Gene-expression profiling using suppression-subtractive hybridization and cDNA microarray in rat mononuclear cells in response to welding-fume exposure

Abstract

Welders with radiographic pneumoconiosis abnormalities have shown a gradual clearing of the X-ray identified effects following removal from exposure. In some cases, the pulmonary fibrosis associated with welding fumes appears in a more severe form in welders. Accordingly, for the early detection of welding-fume-exposure-induced pulmonary fibrosis, the gene expression profiles of peripheral mononuclear cells from rats exposed to welding fumes were studied using suppression-subtractive hybridization (SSH) and a cDNA microarray. As such, Sprague-Dawley rats were exposed to a stainless steel arc welding fume for 2 h/day in an inhalation chamber with a 107.59 ^/ 2.6 mg/m³ concentration of total suspended particulate (TSP) for 30 days. Thereafter, the total RNA was extracted from the peripheral blood mononuclear cells, the cDNA synthesized from the total RNA using the SMART^TM PCR cDNA method, and SSH performed to select the welding-fume-exposure-regulated genes. The cDNAs identified by the SSH were then cloned into a plasmid miniprep, sequenced and the sequences analysed using the NCBI BLAST programme. In the SSH cloned cDNA microarray analysis, five genes were found to increase their expression by 1.9-fold or more, including Rgs 14, which plays an important function in cellular signal transduction pathways; meanwhile 36 genes remained the same and 30 genes decreased their expression by more than 59%, including genes associated with the immune response, transcription factors and tyrosine kinases. Among the 5200 genes analysed, 256 genes (5.1%) were found to increase their gene expression, while 742 genes (15%) decreased their gene expression in response to the welding-fume exposure when tested using a commercial 5.0k DNA microarray. Therefore, unlike exposure to other toxic substances, prolonged welding-fume exposure was found to substantially downregulate many genes.

Keywords

diagnosis cDNA microarray gene expression profile lung fibrosis suppression-subtractive hybridization welding fume

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References

Alizadeh, A. A. , Eisen, M. B. and Davis, R. E. 2000: Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403, 503-511.

Beckett, W. S. 1996: Industries associated with respiratory diseases: welding. In Harber, P. , Schenker, M. B. and Balmes, R. R. , editors, Occupational and environmental ironmental respiratory diseases, St. Louis, MO: Mosby, 704-717.

Bittner, M. , Meltzer, P. , Chen, Y. et al. 2000: Molecular classification of cutaneous malignant melanoma by gene expression profiling. Nature 406, 536-540.

Buerke, U. , Schneider, J. , Rösler, J. and Woitowitz, H. J. 2002: Interstitial pulmonary fibrosis after severe exposure to welding fumes. American Journal of Industrial Medicine 41, 259-268.

Cheng, R. Y. , Zhao, A. , Alvord, W. G. et al. 2003: Gene expression dose-response changes in microarrays after exposure of human peripheral lung epithelial cells to nickel (II). Toxicology and Applied Pharmacology 191, 22-39.

Fubini, B. and Hubbard, A. 2003: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis. Free Radical Biology and Medicine 34, 1507-1516.

Koike, E. , Hirano, S. , Shimojo, N. and Kobayashi, T. 2002: cDNA microarray analysis of gene expression in rat alveolar macrophages in response to organic extract of diesel exhaust particles. Toxicological Sciences 67, 241-246.

Kwag, Y. S. and Paik, N. W. 1997: A study on airborne concentration of welding fumes and metals in confined spaces of a shipyard. Korean Industrial Hygiene Association Journal 7, 113-131.

Lubianova, I. P. 1990: Manifestations of excessive iron accumulation in steel cast iron welders. Gigiena Truda i Professional’nye Zabolevaniia 1990 (12), 11-14.

10.

Nemery, B. 1990: Metal toxicity and the respiratory tract. The European Respiratory Journal 3, 202-219.

11.

NIOSH . 1999: NIOSH manual of analytical methods. Method No. 7300, 7604. Cincinnati, OH: National Institute for Occupational Health.

12.

Roberts, E. , Charboneau, L. , Espina, V. et al. 2004: Application of laser capture microdissection and protein microarray technologies in the molecular analysis of airway injury following pollution particle exposure. Journal of Toxicology and Environmental Health 67, 851-861.

13.

Ruegger, M. 1995: Lung disorders due to metals. Schweizerische Medizinische Wochenschrift 125, 467-474.

14.

Steurich, F. and Feyerabend, R. 1997: Sidero-fibrosis of the lungs after decades of arc welding. Pneumologie 51, 545-549.

15.

Sung, J. H. , Choi, B. G. , Maeng, S. H. et al. 2004: Recovery from welding-fume-exposure-induced lung fibrosis and pulmonary function changes in Sprague Dawley rats. Toxicological Sciences, 82, 608-613.

16.

Ye, J. and Shi, X. 2001: Gene expression profile in response to chromium-induced cell stress in A549 cells. Molecular and Cellular Biochemistry 222, 189-197.

17.

Young, R. R. 2002: Genetic toxicology: web resources. Toxicology 173, 103-121.

18.

Yu, I. J. , Kim, K. J. , Chang, H. K. et al. 2000: Pattern of deposition of stainless welding fume particle inhaled into the respiratory systems of Sprague-Dawley rats exposed to a novel welding fume generating system. Toxicology Letters 116, 103-111.

19.

Yu, I. J. , Song, K. S. , Chang, H. K. et al. 2001: Lung fibrosis in Sprague-Dawley rats, induced by exposure to manual metal arc-stainless steel welding fumes. Toxicological Sciences 63, 99-106.

20.

Yu, I. J. , Song, K. S. , Chang, H. K. et al. 2003: Recovery from manual arc-stainless steel welding-fume exposure induced lung fibrosis in Sprague-Dawley rats. Toxicology Letters 143, 247-259.

21.

Yu, I. J. , Park, J. D. , Park, E. S. et al. 2003: Manganese distribution in brains of Sprague-Dawley rats after 60 days of stainless steel welding-fume exposure. Neurotoxicology 24, 777-785.

22.

Yu, I. J. , Song, K. S. , Maeng, S. H. et al. 2004: Inflammatory and genotoxic responses during 30 day welding-fume exposure period. Toxicology Letters 154, 105-115.