen liver pieces were diluted 1:4 with lysis buffer (10 mL/L Nonidet P-40, 1 g/L SDS, 100 mg/L PMSF, 2 mg/L aprotinin, 2 mg/L leupeptin, 2 mg/L pepstatin A and 1X PBS) and homogenized with an Ultra-Turrax homogenizer (Tekmar). The protein concentrations were measured and adjusted to 25 g/L. Equal volumes of homogenate samples were pooled (n = 5). The samples were denatured by boiling for 5 min in 2X gel loading buffer (173 mL/L glycerol, 1.25 mol/L ß-mercaptoethanol, 52 g/L SDS, 220 mmol/L Tris pH 6.8, 1 mg bromophenol blue). For the analysis of CYP2B1/2B2 and IB proteins, an 8.5% separating and 4% stacking gel was cast. For each pooled sample, 50 µg protein was electrophoresed at 175 V for 55 min. The proteins in the gels were transferred to nitrocellulose membranes (Gibco Life Technologies, Grand Island, NY) at 100 V for 1 h. The membranes were incubated with 5% blocking buffer (10 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 500 µL/L Tween-20, 50 g/L nonfat dry milk) for 1 h at room temperature with shaking. The primary and secondary antibodies were diluted in 5% blocking buffer and incubated with the membrane for 1 h with shaking at room temperature. The membranes were washed 3 times with wash buffer (10 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 500 µL/L Tween-20) between the primary and secondary antibodies. For the detection of CYP2B1/2B2, the following primary and secondary antibodies were used: CYP2B1/2B2 (Oxford Biomedical Research, Oxford, MI) and anti-mouse horseradish peroxidase (HRP) (Santa Cruz Biotechnology). For the detection of IB, the following primary and secondary antibodies were used: IB and anti-rabbit HRP (Santa Cruz Biotechnology). The membranes were probed with antibodies against ß-actin to confirm that equal amounts of protein were loaded in each lane. The Pierce SuperSignal Chemiluminescent Substrate Kit (Pierce, Rockford, IL) was used to detect the proteins.
Spectrophotometric method for determination of -tocopherol.
Vitamin E was analyzed as described by Kayden et al. (29
) with modifications. Briefly, the samples were saponified by mixing WLH (500 µL) with a 20 g/L pyrogallol solution (5 mL) and heated for 2 min in a 70°C shaking water bath. The tubes were removed from the hot water bath, and 0.25 mL of 11 mol/L KOH was added. The tubes were heated in a shaking 70°C water bath for 30 min, then placed in an ice bath. Hexane (2 mL) and water (0.5 mL) were added to the saponified samples and shaken vigorously for 2 min; 1 mL of the hexane layer was transferred to a 4-mL glass test tube for analysis. Standards of 1, 2, 4, 6, 8 and 10 mg/L of -tocopherol were made at the same time. A 2 g/L bathophenanthroline solution (200 µL) was added to all of the samples and standards and thoroughly mixed. The assay proceeded rapidly from this step and was kept away from direct light. Then, 200 µL of 1 mmol/L FeCl3 was added and vortexed. After 1 min, 200 µL of 1 mmol/L H3PO4 solution was added and vortexed again. The tubes were read on a spectrophotometer at 534 nm.
Glutathione equivalent assay.
Deproteinated WLH was used to measure total glutathione using a 96-well plate protocol as described by Baker et al. (30
).
Determination of ascorbic acid.
Total ascorbic acid was determined from liver homogenates precipitated with trichloroacetic acid according to the method of Omaye et al. (31
).
Enzyme assays.
The activity of SOD was determined indirectly by using xanthine and xanthine oxidase to generate O2
and measuring the inhibition of cytochrome c (32
,33
). Catalase activity was determined using whole-liver homogenates as described by Beers and Sizer (34
). Cytosolic fractions from each sample were used for the following enzyme assays. Selenium-dependent and total GSH Px activities were determined according to the method of Paglia and Valentine (35
). GR activity was measured according to the method by Paglia and Valentine (35
). GST were measured as described by Habig and Jakoby (36
) using the broad substrate 1-chloro-2,4-dinitrobenzene. DT-diaphorase activity was measured as described by Lind et al. (37
) with modifications of Hodnick and Sartorelli (38
).
Statistical analysis.
All statistical analyses were conducted using SYSTAT V.8 (SPSS, Chicago, IL) software. Results were first analyzed by two-way ANOVA. Individual differences between means were determined with the Bonferroni post-hoc test. If no significant interactions were found, the PB groups were combined before analysis for vitamin E–induced differences. If significant interactions occurred, control and PB-treated groups were analyzed separately for effects of vitamin E and PB. The results were reported as means ± SEM. Differences were considered significant at = 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-tocopherol acetate levels (Table 2
). Increased -tocopherol acetate in the diet increased hepatic vitamin E levels (Fig. 1
). Hepatic vitamin E concentrations were not decreased significantly by PB (P = 0.067); this is in contrast to several earlier studies that did report decreased hepatic vitamin E because of PB treatment (17
,21
).
|
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). However, a dramatic increase in these enzymes was evident in microsomes from rats that were administered PB; the amount of -tocopherol acetate did not affect protein levels as determined by Western blotting (Fig. 2)
. To further confirm the increase in CYP2B1/2B2 activities due to PB, we measured the dealkylation of the specific substrates benzyloxyresorufin and pentoxyresorufin in microsomal suspensions. Consistent with the increase in protein levels, both BROD and PROD activities were increased (Fig. 3
). Interestingly, rats fed the highest level of -tocopherol acetate (250 mg/kg) had greater BROD activity than the LVE group.
|
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B was increased in rats treated with PB (13
). Furthermore, we have shown that vitamin E can block peroxisome proliferator-mediated NF-B activation in H4IIEC3 rat hepatoma cells (39
). We therefore tested whether PB-mediated NF-B activation could be blocked with dietary vitamin E in rats. In rats that were fed the LVE diet, NF-B DNA binding activity was significantly higher in those that received PB (Fig. 4
). In contrast to the rats fed LVE, no increase in NF-B DNA binding activity was observed in rats fed the MVE and HVE diets (Fig. 4)
, indicating that vitamin E can block PB-mediated NF-B activation.
|
B band that was observed in EMSA. The NF-B band was not affected by the presence of control IgG, but this band was diminished and a supershifted complex was observed in samples containing anti-p50 or anti-p65 antibodies (Fig. 5
). This confirmed that a majority of the NF-B present in the rat liver was composed of p50:p65 heterodimers. NF-B translocation to the nucleus is often associated with the phosphorylation and subsequent degradation of IB molecules. Western analysis was performed to monitor IB and IBß levels in rat livers (Fig. 6
). There were no obvious differences in IB and IBß levels among the six groups. It is possible that Western analysis is not sensitive enough to detect such differences, although it is also possible that NF-B activation by PB is mediated by a mechanism that does not require IB degradation or that degraded IB was replaced with newly synthesized IB.
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and 4
). The activities of GST, GR and DT-diaphorase and the concentration of ascorbic acid were greater in rats administered PB, whereas catalase, total SOD and Se-independent GSH Px activities and glutathione levels were unaffected by PB, irrespective of vitamin E levels (Tables 3
and 4)
. PB treatment decreased Se-dependent GSH Px activity (Table 3)
. Rats fed the HVE diet had higher total glutathione levels than those fed the LVE diet, but only in PB-treated rats (Table 3)
Rats fed the HVE diet had greater catalase activity than rats fed either the LVE or MVE diets, regardless of PB treatment (Table 4)
. Vitamin E, however, did not affect the activities of Se-dependent or independent GSH Px, GST, SOD or DT-diaphorase (Tables 3
and 4)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and hydrogen peroxide (H2O2) (11
). A close correlation has been shown between CYP2B1 level and the rate of oxygen radical generation (40
). We previously showed that hepatic NF-B DNA binding activity was increased in rats treated with PB (13
). Here, we have extended these studies by showing that adequate or high vitamin E levels could block the PB-mediated NF-B activation in rats fed diets low in vitamin E. These data are consistent with a model in which increased ROS, including H2O2, are responsible for elevated levels of NF-B (41
).
NF-B DNA binding was increased 50% in rats fed low vitamin E and treated with PB. However, in a previous study in our laboratory, NF-B DNA binding was increased fourfold by d 10 (13
). The discrepancy between the two studies could be due to the different type of diets that were used, resulting in different levels of oxidants and/or antioxidants. In the current study, the antioxidant mixture and the -tocopherol acetate were ordered fresh, and the prepared purified diet was stored at -80°C until the day of use, whereas the unrefined diet in the previous study was stored at room temperature until use. The purified diet used in our study contained only corn oil stripped of vitamin E, whereas the fat sources in the previous study (13
) were ground meal, including corn, soybean, fish and some animal fat (Purina Lab diet 5001, St. Louis, MO). In addition, unrefined diets contain nonnutritive chemicals (42
), which could contribute to the magnitude of NF-B activation.
In our study, higher dietary vitamin E increased BROD activity, a measurement of CYP2B1/2B2 activities. Several studies have reported increased CYP activity with increased dietary vitamin E (43
,44
) and in animals exposed to increased vitamin E and PB (43
,45
). Lii and colleagues (45
) found that the protein levels of CYP2B1 paralleled the activity of PROD; however, in our study, the protein levels of CYP2B1/2 did not differ among PB-treated rats fed the different levels of -tocopherol acetate.
The activities of CYP2B1/B2 did not correspond with changes in NF-B in our study. One possible explanation is that ROS from sources other than hepatocytes may be involved in changes in NF-B levels. Indeed, transient and resident liver macrophages (Kupffer cells) are activated by PB treatment (46
), and activated macrophages have been shown to release ROS (46
). PB causes an influx of macrophages into the liver, and these macrophages produce elevated quantities of H2O2 in comparison to Kupffer cells (46
). If Kupffer cells are cocultured with hepatocytes and treated simultaneously with lipopolysaccaride (which activates Kupffer cells) and PB, the induction of CYP2B1 mRNA and the activity of PROD are significantly reduced (47
). Cytokines, which are released from macrophages, could be responsible for the down-regulation of CYP2B1/2B2 activities, which could explain the reduced activity in CYP2B1/2B2 in the rats fed the low vitamin E diet. Tumor necrosis factor- and interleukin-1ß have been shown to reduce CYP2B levels (47
–49
). It is possible that the presence of low concentrations of vitamin E activates a greater number of Kupffer cells, leading to the release of cytokines that are capable of decreasing CYP; however, to our knowledge no studies have been conducted on the activation of Kupffer cells in the presence of low vitamin E.
PB significantly affected the antioxidants and antioxidant enzymes in our study: it increased GST, GR and DT-diaphorase activities as well as ascorbic acid levels. Other studies have found similar results (11
,17
,50
–54
). In our study, PB decreased the activity of GSH Px; most studies have shown a decrease (18
,51
,52
) or no change in GSH Px (18
,51
,55
) with PB administration. In our study, PB treatment did not affect total glutathione concentration or catalase or total SOD activities. In other studies, the effects of PB on these three end points varied (6
,11
,18
,51
,55
). Other studies found no effect of dietary -tocopherol (ranging from 0 to 5000 mg/kg) and 500 mg/kg PB on glutathione status after 24 wk or 11 mo (22
, 56
). The effects of PB on antioxidants and antioxidant enzymes are similar to what others have observed (Tables 3
and 4)
, but data are limited on the effects of increasing dietary antioxidants on these other antioxidant end points in rats treated with PB.
Increased dietary -tocopherol did not affect the antioxidants and antioxidant enzymes that were altered by PB treatment; rather, the high vitamin E diet increased catalase and glutathione levels. Total glutathione, which is largely represented by reduced glutathione, was measured in this study (57
). In our study, the increased glutathione may be required to maintain the reduced forms of vitamins E and C (57
). However, normal glutathione levels in rat liver are usually 7–8 µmol/g tissue (58
), but the untreated and PB-treated rats in the LVE group in our study had <7 µmol/g tissue, suggesting that LVE may affect the synthesis of glutathione. Nevertheless, the levels of dietary vitamin E did not affect the glutathione-related enzymes or any of the antioxidant variables affected by PB. It may be that the antioxidants responding to PB treatment are not the result of an increased reactive oxygen environment, but rather PB may increase or decrease the antioxidant enzymes transcriptionally, which has been shown for GST and DT-diaphorase (20
).
Not all studies examining tumor promotion by PB support the oxidative stress hypothesis (19
). Rats that were fed increased dietary -tocopherol in the diet (100 or 5000 mg/kg) compared with those fed none and were initiated with diethylnitrosamine (DEN) and promoted with 500 mg/kg PB for 24 wk had elevated glutathione-related enzyme activities (GST, GSH Px and GR) without an effect on the formation of altered hepatic foci (22
). Other studies have also observed minimal or no effect of supplementing -tocopherol (ranging from 500 to 15,000 mg/kg) to a deficient diet on hepatocarcinogenesis in rats initiated with DEN and promoted with PB for a minimum of 3 mo (21
,56
).
Elevated NF-B activity could contribute to the alterations in cell proliferation and apoptosis observed with PB treatment because NF-B is also a regulator of these two processes (15
,16
,59
). This raises the possibility that NF-B may be involved in the carcinogenic properties of PB. The PB-induced NF-B DNA binding in rats fed the low vitamin E diet was reduced in rats fed adequate (50 mg/kg) and high (250 mg/kg) levels of vitamin E. Therefore, low dietary vitamin E may contribute to NF-B activation, but higher than adequate levels do not appear to be protective. This study further supports our hypothesis that PB administration in rodents leads to oxidative stress, thereby increasing NF-B DNA binding. The relationship between NF-B activation and tumor promotion has not been established for PB, but studies are in progress to address this question.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BROD, benzyloxyresorufin-O-dealkylase; CYP, cytochrome P450; DEN, diethylnitrosamine; DT-diaphorase, NAD(P)H:quinone reductase; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; GR, glutathione reductase; GSH Px, glutathione peroxidase; GST, glutathione S-transferase; HRP, horseradish peroxidase; HVE, high vitamin E; IB, inhibitory B; LVE, low vitamin E; MVE, medium vitamin E; NF-B, nuclear factor-B; PB, phenobarbital; PBREM, PB-responsive enhancer module; PMSF, phenylmethylsulfonyl fluoride; PROD, pentoxyresorufin-O-dealklyase; ROS, reactive oxygen species; SOD, superoxide dismutase; WLH, whole-liver homogenate.
Manuscript received 3 May 2002. Initial review completed 30 May 2002. Revision accepted 10 July 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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