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B Activation Induced by the Peroxisome Proliferator, Wy-14,6431 ,2
Departments of
*
Foods and Nutrition and
Environmental Health Science, University of Georgia, Athens, GA 30602; and the
**
Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, KY 40506
3To whom correspondence should be addressed. E-mail: jfischer{at}fcs.uga.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B activation in livers of rats treated with Wy-14,643. Forty-eight male Sprague–Dawley rats were fed one of four diets (20, 45, 650, 1500 mg Fe/kg diet) for 28 d. At the time of tissue collection, liver iron ranged from 1.4 to 9.9 µmol/g wet tissue in the diet groups. Wy-14,643 (0 or 0.1 g/100 g diet) was added to the diet for the final 10 d of the study. Wy-14,643 doubled the liver weight/body weight ratio (P = 0.0001), which was also increased by iron supplementation (P < 0.01). Iron supplementation increased thiobarbituric acid reactive substances and/or conjugated dienes, but there was no synergism between Wy 14,643 and iron on lipid peroxidation measures. The hepatic DNA binding activity of NF-B was increased in rats administered Wy-14,643. However, differences in liver iron concentration did not alter activation of NF-B in untreated rats or in those treated with Wy-14,643. DNA double-strand breakage was not affected by iron or Wy-14,643. In summary, although moderate changes in iron status altered liver lipid peroxidation, iron did not significantly increase oxidative stress induced by a hepatocarcinogenic peroxisome proliferator.
KEY WORDS: • iron • rats • peroxisome proliferator • oxidative stress • nuclear factor-B
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The liver is the primary site for iron accumulation and, thus, may be at high risk for iron overload-induced oxidative stress and tissue damage. However, the relationship between elevated iron stores and liver carcinogenesis is unclear. The genetic iron overload disease, hereditary hemochromatosis, is linked to an increased risk of liver cancer (2
,3
). Further, some studies have shown that excessive iron accumulation can enhance liver preneoplastic foci or tumors in rats treated with some chemical carcinogens such as diethylnitrosamine (4
), and hexachlorobenzene (5
). Nevertheless, the effects of iron excess on chemically induced liver carcinogenesis vary and depend on the form of iron used, the compounds and protocol selected for cancer initiation and promotion, and the cancer stage studied (4
–8
). Also, it is not clear whether moderate increases in liver iron alter chemically induced carcinogenesis.
One of the proposed mechanisms for iron-enhanced carcinogenesis is by free radical generation resulting in lipid peroxidation of cell membranes and/or DNA damage (2
,3
). Iron is a transition metal and can catalyze the production of the hydroxyl radical from hydrogen peroxide (H2O2) by the Fenton reaction or react with lipid hydroperoxides to form peroxyl and alkoxyl radicals (2
,9
). Although most iron in tissues is bound to proteins such as transferrin and ferritin, it is believed that there is a small amount of catalytically active iron that is present in the cell (2
). When tissue iron stores are high or tissue damage occurs, the pool of catalytically active iron may increase and become available to catalyze radical formation (1
,3
). Both in vitro and in vivo studies have demonstrated that high liver iron concentrations can enhance lipid peroxidation in whole tissue homogenates, microsomes, and mitochondrial fractions (2
). Iron excess may also increase DNA base oxidation and DNA strand breakage (3
,10
). Recently, research has been initiated concerning the role of iron or iron-induced oxidative stress on activation of the transcription factor nuclear factor-kappa B (NF-B)7
(11
). NF-B exists in an inactive state in the cytosol of the cell, usually as two subunits, p50 and p65, complexed with inhibitory proteins (IB). When the inhibitory proteins are degraded, NF-B translocates to the nucleus where it alters transcription of a number of genes involved in cell growth and differentiation, inflammation, and immune function (12
). Currently available data suggest that NF-B activation can be modulated by the redox status of the cell (12
). Reactive oxygen species such as H2O2 and organic hydroperoxides increase NF-B activation, whereas antioxidant compounds, including iron chelators, inhibit it (11
–15
).
The purpose of this study was to examine the effect of iron status on oxidative damage generated by Wy-14,643, a peroxisome proliferator. Peroxisome proliferators (PP) are nongenotoxic hepatic carcinogens in rodents, and include hypolipidemic drugs such as clofibrate, ciprofibrate, and Wy-14,643, and some phthalate ester plasticizers (16
). In rodents treated with PP, livers are enlarged (17
), and peroxisomes are increased in size and number, even after short exposure. Long-term administration of the compounds results in tumor development (16
,18
). The activity of peroxisomal fatty acyl CoA oxidase is elevated by PP administration, generating H2O2 as a reaction by-product (16
,19
). It has been proposed that PP-generated H2O2 leaks into the cytosol and, thus, may contribute to oxidative damage to the cell (1
). Increases in indices of oxidative stress, such as conjugated dienes and lipofuscin deposition, have been correlated with PP-induced carcinogenicity (20
). The peroxisome proliferators ciprofibrate and Wy-14,643 may also elevate hepatic 8-hydroxydeoxyguanosine (8-OHdG) concentration, an indicator of oxidative DNA damage (21
,22
). Recently, it has been documented that PP activate NF-B (13
,19
,23
,24
) and that this is mediated in part by the hydrogen peroxide generating enzyme fatty acyl CoA oxidase (15
). Studies showing that the radical scavenger allopurinol (23
), vitamin E and N-acetylcysteine supplementation (14
) and catalase overexpression (13
) can prevent PP-induced NF-B activation support the theory that the activation is linked to oxidative stress.
This study tested the hypothesis that liver iron concentration would affect lipid peroxidation, DNA double-strand breaks, and NF-B activation after treatment with the peroxisome proliferator Wy-14,643.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male, weanling Sprague–Dawley rats (n = 48; initial weight, 87–105 g; Harlan Sprague–Dawley, Indianapolis, IN) were housed individually in stainless steel wire-bottomed cages in a humidity, temperature (21 ± 1°C), and light (12-h light:dark cycle) controlled facility. All animal procedures were approved by the University of Georgia Institutional Animal Care and Use Committee. Rats were acclimated for 24 h and were then randomly assigned to one of four dietary groups (n = 12 per group) with adjustments made to keep mean group weights similar (Table 1
). Rats were provided free access to distilled water and low, adequate, moderately high or high iron diets (15, 45, 650 and 1500 mg iron/kg diet, respectively) for 28 d. After 18 d, rats in each dietary group were randomly assigned to two subgroups; untreated (n = 6) and treated with the PP Wy-14,643 (n = 6). Wy-14,643 was added to the diets at a concentration of 0.1 g/100 g. Rats were fed an additional 10 d. Body weight and food intake were recorded daily.
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) was used for the study (Table 2
). An iron-deficient AIN-93G mineral mix was obtained from Harlan Teklad (Madison, WI). Diets were supplemented with FeS04 · 7H20 (Sigma, St. Louis, MO) to achieve dietary iron concentrations as noted. Iron concentrations of low, adequate, moderately high or high iron diets, as determined by atomic absorption spectrophotometry, were 21, 46, 551 and 1243 mg/kg diet, respectively (92% recovery). Diets were prepared weekly and stored at -20°C. Rats were provided fresh diet daily.
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Food was withheld overnight before tissue collection. Rats were anesthetized with a 3:2:1 (v/v/v) ratio of ketamine:acepromazine:xylazine (0.8 mL/kg body). Blood was obtained by heart puncture into nonheparinized syringes (Sarstedt, Princeton, NJ) and heparinized capillary tubes were used to collect whole blood for hematocrit determination. The liver and spleen were removed and rinsed with ice-cold saline. The livers were blotted dry, weighed and sectioned. Two 1-g pieces of liver were immediately frozen in liquid nitrogen and stored at -80°C until analyzed for DNA double-strand breaks and NF-B activation. Another 1-g piece of liver was homogenized in potassium phosphate buffer containing 0.001 mol/L EDTA (pH = 7.0) for thiobarbituric acid reactive substances (TBARS) and conjugated dienes assays. The remaining liver sections and spleens were stored at -80°C before mineral analysis. Liver and spleen iron concentrations and hematocrit were used as indicators of iron status.
Tissue analysis.
Lipid peroxidation in fresh whole liver homogenates was assessed by measuring TBARS using the method of Buege and Aust (26
), and protein content of liver homogenates was determined using the method of Lowry et al. (27
). Conjugated dienes were assayed in liver as described by Recknagel and Glende (28
). Briefly, lipid was extracted from fresh liver homogenates with chloroform-methanol (2:1, v/v), and the lipid fraction was dried under nitrogen. In the final step, lipid was resuspended with cyclohexane and the absorbance at 233 nm was recorded spectrophotometrically. Conjugated dienes are expressed per milligram of lipid in the sample.
The iron concentration of diets, liver, and spleen was determined by atomic absorption spectrophotometry (model 5000; Perkin-Elmer, Norwalk, CT) as described by Johnson (29
). The accuracy of the results was verified by determining the iron content of a bovine liver standard obtained from the National Institute of Standards and Technology (Gaithersburg, MD; 92–94% recovery).
DNA double-strand break analysis.
DNA double-strand breaks were measured as described by Westerfield and Black (30
). Approximately 30–35 mg liver was homogenized in ten volumes of buffer (250 mmol/L NaCl, 100 mmol/L EDTA and 100 mmol/L Tris base) and treated with sarcosyl (1% v/v) to lyse the cells. DNA was extracted and purified according to the methods of Maniatis et al. (31
), with modifications described by Westerfield and Black (30
). Extracted DNA was stored in ethanol at -20°C for 12 h to 2 wk before electrophoresis. In preparation for electrophoresis, samples were centrifuged, DNA pellets were washed with 70% ethanol/30% TE buffer (10 mmol/L Tris-HCl and 1 mmol/L EDTA), recentrifuged, dried under nitrogen gas, and resuspended in TE buffer. DNA concentration and purity were assessed with a Beckman 650 spectrophotometer (at 260 and 280 nm; Beckman Instruments, Fullerton, CA). Approximately 1.5 µg DNA was loaded into each well of a 0.75% pulsed-field agarose gel preloaded with ethidium bromide for electrophoresis, with equivalent DNA amounts loaded for each sample during a run. A DNA size standard (Bio-Rad, Hercules, CA) was included on each gel. Samples were resolved in 0.5X TBE buffer at 14°C for 11 h using a field inversion gel electrophoresis system at forward and reverse voltages of 180 and 120 V, respectively (Bio-Rad). The gel was photographed on a UV transilluminator. The negative was then scanned with a densitometer and analyzed with a computer image analysis program (PDI Model 25 densitometer with Quantity One Software; PDI, Huntington Station, NY) to determine the weighted average DNA strand length for each sample, based on band migration and intensity (30
).
NF-B analysis.
The DNA binding activity of NF-B was measured using electrophoretic mobility shift assays (EMSA). Nuclear extracts were made from frozen liver tissue as described by Deryckere and Gannon (32
). A part of the extract was used to assay the protein concentration by using the BCA assay (Pierce, Rockford, IL). The remaining part of the nuclear extract was aliquoted and stored at -80°C. The probe containing an NF-B consensus oligonucleotide (Promega, Madison, WI) (5'-AGT TGA GGG GAC TTT CCC AGG C-3') was end-labeled with P-32 using polynucleotide kinase. Each sample (5 µg) was incubated in a binding buffer for NF-B containing 50 mmol/L KCl, 10 mmol/L HEPES, pH 7.9, 6.5 mmol/L DTT, and 10% glycerol along with 0.5 µg poly (dI:dc) for 10 min on ice, and for 20 min at room temperature with 1 ng (
20,000 dpm) of radioactively labeled NF-B probe. For antibody supershift assays, 4 µg of rabbit anti-p50, anti-p65, or preimmune serum (Santa Cruz Biotech, Santa Cruz, CA) was also added. After incubations, the samples were resolved on a 7% nondenaturing polyacrylamide gel in 0.5X TBE buffer at 150 V for 2 h. The gels were subsequently dried and autoradiographed. The dried acrylamide gels used for autoradiography were then analyzed using a radioanalytic imaging system (Ambis, San Diego, CA).
Statistical analysis.
Treatment means, SEM and ANOVA were calculated using the statistical package SAS (SAS Version 6.12; SAS Institute, Cary, NC). The overall effects and interactions of iron and PP administration on iron concentration, liver weight, lipid peroxidation measures and DNA strand breaks were determined with two-way ANOVA. Fisher’s least significant difference test was used for post-hoc comparisons. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. Dietary iron concentrations did not affect body weight. Relative liver weight was nearly doubled by Wy-14,643 treatment (P = 0.0001; Table 1
) at all dietary iron concentrations and was positively affected by dietary iron concentrations that ranged from below adequate to high levels (P < 0.01; Table 1
).
Liver iron of rats fed moderately high and high iron diets was approximately two and four times that of rats fed adequate iron diets, respectively. Low iron diets reduced liver iron concentrations by 45% (Table 3
). Although Wy 14,643 treatment increased total liver iron, liver iron concentrations (g wet tissue) were either unchanged or decreased by the compound, depending on dietary iron level. There was a significant interaction between iron and Wy-14,643, such that Wy-14,643 reduced liver iron concentrations 22% in rats fed 1500 mg Fe/kg diet but had no effect at other levels of iron intake. However, rats fed the highest level of iron and treated with the drug still had liver iron concentrations that were almost four times that of control rats. Spleen iron concentrations further verified that four different levels of iron accumulation were achieved in these rats. Although spleen iron concentration was higher in rats treated with Wy-14,643, this was most likely due to a decrease in spleen weight due to Wy-14,643 treatment (P = 0.0001, data not shown). Iron intake altered hematocrit (P < 0.02), although Wy-14,643 treatment did not affect it (data not shown). Hematocrit was not reduced by the low iron diet, but rats fed 1500 mg Fe/kg diet had slightly higher hematocrit (0.49 ± 0.004; P < 0.05) than rats fed adequate iron (0.45 ± 0.008; P < 0.05).
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). Although Wy-14,643 treatment was associated with higher levels of TBARS, it resulted in slightly lower conjugated dienes (P < 0.05).
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Another possible result of oxidative stress is the activation of transcription factors such as NF-B. EMSA were therefore performed to determine whether hepatic NF-B activity could be influenced by dietary iron in Wy-14,643-treated and control rats. The DNA binding activity of NF-B was greater in rats administered Wy-14,643 (Fig. 1
), as has been observed previously with Wy-14,643 and other PP (13
,19
,24
). Dietary iron did not affect NF-B activation in rats that did not receive Wy-14,643 (Fig. 2
) or in Wy-14,643-treated rats (Fig. 3
). To confirm that the bands observed contained NF-B, we performed supershift analyses. Preimmune serum did not affect the NF-B band, whereas antibodies to p50 and/or p65 decreased the NF-B band and resulted in the appearance of a new, supershifted complex (Fig. 4
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), and some studies have suggested that iron status may alter chemically induced carcinogenesis by modulating free radical production and lipid peroxidation (3
). The objective of this study was to determine whether iron status affects oxidative stress after treatment with a carcinogen suspected to generate reactive oxygen species.
Four different concentrations of liver iron were achieved through dietary manipulation. Liver iron concentrations for rats fed moderately high and high iron diets were approximately two and four times levels found in rats fed adequate iron diets. This magnitude of iron accumulation is not as great as that found in rodent models of homozygotic hereditary hemochromatosis (2
) but instead represents moderate iron accumulation. The low iron diets used in this study diminished liver iron concentrations to 60% of control, but did not alter hematocrit, representing a moderate reduction in iron stores.
Although Wy-14,643 treatment consistently increased total liver iron, this effect was somewhat less at the highest level of dietary iron intake, as shown by the iron concentrations within this group. Little is known about the effect of PP on iron homeostasis. However, Hertz et al. (33
) reported that plasma serum transferrin and liver transferrin mRNA were reduced in rats after administration of a PP, which could alter iron transport capacity. Future studies should focus on the impact of these compounds on iron transport and cellular iron uptake.
Enhanced lipid peroxidation has been correlated with PP-induced carcinogenesis by some (20
). We hypothesized that the combination of high liver iron concentrations and Wy-14,643-induced H2O2 production would stimulate lipid peroxidation by the Fenton reaction, whereas low liver iron concentrations would diminish it. Conjugated dienes are a marker of an early stage of lipid peroxidation, immediately after abstraction of a hydrogen atom from a polyunsaturated fatty acid, whereas the TBARS assay assesses a later stage in which lipid peroxides are decomposed (1
). In this study, iron concentration was positively associated with increases in both TBARS and conjugated dienes, which is consistent with previous reports (2
). If excess iron levels had catalyzed hydroxyl radical production from Wy-14,643-generated H2O2, as hypothesized, the expected result should have been a synergistic effect between iron and Wy-14,643 on both TBARS and conjugated dienes. Although iron and Wy-14,643 increased TBARS, the effect was additive rather than synergistic. Further, conjugated dienes were actually lower in Wy-14,643 treated rats than in untreated rats, regardless of iron concentration. This suggests that iron and Wy-14,643 did not interact and are affecting lipid peroxidation markers through different pathways. There are several possible explanations for these results. First, it is possible that reactive iron and H2O2 generated by the drug were not located in the same cellular compartment. Second, catalase activity slightly increases after Wy-14,643 administration (20
) and may be sufficient in early stages of treatment to lower H2O2 levels, thereby reducing or preventing lipid peroxidation. Lipid peroxidation may only be increased by long-term administration of PP. In support of this, Conway et al. (20
) reported an initial reduction in conjugated dienes after short-term (8 and 18 d) treatment with a PP, with elevated conjugated dienes appearing after 5–6 mo of treatment. Finally, iron may increase lipid peroxidation in vivo primarily by catalyzing peroxyl and alkoxyl radical production from lipid hydroperoxides, as has been reviewed by Winterbourn (9
). It is unclear why Wy-14,643 affected the TBARS assay, without a concurrent change in conjugated dienes. This may be because TBARS represents a different stage of lipid peroxidation than conjugated dienes, or it may be because in the TBARS assay, chromagens other than malondialdehyde that are generated by Wy-14,643 treatment react.
High levels of H2O2 can cause DNA strand breaks and oxidized DNA adducts such as 8-OHdG in many cell types, including hepatocytes (1
,34
). Therefore, many have examined the impact of H2O2-generating PP on DNA damage in the liver (21
,22
,35
). Increases in liver 8-OHdG have been reported after as few as 21 d of PP administration (22
). High concentrations of iron can produce oxidized DNA adducts, and DNA single- and double-strand breaks in vitro (1
,3
). Further, Kang et al. (10
) reported an increase in liver 8-OHdG in a rodent model of hereditary hemochromatosis. In this study, we tested the hypothesis that elevating tissue iron concentrations in rats treated with Wy-14,643 would increase the DNA double-strand breaks. However, Wy-14,643 did not affect DNA double-strand breaks, regardless of iron level, and elevations in liver iron due to dietary iron level had no effect on strand breaks. Tissue iron concentrations may need to be much higher than levels achieved in this study to cause DNA damage in vivo, even in a model of chemical carcinogenesis. Kang et al. (10
) reported elevated levels of 8-OHdG in rats fed 3% carbonyl iron resulting in liver iron levels that were 16 times higher than control, which was much higher than iron concentrations of this study. It is also possible that 10 d of Wy-14,643 administration is not sufficient to cause DNA strand breakage.
The final measure assessed in this study was NF-B activation. NF-B can activate a number of genes implicated in carcinogenesis, including genes that encode growth factors and components of the immune system (12
). It is a redox-sensitive transcription factor (1
), which has prompted research into the role of iron in NF-B activation. Several studies have shown that iron chelation can diminish NF-B activation in vitro. For example, the addition of the iron chelators deferoxamine mesylate plus ferrozine to alveolar epithelial (A549) cells prevented NF-B activation induced by particulate air pollution (36
). Also, Tsukamoto et al. (11
) reported an association between iron concentration of hepatic macrophages and lipopolysaccharide-induced NF-B activation, whereas Youdim et al. (37
) reported that iron chelators reduce NF-B activation in neural tissue. We theorized that iron status would modulate oxidative stress and alter Wy-14,643-induced NF-B activation. In our study, NF-B activation was clearly enhanced by Wy-14,643, as reported previously (24
). However, iron status did not influence NF-B activation in Wy-14,643-treated or untreated rats. The superoxide radical, H2O2, and organic hydroperoxides all can activate NF-B (12
). Our results suggest that Wy-14,643-generated products such as H2O2 may be sufficient to induce NF-B activation, and iron-induced hydroxyl radical formation does not play a role in this activation. It also could be that extremes of iron overload or deficiency are needed to alter NF-B activation, and this should be an area for further research.
Some studies suggest that iron status may influence chemical carcinogenesis. Our work showed that moderately high liver iron concentrations do contribute to lipid peroxidation, as previously documented. However, iron and the PP Wy-14,643 did not act synergistically to increase lipid peroxidation, DNA double-strand breaks or NF-B activation. Future studies should examine whether extreme iron deficiency or iron overload affect PP-generated oxidative stress, as well as the long-term effect of iron status on PP-induced hepatocarcinogenesis.
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FOOTNOTES |
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2 Presented in part at Experimental Biology 1999, April 1999, Washington, DC. [Fischer, J., Glauert, H., Black M., Larmonier, N., Sweeney, M. & Yin, T. (1999) High iron intake does not enhance activation of hepatic NF-B by Wy-14,643, a peroxisome proliferator. FASEB J. 13: A917(abs.)].
4 Present address: Integrated Graduate Program in the Life Sciences, Northwestern University, Chicago, IL.
5 Present address: Marine Extension Service, University of Georgia, Savannah, GA.
6 Present address: INSERM U517, IFR 100, Faculty of Medicine, Dijon, France.
7 Abbreviations used: 8-OHdG, 8-hydroxydeoxyguanosine; EMSA, electrophoretic mobility shift assays; NF-B, nuclear factor B; PP, peroxisome proliferator; TBARS, thiobarbituric acid reactive substances.
Manuscript received 29 November 2001. Initial review completed 15 January 2002. Revision accepted 10 June 2002.
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