TY - JOUR
AU1 - Wijeweera, Jayanthika B.
AU2 - Gandolfi, A. Jay
AU3 - Parrish, Alan
AU4 - Lantz, R. Clark
AB - Abstract Arsenic is a known human carcinogen. These studies were designed to examine the impact of low arsenite concentrations on immediate early gene expression in precision-cut rat lung slices. Precision-cut lung slices are a versatile in-vitro system for toxicity studies, as they preserve the architecture and cellular heterogeneity of the lung. Since 0.1–100 μM arsenite did not compromise slice viability at 4 hours, effects of arsenite on the expression of c-jun/AP-1, NFκB, HSP 32, HSP 72, HSP 60, and HSP 90 were studied, using these concentrations of arsenite at 4 h. Nuclear c-jun was increased by 10 and 100 μM arsenite, while NFκB was not affected. Gel-shift assays indicated that 10 μM arsenite resulted in an enhanced DNA-binding activity of both AP-1 and NFκB. Confocal microscopic analysis of AP-1 indicated nuclear localization of this transcription factor, mainly in type-II epithelial cells and alveolar macrophages. Nuclear localization of NFκB was lower than that observed for AP-1, while most of the NFκB was localized to cytoplasm of type-II epithelial cells and alveolar macrophages. HSP 32 was increased by 1.0 and 10 μM arsenite, while HSP 72 was increased by only 100 μM arsenite. HSP 60 and HSP 90 were not changed by arsenite. These studies indicate that noncytotoxic concentrations of arsenite are capable of affecting signal transduction pathways and gene expression in the lung. sodium arsenite, in vitro, precision-cut lung slices, NFκB, AP-1, stress proteins Arsenic is a known human carcinogen. Inhalation exposure to arsenic occurs in industrial settings such as lead, copper, and zinc smelting, from fossil fuel combustion in power plants, in the semiconductor industry, as well as in pesticide production. Nonoccupational exposure to arsenic can take place through ingestion of contaminated food and water. In the U.S., high concentrations of arsenic in drinking water are found in the western and southwestern states and in Alaska. Exposure to high concentrations of arsenic in drinking water causes lung, skin, liver, and kidney cancers. Although the current EPA maximum contaminant level (MCL) for arsenic in drinking water is 50 μg/l (proposed level of arsenic in drinking water is 5 μg/l), in some parts of the country, 50–100 μg/l of arsenic has been detected in drinking water (Davis et al., 1994). In both occupational and drinking water, humans can be exposed to As(V) and/or As(III) forms of arsenic. For occupational inhalation, these can include slightly soluble arsenic trioxide, arsenic trisulfide, and calcium arsenate, while drinking-water exposure is from soluble salts (sodium arsenate and arsenite). Interconversion of the oxidative states can take place within the body. However, the As(III) compounds are the more toxic. The mechanism(s) responsible for causing cancer by arsenic in humans is not fully known, because it is not mutagenic in bacteria or mammalian cells (Lofroth and Ames, 1978; Rossman et al., 1980). Furthermore, there are no suitable animal models for investigating carcinogenicity of arsenic compounds. Several mechanisms by which arsenic may cause genetic damage have been proposed, including the generation of reactive oxygen species (Applegate et al., 1991; Wang et al., 1996; Yamanaka et al., 1991). Arsenic has been classified as an atypical carcinogen, as it does not fall into the category of initiator or promoter (Barrett et al., 1989; Brown and Kitchin, 1996). However, recent investigations reveal that arsenite activates NADH oxidase to produce superoxide, resulting in DNA strand breaks and large deletion mutations in mammalian cells (Hei et al., 1998; Lynn et al., 2000). It can also cause inhibition of DNA repair by inhibiting DNA ligase (Lynn et al., 1997). Since no animal models are available for the investigation of mechanisms by which arsenic causes cancer, in vitro systems become important in studying mechanisms of toxicity and elucidating early changes in gene expression after arsenic exposure. Precision-cut lung slices are a versatile in vitro system for toxicity studies, since they preserve the structural architecture of the lung as well as the cellular heterogeneity. They also preserve cell-cell interactions and cell-matrix interactions. Lung slices from one animal can be used for one experiment as well as control and treatment group, thus reducing variability. Precision-cut lung slices have been used previously in various toxicological studies (Fisher et al., 1994; Jones et al., 1992; Morin et al., 1999; Price et al., 1995). Exposure to arsenic results in the activation of several signaling pathways: the mitogen-activated protein kinase (MAPK), and the NFκB signaling pathways. The activation of these pathways, which leads to the expression of stress proteins, is considered to be an important stress response that enables cells to survive, to undergo proliferation, or to experience apoptosis. There are 4 major MAPK pathways: extracellular signal-regulated kinase or ERK (Boulton et al., 1991), c-jun N-terminal kinase or stress-activated protein kinase, abbreviated JNK/SAPK (Davis, 1994), p38 MAP kinase (Stein et al., 1997), and big MAP kinase or BMK/ERK5 (Lee et al., 1995). MAPK pathways regulate the expression of transcription factors such as AP-1, ATF-2, ELK-1 (Karin, 1995), and HSF (Lee and Corry, 1998). AP-1 and ATF-2 are responsible for the expression of protooncogenes c-jun (Karin et al., 1997), and ELK-1 for the expression of fos (Cavigelli et al., 1995). AP-1 complex can consist of multiple protein complexes (Cohen et al., 1989). It can be a heterodimer of the jun family of transcription factors: c-jun, jun-B, jun-D, or the fos-family of transcription factors: c-fos, fos-B, Fra1 and Fra2 (Angel and Karin, 1991). AP-1 can also consist of jun-jun homodimers (Karin, 1995). NFκB is activated due to oxidative stress and is involved in the expression of antioxidant enzymes such as superoxide dismutase (Iwanaga et al., 1988) and γ-glutamylcysteine synthetase, which is the rate-limiting enzyme of glutathione synthesis (Meyrick and Magnuson, 1994). In unstimulated cells, NFκB is localized in the cytoplasm, bound to its inhibitory subunit IκB (Beg et al., 1992). Phosphorylation of IκB by IκB kinase (Brown et al., 1995) results in the dissociation of IκB from NFκB and ubiquitination of IκB (Scherer et al., 1995). As a result, the nuclear localization signal on NFκB becomes unmasked and it migrates into the nucleus and binds to DNA (Jung et al., 1995). The exact mechanisms by which reactive oxygen species activate NFκB are not fully known. However, there is evidence to believe that the redox-sensitive enzyme, thioredoxin peroxidase, regulates the activity of NFκB by modulating the phosphorylation of IκB (Jin et al., 1997). The purpose of the present study was to examine the acute toxicity of low concentrations of sodium arsenite in lung slices, and to determine whether arsenite exposure results in the expression and activation of transcription factors c-jun/AP-1 and NFκB. These transcription factors are involved in the regulation of genes induced under stressful conditions, such as stress proteins HSP-32 and HSP 72. MATERIALS AND METHODS Chemicals. Arsenite and gentamycin sulfate were purchased from Sigma Chemical Co. (St. Louis, MO). Waymouth MB751/2 was obtained from GIBCO Laboratories (Grand Island, NY). Fungibact was purchased from Irvine Scientific (Santa Ana, CA). Chemicals for Western blotting were from BioRad (Hercules, CA). Horseradish peroxidase conjugated secondary antibodies were from Vector Laboratories (Burlingame, CA). Oligonucleotide probes for NFκB and AP-1 was from Promega (Madison, WI). All the other chemicals were from Sigma Chemical Co. Animals. Male Sprague-Dawley rats (275–300 g) were purchased from Harlen (Indianapolis, IN). The animals were allowed to acclimatize for 1 week before use. Food (standard laboratory chow) and water were freely available to the rats. Preparation and incubation of lung slices. Rats were sacrificed by carbon dioxide asphyxiation. The skin was dissected from the abdomen to the neck and the pleural cavity and trachea were exposed. The trachea was cannulated, and warm 5% agarose solution (40°C) in Waymouth MB751/2 was instilled as described earlier (Stefaniak et al., 1992). The trachea and lungs were immediately removed en bloc and placed in ice-cold Krebs-Henseleit buffer (pH 7.4) for the agarose to gel. Tissue cores of 10-mm diameter were prepared using a sharpened stainless steel tube. Precision-cut lung slices were prepared using a Krumdieck tissue slicer (Alabama Research and Development Corporation, Munsford, AL) and ice-cold Krebs-Hensleit buffer (pH 7.4) gassed with 95% O2:5% CO2. Slice thickness was 500–600 μ. Three lung slices were loaded onto a semicircular screen with 2 steel rings at each end, and the screen was placed in a glass scintillation vial containing 1.7 ml Waymouth MB751/2 to which 0.5% gentamycin and Fungibact solution (10 ml/l) has been added. The vials were incubated at 37°C for 1–24 h on a dynamic roller incubator, with continuous passage of 95% O2:5% CO2 (1 L/min). Sodium arsenite was added after a 1-h preincubation period. Different concentrations of sodium arsenite (0.1, 1.0, 10, and 100 μM) were obtained by diluting a stock solution (10 mM, pH 7.4) in Waymouth medium. Control slices were incubated in the medium only. Viability. Analyzing the intracellular K+ content assessed viability of lung slices. Three slices (from the same vial and from the same animal) were pooled into 1 ml of deionized water and homogenized by sonication. Proteins were precipitated by adding 40 μl of perchloroacetic acid to 800 μl of homogenate and the samples were centrifuged at 14,000 rpm for 15 min. The supernatant fraction was used for K+ assay by flame photometry, as described earlier (Azri et al., 1990). The pellet was dried overnight and dissolved in 1 ml of 1 M sodium hydroxide. The protein content was analyzed by the Bradford Method (Bradford, 1976). The results were expressed as μmol K+/mg protein. Histopathology. Lung slices were incubated for 4 h with 10-μM arsenite. Control slices were incubated without arsenite. The slices were fixed in 10% neutral buffered formalin, embedded in paraffin, and processed for light microscopy. Sections were stained with hematoxylin and eosin. Western blot analysis of stress proteins. After incubation for 4 h with 0–100 μM arsenite, 3 lung slices from the same vial were homogenized in 300 μl lysis buffer (1% SDS, 1.0 mM sodium orthovanadate, 10 mM Tris–HCl pH 7.4) containing 1 μl/ml Protease Inhibitor Cocktail (Sigma Chemical Co.). Sliced homogenate was centrifuged at 14,000 rpm for 15 min. and the supernatant fraction was used for the analysis. Protein content was determined by Bradford Assay (Bradford, 1976). Protein (40 μg) was mixed with an equal volume of sample buffer (BIO RAD, Hercules, CA), containing 10% β-mercaptoethanol and boiled for 5 min. Samples were then loaded onto a 10% gel, and the proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS–PAGE, 130 V) until the dye front reached the bottom of the gel. The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (400 mÅ for 30 min), and the membranes were blocked overnight with 5% nonfat milk in TBST (0.1 M Tris base pH 7.5, 0.1 M NaCl, 0.1% Tween-20). Immunoperoxidase staining for HSP 72, HSP 60, HSP 32, and HSP 90 were performed using specific monoclonal antibodies for each stress protein (StressGen, Victoria, BC, Canada), and horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using a DAB Substrate Kit for Peroxidase (Vector Laboratories, Burlingame, CA), which utilizes the enzymatic conversion of 3,3′-diaminobenzidine (DAB) to a chromogen by horseradish peroxidase. Densitometric analysis of the bands was performed using Scion Image (NIH). Preparation of nuclear proteins. Nuclear protein was isolated, as previously described (Parrish et al., 1999), from sodium arsenite-treated or control lung slices incubated for 4 h. Six slices from the same condition were pooled and homogenized in 1 ml of HEGD buffer (25 mM HEPES, 1.5 mM EDTA, 10% glycerol, 0.15 mg/ml dithiothreitol, 0.1 mg/ml phenylmethylsulfonyl fluoride, and 1 μl/ml Protease Inhibitor Cocktail (Sigma Chemical Co.) using a glass homogenizer, and was centrifuged at 14,000 rpm for 15 min. The supernatant was aspirated and the pellet resuspended in 80 μl of ice-cold HEGDK buffer (HEGD buffer containing 0.5-M KCl) and incubated on ice for 1 h. The samples were microfuged at 14,000 rpm for 15 min, and the nuclear protein extract in the supernatant was transferred to a fresh tube. The nuclear protein extracts were stored at –20°C and used within a few days. Gel-shift assay for AP-1 and NFκB. Gel-shift assay was performed using a Digoxigenin Kit # 1635 (Boehringer Mannheim) according to the manufacturer's directions. NFκB (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) and AP-1 (5′–d[CGC TTG ATG AGT CAG CCG GAA]-3′) double-stranded consensus oligonucleotides (Promega, Madison, WI) were 3′ end-labeled with Dig-11-ddUTP using terminal transferase. Nuclear protein (10 μg) from control or 10 μM arsenite-treated lung slices was incubated for 15 min. at room temperature with 4 μl binding buffer, 1 μl poly [D(I-C)], 1 μl poly L-lysine, 1 μl digoxigenin labeled AP-1 or NFκB (400 pg/μl), and nuclease-free water to make up to 20 μl. Two hundred-μM H2O2-treated lung slices were used as a positive control. The specificity of the reaction was determined by the addition of excess unlabeled oligonucleotide for competition. The samples were loaded onto a 5% nondenaturing polyacrylamide gel for electrophoresis, and after electroblotting onto a nylon membrane, oligonucleotides were cross-linked to nylon membrane using a UV crosslinker. The digoxigenin-labeled oligonucleotides on the membrane were bound by anti-digoxigenin antibody linked to alkaline phosphatase. Chemiluminescence is produced when the substrate CSPD (disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5chloro)tricyclo[3.3.1.137]decan}-4-yl)phenyl phosphate) is dephosphorylated by alkaline phosphatase and is recorded on x-ray film (Kodak Biomax) for 15–30 s. Western blot analysis of nuclear c-jun/AP-1 and NFκB proteins. Nuclear proteins from lung slices exposed to 0, 0.1, 1.0, 10, or 100 μM of sodium arsenite for 4 h were prepared as mentioned above. Western blot analysis was performed as described for the stress proteins, with a few modifications. Ten μg protein was loaded onto a 10% gel and SDS/PAGE was performed. Proteins were transferred onto PVDF membranes, and the membranes were incubated with primary antibodies to NFκB (Chemicon, Temecula, CA) or c-jun/AP-1 (Oncogene, Boston, MA). NFκB is a mouse monoclonal antibody to the p65 subunit, which recognizes an epitope overlapping the nuclear localization signal. Thus it recognizes the activated form of NFκB. C-jun/AP-1 is a rabbit polyclonal antibody raised against an amino acid sequence in the DNA binding domain of jun protein, and it recognizes the activated form of AP-1 as well as c-jun protein. For the detection of NFκB, the membranes were incubated with biotinylated goat anti-mouse antibody for 1 h. The membranes were then incubated with streptavidin-HRP complex (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h. For the detection of c-jun/AP-1, membranes were incubated with HRP-labeled goat anti-rabbit antibody for 1 h. Protein bands were detected by using enhanced chemiluminescence reagents (ECL Plus Kit, Amersham Pharmacia Biotech) and exposure to x-ray films (Kodak Biomax). The density of the bands was analyzed using Scion Image (NIH). Localization of c-jun/AP-1 and NFκB by confocal microscopic immunofluorescence detection. Lung slices were incubated for 4 h with 10 μM arsenite. Control slices were incubated without arsenite. The slices were fixed in 10% neutral buffered formalin, embedded in paraffin, and 5-μ sections were cut. Lung sections were deparaffinized and antigen retrieval was performed by placing slides in citrate buffer (pH. 6.1) and heating in a microwave oven on high power for 3–5 min or until boiling point was reached. The slides were microwaved for another 5 min on Defrost setting and allowed to cool to room temperature (25–30 min). The tissues were permeabilized by incubation with 0.1% NP-40 (Sigma Chemical Co.) in phosphate-buffered saline. The sections were incubated with anti-NFκB against the p65 sub unit (which detects activated form of NFκB), or anti-c-jun/AP-1 (which detects activated AP-1 or c-jun protein) at 37°C for 1 h. This was followed by incubation with the appropriate biotinylated secondary antibody for 1 h at 37°C. After the washing step, sections were incubated with Cy-5 conjugated to Streptavidin, and RNA was digested using DNase-free RNase. The nuclei were stained with YOYO iodide. The sections were mounted in DAKO mounting medium and stored at 4°C. Samples were viewed using a Leica TCS confocal microscope with a Kr/Ar laser. Statistics. Values represent mean ± standard error of 3–4 animals. Three slices from the same vial were pooled for each condition unless mentioned otherwise. Data was analyzed by ANOVA, followed by Dunnett's multiple comparison test. P < 0.05 was considered significant. RESULTS Viability In control lung slices, viability was maintained for up to 24 h (Fig. 1). In lung slices treated with 0.1 μM arsenite, K+ was maintained up to 24 h. In slices exposed to 1.0–10 μM arsenite, K+ contents were maintained at control levels for up to 6 h. At 6 h, 100 μM sodium arsenite resulted in a 50% decrease of intracellular K+, which was maintained for up to 24 h. Histopathology There were no observable differences in structure between control and 10 μM arsenite-treated lung slices after 4 h under light microscopic (Figs. 2A and 2B) or confocal microscopic examination (Figs. 8A, 8B, 9A, and 9B). Expression of Stress Proteins Representative Western blots of stress proteins are shown in Figure 3. Exposure to 1 and 10 μM arsenite resulted in a 3-fold and 4.4-fold increase, respectively, of HSP 32 content in lung slices (Figs. 3 and 4A), while, 100-μM arsenite decreased HSP 32 to control levels. HSP 72 was increased only by 100-μM arsenite (Figs. 3 and 4B), which resulted in a 3.2-fold increase of this stress protein. There was no change in the expression of HSP 60 (Figs. 3 and 4C) or HSP 90 (Figs. 3 and 4D) due to arsenite. Gel-Shift Assay of AP-1 and NFκB DNA binding activity of both AP-1 (Fig. 5A) and NFκB (Fig. 5B) were increased by 10-μM arsenite at 4 h. At 2 h, DNA binding of these transcription factors was not observed (data not shown). The intensity of the shifted band for NFκB was less than that observed for AP-1. H2O2 also resulted in an increase in the binding activity of both of these transcription factors. Treatment with excess unlabeled AP-1 or NFκB consensus oligonucleotides resulted in a decrease in the intensity or abolishment of the supershifted bands seen after arsenite treatment. Western Blot Analysis of Nuclear c-jun and NFκB Proteins Arsenite resulted in a concentration-dependent increase in c-jun. Treatment with 0.1 and 1.0 μM arsenite resulted in 1.2- and 2-fold increases in c-jun protein, which were not significant. Treatment with 10- and 100-μM arsenite resulted in a significant increase in this protein (Figs. 6A and 7A). Ten-μM arsenite resulted in a 3-fold increase in c-jun, while 100-μM arsenite increased it by 4-fold. Although the antibody used can detect c-jun protein or the AP-1 complex, an increase in the protein band corresponding to c-jun (39 KDa) was observed. Probably the denaturing conditions needed for Western blot analysis may have resulted in the dissociation of the AP-1 complex. Western blot analysis of NFκB indicated a protein band corresponding to 65 KDa, probably because of the dissociation of p65 and p50 due to denaturing conditions employed in Western blotting. Treatment with arsenite did not result in an increase in nuclear NFκB content (Figs. 6B and 7B), probably due to low contents of the activated form of NFκB in the nuclei. In fact, unlike c-jun/AP-1, in order to visualize NFκB (p65), it was necessary to use an indirect staining method employing biotinylated secondary antibody and streptavidin-HRP complex to increase the sensitivity of the assay. Localization of c-jun/AP-1 and NFκB by Confocal Microscopy Nuclear localization of c-jun/AP-1 was observed mainly in the nuclei of alveolar type-II cells and macrophages, indicating activation and nuclear translocation of this transcription factor. Cytoplasm of the above cells had very little staining for c-jun/AP-1 (Figs. 8B and 8D). Bronchial epithelial cells too exhibited positive staining for AP-1. Only minimal staining was seen in control tissue (Figs. 8A and 8C). NFκB was observed in the nuclei and cytoplasm of type II epithelial cells and macrophages (Figs. 9B and 9D). However, the intensity of staining for NFκB was less as compared to that of c-Jun/AP-1. Cytoplasm of type-II epithelial cells and macrophages seemed to accept more staining for NFκB than did the nuclei. In control tissue, only minimal staining was observed (Figs. 9A and 9C). DISCUSSION Leakage of intracellular K+ results when the cell membrane is damaged as well as a result of damage to cell membrane associated Na/K/ATPase pump. In most of the studies on tissue slices K+ leakage is used as an index of toxicity because leakage of intracellular enzymes is difficult to measure due to smaller number of cells in lung slices. Treatment with low concentrations (0.1–10 μM) of arsenite did not cause a decrease in viability in lung slices until after 6 h, as indicated by intracellular K+ levels. An increase in the expression of the stress proteins HSPs 32 and 72 were observed with noncytotoxic concentrations of arsenite. There was activation and increased expression of the transcription factor AP-1 and activation of the transcription factor NFκB. These results indicate that low, environmentally relevant concentrations of arsenite can result in the activation of transcription factors and increase stress protein expression in the lungs. Stress proteins are constitutively expressed at low levels in eukaryotic as well as prokaryotic cells and are well conserved during evolution. Their expression is increased due to a variety of cellular stresses such as heat shock, xenobiotics, radiation, metals, ischemia, cytokines, and oxygen-free radicals. Stress proteins play a role in protecting cells from irreversible damage and help recovery of cells after a toxic insult. Expression of HSP 32 is particularly increased as a response to hyperoxic lung injury in vivo (Lee et al., 1996). Exposure of rats to arsenite for 12 weeks resulted in a depletion of glutathione (GSH), and an increase in oxidized glutathione (GSSG) and malondialdehyde in liver and brain (Flora, 1999), indicating oxidative injury. In other studies, arsenite has been shown to cause damage to cellular macromolecules by generating reactive oxygen species (Chen et al., 1998; Liu and Jan, 2000; Rhee et al., 2000). Reactive oxygen species include, H2O2, superoxide anion (O•−2), hydroxyl radical (OH•), as well as organic peroxides and radicals. These can damage cells by lipid peroxidation and DNA and protein oxidation. In bovine aortic cells, arsenite caused DNA strand breaks. These could be decreased by nitric oxide synthase inhibitors, superoxide scavengers, and peroxynitrite scavengers, indicating that arsenite increases superoxide and nitric oxide formation, and nitric oxide and superoxide probably react to produce peroxynitrite (Liu and Jan, 2000). In the present study, concentrations as low as 1 μM arsenite, which did not cause K+ leakage, induced HSP 32. Since both, HSP 32 (Lee et al., 1996) and HSP 72 (Gomer et al., 1996) are increased after oxidative stress, arsenic may also induce these genes by inducing oxidative stress. However, other than induction of HSP-32, we have not examined indices of oxidative stress in this study. HSP 32 plays a role in protecting cells against the damaging effects of oxygen-free radicals. It is the rate-limiting enzyme in heme degradation. Heme, a prooxidant, is converted by HSP 32 into biliverdin and bilirubin, which are potent physiological antioxidants (Stocker et al., 1987, 1990). Iron, which is released from heme during its degradation, may generate oxygen-free radicals by Fenton-type reactions (Halliwell and Gutteridge, 1984). However, iron induces ferritin production (Munro, 1993), and ferritin binds free iron and further protects cells from oxidant-mediated cellular damage (Balla et al., 1992; Cermak et al., 1993). In the present study, an increase in HSP 72 was evident only after 100 μM arsenite, unlike HSP 32, which was increased by 10 μM arsenite. One hundred μM arsenite resulted in a decrease of HSP 32 to control levels. Similar observations were found in the chicken hepatoma cell line, LMH, where dose-dependent induction in HSP 32 occurred as observed by luciferase reporter gene expression. In the above studies, the most effective dose for HSP 32 induction was 75 μM, and higher doses resulted in a decrease in HSP 32 (Elbirt et al., 1998). Other studies have investigated the expression of stress proteins in liver slices and renal slices. In liver slices, 10 μM arsenite increased HSP 72 and HSP 90 (Wijeweera et al., 1995). Treatment with 0.1–10 μM arsenite induced HSP 32, but not HSP 72, HSP 90, or HSP 60, in renal slices (Parrish et al., 1999). In our studies, no increase in either HSP 60 or HSP 90 contents were seen after arsenite exposure. This indicates that there are tissue-specific differences in the induction of stress proteins by arsenite. The fact that HSP 32 was induced by lower concentrations of arsenite, compared to HSP 72 in our studies, suggests that HSP 32 is much more sensitive to macromolecular damage caused by arsenite. It has been suggested that HSP-72 expression increases after a certain threshold of cellular damage (Tacchini et al., 1993). Only 100-μM arsenite increased HSP 72 in our studies, indicating this concentration of arsenite may have caused subtle changes in the structure of cellular proteins. Damaged or abnormal proteins serve as a signal for the induction of HSP 72 (Ananthan et al., 1986). The function of HSP 72 is to bring about the proper folding of proteins and to prevent the formation of protein aggregates that result when hydrophobic regions of damaged proteins are exposed (Aufricht et al., 1998). Arsenite can certainly cause damage to cellular proteins by its reactivity towards thiol groups, as well as due to the production of reactive oxygen species that are generated during its cellular metabolism. From our studies, it is apparent that HSP 32 has a lower threshold for increased expression and is much more sensitive to damage to cellular macromolecules than HSP 72. Western blot analysis of nuclear proteins from lung slices indicated that arsenite increased c-jun/AP-1 contents in a concentration-dependent manner. Confocal microscopic analysis indicated nuclear localization of c-jun/AP-1 in type-II epithelial cells and macrophages. Gel-shift assays indicated an increased nuclear presence of c-jun/AP-1 and NFκB following arsenic exposure. However, an arsenite-induced increase in nuclear NFκB, as indicated by Western blot analysis, was not detected. Confocal microscopy indicated that activated NFκB was localized mainly to the cytoplasm of type-II epithelial cells and macrophages. Very little nuclear staining was seen. This indicates that NFκB is activated in the cytoplasm. However, the levels of nuclear NFκB were too low to detect with Western blot analysis. Confocal microscopy provided information on site-specific localization of AP-1 and NFκB in lung slices after arsenite treatment. Normal tissue homeostasis requires a balance between cell proliferation and apoptosis, and an imbalance between these two processes may lead to cancer (Manning and Patierno, 1996; Thompson, 1995). Apoptosis is important in removing genetically damaged cells. However, after a toxic insult, the surrounding surviving cells that contain damaged DNA may be stimulated to proliferate, resulting in neoplastic growth (Manning and Patierno, 1996). Both AP-1 and NFκB appear to be critically involved in genes that regulate cell proliferation and apoptosis (Baeuerle, 1991; Karin, 1995; Lenardo and Baltimore, 1989). Many of the environmental carcinogens such as arsenite can stimulate apoptosis (Hossain et al., 2000) as well as proliferation (Simeonova et al., 2000). Whether proliferation or apoptosis occurs is determined by the balance of several cell-signaling pathways leading to the activation of c-fos and c-jun (Xia et al., 1995) as well as activation of NFκB (Baldwin, 1996; Ghosh et al., 1998). Furthermore, subunits of AP-1 and NFκB can cross talk and both of these factors may be involved in cell transformation (Denhardt, 1996; Stein et al., 1993). HSP 32 expression is under the regulation of the transcription factors AP-1 and NFκB, and both AP-1 and NFκB are known to be expressed under conditions of oxidative stress (Meyer et al., 1993; Pinkus et al., 1996; Schulze-Osthoff et al., 1995). Both human (Lavrovsky et al., 1994, 1996; Shibahara et al., 1989) and rodent (Alam, 1994; Alam and Den, 1992, 1995; Bergeron et al., 1998; Kurata et al., 1996; Lavrovsky et al., 2000) HSP 32 gene promoters have NFκB binding sites. Oxidant-induced lung injury in rat increased HSP 32 and AP-1 binding activity to the promoter region of HSP 32 gene (Lee et al., 1996). Both HSP 32 and HSP 72 expression is under the control of another transcription factor, HSF (Stuhlmeier, 2000). HSF binds to heat shock element (HSE) in the upstream promoter region of stress proteins and increase their transcription (Sorger et al., 1987; Wu, 1984). Several stressors including arsenite are known to activate HSF (Kato and Okamoto, 1997; Larson et al., 1988). Although we did not study expression and activation of HSF, probably arsenite may have activated it in lung slices. Activation of HSF by phosphorylation can be mediated by several members of the MAP kinase family, including ERK, JNK, and P38 (Kim et al., 1997; Lee and Corry, 1988). Thus, stress protein expression is under a complex regulatory mechanism, which requires integration of signals from different signal transduction pathways that converge upon AP-1, NFκB, and HSF. This may explain why HSP 32 decreased, although AP-1 contents were increased by 100-μM arsenite in our study. Recent investigations indicate that HSP 72 protein can protect cells from damage caused by various stresses by multiple mechanisms. HSP 72 can function as a chaperonin by refolding denatured proteins (Gething and Sambrook, 1992). In addition, HSP 72 can regulate signal transduction pathways: it can inhibit activation of NFκB by blocking IκB dissociation and degradation (Yoo et al., 2000). Dissociation of IκB from p65/p50 NFκB complex is needed for the activation and nuclear translocation of this transcription factor (Siebenlist et al., 1994). Thus, HSP 72 can prevent the expression of NFκB-dependent genes. Furthermore, HSP 72 plays a role in the activation as well as in the deactivation of JNK, which is responsible for the phosphorylation and activation of c-jun/AP-1. This activation of JNK is due to the inhibition of JNK phosphatase by HSP 72. When JNK phosphatase is inhibited, the background activity of JNK activating kinase, SEK1, is sufficient to account for JNK activation (Meriin et al., 1999). Increased expression of HSP 72 may protect cells against apoptotic or necrotic cell death. Recent studies indicate that the overexpression of HSP 72 forms a negative-feedback loop involved in the inhibition of apoptosis. This inhibitory effect on apoptosis is due to the prevention of JNK activation by high levels of HSP 72 protein (Gabai et al., 1997). Necrotic cell death is associated with ATP loss, aggregation of cytoskeletal proteins, and bleb formation (Gabai et al., 1993). Protein aggregation due to ATP depletion was inhibited (Kabakov and Gabai, 1995) in tumor cells that accumulated HSP 72 after mild heat shock. Arsenite did not increase HSP 60 in lung slices. Although HSP 90 was found to be much more abundant in control slices than the other stress proteins, its expression was not changed by arsenite treatment. HSP 60 is a mitochondrial stress protein which functions as a chaperonin to assist the correct folding of cytoplasmic polypeptides that are targeted for import into the mitochondria (Hartl and Martin, 1995). Exposure of human proximal tubular cells to 100 μM arsenite for 4 h resulted in an increase in HSP-60 content after the cells were allowed to recover for 12 h, and increased levels of this protein were observed for up to 48 h (Somji et al., 2000). It appears that HSP 60 only increases after the stress factor is removed and the cells are allowed to recover. Similar to HSP 72, HSP 90 protein functions as a chaperonin to bring about proper folding of proteins and prevent aggregation of unfolded proteins (Freeman and Morimoto, 1996; Young et al., 1997). While arsenite induces changes in the expression of HSP 90 in the liver (Wijeweera et al., 1995), such changes were not observed in the lung. Thus, the present study indicates that precision-cut lung slices are a suitable in vitro system for studying perturbations in gene expression after a toxic insult. Low levels of arsenite resulted in the activation and expression of transcription factors and expression of stress proteins in lung slices. Confocal microscopic analysis provided information on the site-specific localization of transcription factors AP-1 and NFκB. FIG. 1. View largeDownload slide Viability. Lung slices were incubated with 0–100 μM sodium arsenite for 2–24 h after a 1-h preincubation in the culture medium. Three slices from the same vial were sonicated in 1 ml deionized water. Proteins were precipitated by adding 40 μl of perchloroacetic acid to an 800-μl homogenate, and the samples were centrifuged at 14,000 rpm for 15 min. The supernatant fraction was used for K+ assay by flame photometry. The pellet was dried overnight and dissolved in 1 ml of 1-M sodium hydroxide, and the protein content was analyzed by the Bradford method. FIG. 1. View largeDownload slide Viability. Lung slices were incubated with 0–100 μM sodium arsenite for 2–24 h after a 1-h preincubation in the culture medium. Three slices from the same vial were sonicated in 1 ml deionized water. Proteins were precipitated by adding 40 μl of perchloroacetic acid to an 800-μl homogenate, and the samples were centrifuged at 14,000 rpm for 15 min. The supernatant fraction was used for K+ assay by flame photometry. The pellet was dried overnight and dissolved in 1 ml of 1-M sodium hydroxide, and the protein content was analyzed by the Bradford method. FIG. 2. View largeDownload slide Histopathology. Lung slices were exposed to 0 or 10 μM arsenite for 4 h after a 1-h preincubation in the culture medium. Slices were fixed in 10% buffered formalin, processed for light microscopy, and stained with H&E. (A) Control, (B) 10 μM arsenite; magnification 10×. FIG. 2. View largeDownload slide Histopathology. Lung slices were exposed to 0 or 10 μM arsenite for 4 h after a 1-h preincubation in the culture medium. Slices were fixed in 10% buffered formalin, processed for light microscopy, and stained with H&E. (A) Control, (B) 10 μM arsenite; magnification 10×. FIG. 3. View largeDownload slide Representative Western blot analysis of stress proteins. Lung slices were exposed to 0–100 μM sodium arsenite for 4 h after a 1-h preincubation in the culture medium. Three lung slices from the same vial were pooled into 300 μl of lysis buffer and homogenized. Sliced homogenate was centrifuged at 14,000 rpm for 15 min, and the supernatant fraction was used for the analysis. Forty μg of protein was separated by SDS-PAGE and transferred onto a PVDF membrane. Immunoperoxidase staining was performed using specific monoclonal antibodies for each stress protein and horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using DAB Substrate Kit for Peroxidase. Lane 1, control; lane 2, 0.1 μM arsenite; lane 3, 1.0 μM arsenite; lane 4, 10 μM arsenite; and lane 5, 100 μM arsenite. FIG. 3. View largeDownload slide Representative Western blot analysis of stress proteins. Lung slices were exposed to 0–100 μM sodium arsenite for 4 h after a 1-h preincubation in the culture medium. Three lung slices from the same vial were pooled into 300 μl of lysis buffer and homogenized. Sliced homogenate was centrifuged at 14,000 rpm for 15 min, and the supernatant fraction was used for the analysis. Forty μg of protein was separated by SDS-PAGE and transferred onto a PVDF membrane. Immunoperoxidase staining was performed using specific monoclonal antibodies for each stress protein and horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using DAB Substrate Kit for Peroxidase. Lane 1, control; lane 2, 0.1 μM arsenite; lane 3, 1.0 μM arsenite; lane 4, 10 μM arsenite; and lane 5, 100 μM arsenite. FIG. 4. View largeDownload slide Densitometric analysis of stress proteins. (A) HSP 32, (B) HSP 72, (C) HSP 60, (D) HSP 90. Lane 1, control; lane 2, 0.1 μM arsenite; lane 3, 1.0 μM arsenite; lane 4, 10 μM arsenite; and lane 5, 100 μM arsenite; * indicates difference from control. FIG. 4. View largeDownload slide Densitometric analysis of stress proteins. (A) HSP 32, (B) HSP 72, (C) HSP 60, (D) HSP 90. Lane 1, control; lane 2, 0.1 μM arsenite; lane 3, 1.0 μM arsenite; lane 4, 10 μM arsenite; and lane 5, 100 μM arsenite; * indicates difference from control. FIG. 5. View largeDownload slide Gel shift assay of: (A) AP-1, and (B) NFκB. Lung slices were exposed to 0 or 10 μM arsenite for 4 h after a 1-h preincubation in the culture medium. Nuclear protein was isolated from lung slices and a gel-shift assay was performed as described under Materials and Methods. Lane 1, labeled probe only; lane 2, labeled probe + untreated sample; lane 3, labeled probe + 10 μM arsenite-treated sample; lane 4, labeled probe + 10 μM arsenite-treated sample + excess unlabeled probe; lane 5, labeled probe + H2O2-treated sample; and lane 6, labeled probe + H2O2-treated sample + excess unlabeled probe. Arrow indicates the position of the supershifted band. FIG. 5. View largeDownload slide Gel shift assay of: (A) AP-1, and (B) NFκB. Lung slices were exposed to 0 or 10 μM arsenite for 4 h after a 1-h preincubation in the culture medium. Nuclear protein was isolated from lung slices and a gel-shift assay was performed as described under Materials and Methods. Lane 1, labeled probe only; lane 2, labeled probe + untreated sample; lane 3, labeled probe + 10 μM arsenite-treated sample; lane 4, labeled probe + 10 μM arsenite-treated sample + excess unlabeled probe; lane 5, labeled probe + H2O2-treated sample; and lane 6, labeled probe + H2O2-treated sample + excess unlabeled probe. Arrow indicates the position of the supershifted band. FIG. 6. View largeDownload slide Representative Western blot analysis of A, c-jun; B, NFκB. Nuclear protein from lung slices exposed to 0–100 μM sodium arsenite for 4 h was subjected to SDS/PAGE and proteins were transferred onto a PVDF membrane. For the detection of c-jun, membrane was incubated with primary antibody to c-jun/AP-1, followed by secondary antibody conjugated to horseradish peroxidase. For the detection of NFκB, the membrane was incubated with primary antibody to NFκB (p65 subunit) followed by biotinylated secondary antibody and streptavidin-HRP complex. Protein bands were detected using enhanced chemiluminescence (ECL) reagents. Lane 1, control; lane 2, 0.1 μM arsenite; lane 3, 1.0 μM arsenite; lane 4, 10 μM arsenite; and lane 5, 100 μM arsenite. FIG. 6. View largeDownload slide Representative Western blot analysis of A, c-jun; B, NFκB. Nuclear protein from lung slices exposed to 0–100 μM sodium arsenite for 4 h was subjected to SDS/PAGE and proteins were transferred onto a PVDF membrane. For the detection of c-jun, membrane was incubated with primary antibody to c-jun/AP-1, followed by secondary antibody conjugated to horseradish peroxidase. For the detection of NFκB, the membrane was incubated with primary antibody to NFκB (p65 subunit) followed by biotinylated secondary antibody and streptavidin-HRP complex. Protein bands were detected using enhanced chemiluminescence (ECL) reagents. Lane 1, control; lane 2, 0.1 μM arsenite; lane 3, 1.0 μM arsenite; lane 4, 10 μM arsenite; and lane 5, 100 μM arsenite. FIG. 7. View largeDownload slide Densitometric analysis of c-jun and NFκB. Nuclear protein from lung slices exposed to 0–100 μM sodium arsenite for 4 h was subjected to SDS/PAGE and proteins were transferred onto a PVDF membrane. The membrane was incubated with primary antibody to (A) c-jun/AP-1, or (B) NFκB, followed by secondary antibody conjugated to horseradish peroxidase. Protein bands were detected using enhanced chemiluminescence (ECL) reagents. Densitometric analysis was performed using Scion Image (NIH); * indicates difference from control. FIG. 7. View largeDownload slide Densitometric analysis of c-jun and NFκB. Nuclear protein from lung slices exposed to 0–100 μM sodium arsenite for 4 h was subjected to SDS/PAGE and proteins were transferred onto a PVDF membrane. The membrane was incubated with primary antibody to (A) c-jun/AP-1, or (B) NFκB, followed by secondary antibody conjugated to horseradish peroxidase. Protein bands were detected using enhanced chemiluminescence (ECL) reagents. Densitometric analysis was performed using Scion Image (NIH); * indicates difference from control. FIG. 8. View largeDownload slide Localization of AP-1 by confocal microscopy. Lung slices were incubated with 0 or 10 μM sodium arsenite for 4 h after a 1-h preincubation in the culture medium. After fixing in 10% formalin, slices were embedded in paraffin wax and 5-μm sections were prepared. Sections were incubated with anti-c-jun/AP-1 antibody specific to the DNA binding domain of c-jun, followed by biotinylated secondary antibody and streptavidin-Cy5. Nuclei were counterstained with YOYO iodide after RNase digestion. (A) Control, (B) 10 μM arsenite, (C) control, (D) 10 μM arsenite. A and B are overlays of transmitted light with YOYO nuclear and c-jun/AP-1 staining. C and D are c-jun/AP-1 staining only. Red indicates c-jun/AP-1, green indicates nuclei, and yellow indicates co-localization. Type II, type II epithelial cell; Mac, alveolar macrophage; and Endo, endothelial cell. FIG. 8. View largeDownload slide Localization of AP-1 by confocal microscopy. Lung slices were incubated with 0 or 10 μM sodium arsenite for 4 h after a 1-h preincubation in the culture medium. After fixing in 10% formalin, slices were embedded in paraffin wax and 5-μm sections were prepared. Sections were incubated with anti-c-jun/AP-1 antibody specific to the DNA binding domain of c-jun, followed by biotinylated secondary antibody and streptavidin-Cy5. Nuclei were counterstained with YOYO iodide after RNase digestion. (A) Control, (B) 10 μM arsenite, (C) control, (D) 10 μM arsenite. A and B are overlays of transmitted light with YOYO nuclear and c-jun/AP-1 staining. C and D are c-jun/AP-1 staining only. Red indicates c-jun/AP-1, green indicates nuclei, and yellow indicates co-localization. Type II, type II epithelial cell; Mac, alveolar macrophage; and Endo, endothelial cell. FIG. 9. View largeDownload slide Localization of NFκB by confocal microscopy. Lung slices were incubated with 0 or 10 μM sodium arsenite for 4 h after 1-h preincubation in the culture medium. After fixing in 10% formalin, slices were embedded in paraffin and 5 μm sections were prepared. Sections were incubated with anti-NFκB specific to the p65 subunit, followed by biotinylated secondary antibody and streptavidin-Cy5. Nuclei were counterstained with YOYO iodide after RNase digestion. (A) Control; (B), 10 μM arsenite, (C) control, and (D) 10 μM arsenite. A and B are overlays of transmitted light with YOYO nuclear and NFκB staining. C and D had NFκB staining only. Red indicates NFκB, green indicates nuclei, and yellow indicates co-localization. Type II, type II epithelial cell. FIG. 9. View largeDownload slide Localization of NFκB by confocal microscopy. Lung slices were incubated with 0 or 10 μM sodium arsenite for 4 h after 1-h preincubation in the culture medium. After fixing in 10% formalin, slices were embedded in paraffin and 5 μm sections were prepared. Sections were incubated with anti-NFκB specific to the p65 subunit, followed by biotinylated secondary antibody and streptavidin-Cy5. Nuclei were counterstained with YOYO iodide after RNase digestion. (A) Control; (B), 10 μM arsenite, (C) control, and (D) 10 μM arsenite. A and B are overlays of transmitted light with YOYO nuclear and NFκB staining. C and D had NFκB staining only. Red indicates NFκB, green indicates nuclei, and yellow indicates co-localization. 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TI - Sodium Arsenite Enhances AP-1 and NFκ B DNA Binding and Induces Stress Protein Expression in Precision-Cut Rat Lung Slices
JF - Toxicological Sciences
DO - 10.1093/toxsci/61.2.283
DA - 2001-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/sodium-arsenite-enhances-ap-1-and-nf-b-dna-binding-and-induces-stress-riB4OSuib8
SP - 283
EP - 294
VL - 61
IS - 2
DP - DeepDyve
ER -