* Department of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan;
Research Center for Environmental Risk, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan;
Institute of Community Medicine, University of Tsukuba, Tsukuba, Japan; and
Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan
Received October 22, 2001; accepted January 21, 2002
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ABSTRACT |
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Key Words: cDNA microarray; alveolar macrophages; organic extract; diesel exhaust particles; heme oxygenase; thioredoxin peroxidase 2; glutathione S-transferase P subunit; NAD(P)H dehydrogenase; proliferating cell nuclear antigen.
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INTRODUCTION |
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There are many possibilities in the effects of DEP on AM. DEP could give oxidative stress, which might induce gene expression of antioxidative enzymes, the gene expression related to DNA-damage, production of cytokines and chemokines. It is very important to identify the alteration of gene expressions in the early response to characterize the stimuli of the chemicals and to predict the subsequent responses. The objective of this study is a comprehensive analysis of the alterations of gene expression in AM. There have been no reports, however, of any systematic analysis of such extracts on gene expression in AM. Therefore, a complete analysis, including the transcriptome and proteome, is needed to elucidate the toxic effect of air pollutants on pulmonary cells.
The recently developed cDNA microarray or DNA-chip technology allows expression monitoring of hundreds or thousands of genes simultaneously and provides rapid and immediate information for identifying genes (Schena et al., 1995; Shalon et al., 1996
).
In this study, we investigated the alterations of gene expression in rat AM after short-term exposure of them to a DEP extract in order to elucidate the early response of these lung cells to the toxicological effects of the extract. The gene expression in AM was evaluated using an Atlas Rat Toxicology Array II, which includes 450 rat cDNAs, and the results were further confirmed by Northern blot analysis.
Our results showed that exposure to DEP extract elevates the gene expression of antioxidative enzymes and proliferating cell nuclear antigen (PCNA), the latter of which contributes to repair of the DNA damage and to cell proliferation. Therefore, the increased expression of these genes may play a role in the pulmonary defense against oxidative stress caused by DEP.
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MATERIALS AND METHODS |
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Animals.
Specific pathogen-free, male Sprague-Dawley rats (6-weeks-old) were obtained from Japan Charles River Inc. (Yokohama, Japan) and were used at 810 weeks of age. The animals were housed in a clean air-conditioned room and given sterile distilled water and commercial food (CE-2, Clea Japan, Tokyo, Japan).
Collection of alveolar macrophages.
Rats were anesthetized with sodium pentobarbital (Dainippon Pharmaceutical Co., Osaka, Japan) given intraperitoneally (50 mg/kg), and exsanguinated from the abdominal aorta. A cannula was inserted into the trachea and secured with a suture. The lungs were lavaged 10 times with 10 ml of 37°C endotoxin-free saline (Otsuka, Pharm. Co., Naruto, Japan). Cells were collected by centrifugation at 400 x g for 10 min at 4°C; washed in R10, which is RPMI 1640 medium (Dainippon Pharmaceutical Co., Osaka, Japan) containing 10% heat-inactivated fetal bovine serum (FBS; Dainippon Pharmaceutical Co., Osaka, Japan), 100 µg/ml penicillin, and 100 U/ml streptomycin (Sigma, St. Louis, MO); and resuspended in R10. The viable cells count and differential cells count were conducted by the trypan blue (Gibco BRL, Rockville, MD) exclusion method and Diff-Quik stain, respectively. Viability of the bronchoalveolar lavage cells from normal rats were > 98%, and these cells were > 99% AM. For the cDNA microarray analysis, AM were pooled from 10 rats, and the cells were divided for culture with or without the DEP extract.
Exposure to DEP extract.
A 10-ml aliquot of the cell suspension (1 x 106 viable cells/ml) was placed in culture dishes (100 mm diameter, Becton Dickinson, Co., NJ), and the cells were precultured in R10 for 20 h (2 dishes per group). It is important to identify the alteration of gene expression in the early response to characterize the stimuli of the chemicals and to predict the subsequent responses. Therefore, the AM were exposed to the DEP extract (10 µg/ml in R10 containing 0.1% DMSO) or R10 containing 0.1% DMSO for 6 h at 37°C in an atmosphere of 5% CO2, 95% air.
cDNA Expression Array
Total RNA isolation and cDNA probe synthesis.
Total RNA was extracted from AM exposed to the DEP extract by using TRIZOL (GIBCO BRL, Rockville, MD) according to the manufacturer's instructions. Using 50 µg of total RNA, poly (A)+ RNA enrichment and radiolabeled cDNA probe synthesis were carried out using an Atlas Pure Total RNA Labeling System (CLONTECH Laboratories, Palo Alto, CA). The 33P-labelled cDNA probes were separated from unincorporated nucleotides and small cDNA fragments by using a spin column (Atlas NucleoSpin Extraction Kit, CLONTECH Laboratories).
Hybridization.
We used the Atlas Rat Toxicology Array II (CLONTECH Laboratories), which includes 450 rat cDNAs immobilized in duplicate dots on a nylon membrane. The cDNA microarray analysis was performed according to the instruction manual (CLONTECH). The radioactivity on the membrane was analyzed and quantified by using a bioimaging analyzer (BAS 2000, Fuji, Tokyo, Japan). Expression levels of the housekeeping genes, ß-actin and glyceraldehyde 3-phosphate dehydrogenase, were used as standards for normalizing the expression levels of the genes of interest.
Northern blot analysis.
The mRNA levels of heme oxygenase (HO)-1, thioredoxin peroxidase 2 (TDPX-2), glutathione S-transferase P subunit (GST-P), NAD(P)H dehydrogenase, and PCNA were analyzed by Northern blot analysis. AM were obtained from 2 different groups of rats. The cells (5 x 105 viable cells/ml) were precultured for 20 h in R10 and then exposed to the DEP extract (2.5, 5, 10 µg/ml in R10 containing 0.1% DMSO) or R10 containing 0.1% DMSO for 6 h at 37°C in an atmosphere of 5% CO2, 95% air.
Total RNA was extracted from the AM by using TRIZOL reagent. The RNA was denatured and separated on a formaldehyde-denatured agarose gel (1%) by electrophoresis. The RNA was transferred to a nylon membrane (Hybond-N; Amersham) by use of a vacuum blotter (Model 785; Bio-Rad, Hercules, CA) and crosslinked to the membrane by UV. The membrane was prehybridized in ExpressHyb solution at 68°C for 30 min, and hybridized with 32P-labeled cDNA probes overnight at 68°C. The probes were labeled with [-32P] dCTP (3000 Ci/mmol, Amersham) by using the REDIPRIME DNA labeling system (Amersham). The radioactivity on the membranes was analyzed and quantified with a bioimaging analyzer. Expression levels of the mRNAs were normalized against ß-actin mRNA.
Probes for Northern blot analysis.
Atlas cDNA (Atlas cDNA Clones Custom Order, CLONTECH), which was blunt-end ligated into the SmaI site of pAtlas plasmid was used to prepare the rat TDPX-2, GST-P, NAD(P)H dehydrogenase, and PCNA probes. The cDNA probes were obtained by digestion of each plasmid with KpnI and SacI. The corresponding cDNA was excised from agarose gels and extracted by using a GENECLEAN Spin Kit (BIO 101, Vista, CA). Preparation of the rat HO-1 and ß-actin probes was described previously (Kitajima et al., 1999).
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RESULTS |
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DISCUSSION |
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The results of our present study demonstrate that the gene expression of antioxidative enzymes such as HO, TDPX-2, GST-P, and NAD(P)H dehydrogenase in AM was elevated by the exposure to the DEP extract. HO is prominently induced under various oxidative stress conditions in many different cell types (Keyse and Tyrrell, 1989). A crude DEP total extract, aromatic and polar DEP fractions, and a benzo[a]pyrene quinone all induced HO-1 expression in macrophages (Li et al., 2000
). Induction of HO-1 is a cytoprotective mechanism against oxidative cellular injury (Horvath et al., 1998
); and it was also reported that HO-1-deficient mice were more sensitive to chemical-induced oxidative stress than normal mice (Poss and Tonegawa, 1997
). It is known that HO-2 is constitutively expressed in mammalian tissues, whereas HO-1 is inducible in response to oxidative stress (Fernandez and Bonkovsky, 1999
). Because the increase in HO-2 mRNA level was marginal in the cDNA microarray analysis, HO-1 mRNA but not HO-2 mRNA levels were examined in Northern blotting. GST-P, one of the subunits of GST, detoxifies oxygenated xenobiotics and electrophilic compounds. It was reported that instillation of hematite and benzo[a]pyrene increased the amount of
-GST in bronchoalveolar lavage fluid and that instillation of benzo[a]pyrene alone caused a significant elevation of the
-GST level in serum and urine in rats (Boutin et al., 1998
). NAD(P)H dehydrogenase was shown to be coordinately induced with other electrophile-processing phase II enzymes, such as GST (Talalay and Benson, 1982
; Wattenberg, 1985
). TDPX-2 is a member of the peroxiredoxin family of peroxidases, a recognized class of antioxidant proteins, as are heme-binding protein 23 kDa (HBP23) and macrophage 23-kDa stress protein (Chae et al., 1994
). TDPX was found to be essential for the transcriptional induction of other components of the thioredoxin system, i.e., thioredoxin and thioredoxin reductase, in response to oxidative stress such as H2O2 (Ross et al., 2000
). HBP23 mRNA in rat hepatocytes was increased by treatment with heme and cadmium (Iwahara et al., 1995
). It has been also suggested that the coordinate gene regulation pattern of HBP23 and HO-1 plays a physiological role against oxidative stress (Immenschuh et al., 1997
).
Antioxidants prevent the formation of free radicals, convert oxidants to less toxic species, compartmentalize reactive species away from vital cellular structures, and repair molecular injury induced by free radicals (Sies, 1987) or by modification of enzyme molecules (Kondo et al., 1984
). The antioxidant capacity of AM is important not only to maintain their function in lung defense, but also to potentially protect the lungs from oxidative damage. It has been suggested that the antioxidant response element (ARE) pathway may contribute to antioxidant defense in the lungs (Camhi et al., 1995
). For example, a crude total DEP extract, aromatic and polar DEP fractions, and a benzo[a]pyrene quinone all induced HO-1 expression in macrophages via ARE in the promoter of this gene (Li et al., 2000
). Oxidative stress agents were shown to cause the transfer of NF-E2-related factor 2 (Nrf2) from the cytoskeleton to the nucleus, where Nrf2 interacted with ARE, and to induce the transcription of genes that encode antioxidant functions (Ishii et al., 1993
; Itoh et al., 1997
). Thus, DEP extract-induced antioxidative enzymes may be regulated by the activation of transcription factors including Nrf2, nuclear factor-
B, activator protein-1, and heat shock factor via the ARE pathway.
The present study also demonstrates that the gene expression of PCNA in AM was elevated by the exposure to the DEP extract. PCNA is known to be involved in DNA replication and repair, and was induced by DNA damage and other stress (Smith et al., 1994). Expression of the PCNA gene was reportedly activated in the lungs by DEP exposure in vivo (Sato et al., 1999
). Acute exposure to ozone also induced expression of PCNA in both type II cells and AM (Prokhorova et al., 1998
). Activation of the PCNA gene may have contributed to DNA repair and cell proliferation caused by exposure to a DEP extract.
Therefore, exposure to the DEP extract (10 µg/ml) for 6 h increased the gene expression of antioxidative enzymes and PCNA but not that of cytokines, chemokines, or chemical mediators associated with lung inflammation. These AM-mediated functions may suppress the lung inflammation induced by chemical compounds in DEP. It remains to be elucidated whether changes in message expression are also translated into respective protein expression.
In summary, exposure to DEP extract particularly elevated the gene expression of HO, TDPX-2, GST-P, NAD(P)H dehydrogenase, and PCNA genes in rat AM. These results suggest that AM may play a crucial role in pulmonary defense via acute inflammation by various pollutants through the induction of antioxidative enzymes and PCNA. Our results also demonstrate that cDNA microarray analysis can provide useful information to investigate the biological response to pulmonary toxicants.
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NOTES |
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