Global Gene Expression Profiling
Accumulating evidence demonstrates that particulate air pollutants can cause both pulmonary and airway inflammation. However, few data show that particulates can induce systemic inflammatory responses. We conducted an exploratory study using microarray techniques to analyze whole-blood total RNA in boilermakers before and after occupational exposure to metal fumes. A self-controlled study design was used to overcome the problems of larger between-individual variation interferences with observations of relatively smaller changes caused by environmental exposure. Moreover, we incorporated the dichotomous data of absolute gene expression status in the microarray analyses. Compared with nonexposed controls, we observed that genes with altered expression in response to particulate exposure were clustered in biologic processes related to inflammatory response, oxidative stress, intracellular signal transduction, cell cycle, and programmed cell death. In particular, the preinflammatory cytokine interleukin 8 and one of its receptors, chemokine receptor 4, seemed to play important roles in early-stage response to heavy metal exposure and were down-regulated. Furthermore, most observed expression variations were from nonsmoking exposed individuals, suggesting that smoking profoundly affects whole-blood expression profiles. Our study is the first to demonstrate that with a paired sampling study design of pre- and postexposed individuals, small changes in gene expression profiling can be measured in whole-blood total RNA from a population-based study. This technique can be applied to evaluate the host response to other forms of environmental exposures.
Exposure to ambient particulate air pollution is associated with increases in morbidity and mortality from respiratory and cardiovascular diseases (Godleski et al. 2000). The welding process generates high levels of metal fume containing respirable particles. Epidemiologic studies have shown that acute exposure to welding fume is associated with metal-fume fever (Mueller and Seger 1985) and increased reversible respiratory symptoms (El-Zein et al. 2003a; Wolf et al. 1997). There was an increased prevalence of inflammatory lung diseases, such as asthma and chronic bronchitis, among welders (El-Zein et al. 2003b). Additionally, accumulating epidemiologic evidence in the last decade has pointed to the associations of particulate exposure with adverse cardiovascular effects (Dockery et al. 1993; Mann et al. 2002; Peters et al. 2000, 2001a; Pope et al. 2002). Limited evidence indicates that welding-fume exposure also may be associated with increased cardiovascular events (Sjogren et al. 2002).
It has been proposed that inhaled particulates from air pollution may cause systemic alterations by the release of inflammatory cytokines subsequent to pulmonary inflammation, which plays an important role in the pathogenesis of atherosclerosis and coronary diseases. Indeed, elevated ambient particulate levels have been shown to be associated with increased levels of inflammatory markers, such as white blood cell (WBC) counts (Schwartz 2001), C-reactive protein (CRP; Peters et al. 2001b; Seaton et al. 1999), and fibrinogen (Pekkanen et al. 2000; Schwartz 2001) in both cross-sectional and longitudinal epidemiologic observations. In the experimental setting, animal studies have revealed that concentrated ambient particulate exposures increase the total WBC counts and the differential count of circulating neutrophils (Clarke et al. 2000; Gordon et al. 1998) in both healthy animals and those with pulmonary hypertension. Intratracheal instillation of residual oil fly ash (ROFA) can induce a significant elevation of plasma fibrinogen in cardiopulmonary-compromised rats (Gardner et al. 2000). Suwa et al. (2002) in their important work showing progressive atherosclerosis related to particulate exposure in hyperlipidemic rabbits also noted an increase in circulating polymorphonuclear leukocyte counts caused by exposures to particulate matter (PM) with a mass median aerodynamic diameter ≤ 10 µm (PM10).
However, most previous studies evaluated only downstream markers for systemic inflammatory responses. Direct human evidence is still lacking that shows particulates can induce systemic inflammation, although previous human studies and animal experiments did generate data, suggesting the involvement of inflammatory responses in particulate-mediated acute cardiac events. If particulate-mediated systemic inflammation were responsible for the observed adverse effects on the cardiovascular system, we would expect to see corresponding changes in mRNA expression for particulate-mediated systemic inflammation. The study described in this article addresses this mechanistic gap by investigating the systemic inflammatory response to welding-fume exposure using cDNA microarray technology on whole-blood total RNA. Blood samples were collected from welders and nonwelding controls before and after the work shift. We hypothesized that welding-fume exposure would be associated with systemic inflammation, as indicated by the findings that genes involved in systemic inflammation have significantly altered expressions. Furthermore, previous epidemiologic studies have shown that cigarette smoking significantly affects CRP, fibrinogen, and WBC levels (Frohlich et al. 2003; Smith et al. 2003). Therefore, we also hypothesized that smoking status would significantly affect the association between welding fume and the various systemic inflammatory gene expressions.
Microarray technology provides a format for the simultaneous measurement of the expression of thousands of genes in a single experimental assay and quickly becomes one of most the powerful and versatile tools for genomics and biomedical research (Murphy 2002). Peripheral blood is an essential tissue type for biomedical and clinical research because of its critical roles in immune response and metabolism. Furthermore, considering the simplicity and ease of collection, peripheral blood is also essential for discovery of biomarkers of hematologic diseases and surrogate markers of a wide range of nonhematologic disorders. Thus, applying microarray technology on peripheral blood may provide new insights of variations in global gene expression specifically associated with states of normal and disease and has the potential of applying the technology in disease detection and diagnosis. However, with the challenges unique to the blood sample, including complex composition of heterogeneous cell types and ex vivo changes of expression profiles induced by different handling and processing methods, it is difficult to apply microarray technology on whole-blood total RNA, and there are few previous publications of such research. To this end, this study is also an exploratory research with the purpose of developing proper methods for applying microarray technology on whole-blood total RNA.
Accumulating evidence demonstrates that particulate air pollutants can cause both pulmonary and airway inflammation. However, few data show that particulates can induce systemic inflammatory responses. We conducted an exploratory study using microarray techniques to analyze whole-blood total RNA in boilermakers before and after occupational exposure to metal fumes. A self-controlled study design was used to overcome the problems of larger between-individual variation interferences with observations of relatively smaller changes caused by environmental exposure. Moreover, we incorporated the dichotomous data of absolute gene expression status in the microarray analyses. Compared with nonexposed controls, we observed that genes with altered expression in response to particulate exposure were clustered in biologic processes related to inflammatory response, oxidative stress, intracellular signal transduction, cell cycle, and programmed cell death. In particular, the preinflammatory cytokine interleukin 8 and one of its receptors, chemokine receptor 4, seemed to play important roles in early-stage response to heavy metal exposure and were down-regulated. Furthermore, most observed expression variations were from nonsmoking exposed individuals, suggesting that smoking profoundly affects whole-blood expression profiles. Our study is the first to demonstrate that with a paired sampling study design of pre- and postexposed individuals, small changes in gene expression profiling can be measured in whole-blood total RNA from a population-based study. This technique can be applied to evaluate the host response to other forms of environmental exposures.
Exposure to ambient particulate air pollution is associated with increases in morbidity and mortality from respiratory and cardiovascular diseases (Godleski et al. 2000). The welding process generates high levels of metal fume containing respirable particles. Epidemiologic studies have shown that acute exposure to welding fume is associated with metal-fume fever (Mueller and Seger 1985) and increased reversible respiratory symptoms (El-Zein et al. 2003a; Wolf et al. 1997). There was an increased prevalence of inflammatory lung diseases, such as asthma and chronic bronchitis, among welders (El-Zein et al. 2003b). Additionally, accumulating epidemiologic evidence in the last decade has pointed to the associations of particulate exposure with adverse cardiovascular effects (Dockery et al. 1993; Mann et al. 2002; Peters et al. 2000, 2001a; Pope et al. 2002). Limited evidence indicates that welding-fume exposure also may be associated with increased cardiovascular events (Sjogren et al. 2002).
It has been proposed that inhaled particulates from air pollution may cause systemic alterations by the release of inflammatory cytokines subsequent to pulmonary inflammation, which plays an important role in the pathogenesis of atherosclerosis and coronary diseases. Indeed, elevated ambient particulate levels have been shown to be associated with increased levels of inflammatory markers, such as white blood cell (WBC) counts (Schwartz 2001), C-reactive protein (CRP; Peters et al. 2001b; Seaton et al. 1999), and fibrinogen (Pekkanen et al. 2000; Schwartz 2001) in both cross-sectional and longitudinal epidemiologic observations. In the experimental setting, animal studies have revealed that concentrated ambient particulate exposures increase the total WBC counts and the differential count of circulating neutrophils (Clarke et al. 2000; Gordon et al. 1998) in both healthy animals and those with pulmonary hypertension. Intratracheal instillation of residual oil fly ash (ROFA) can induce a significant elevation of plasma fibrinogen in cardiopulmonary-compromised rats (Gardner et al. 2000). Suwa et al. (2002) in their important work showing progressive atherosclerosis related to particulate exposure in hyperlipidemic rabbits also noted an increase in circulating polymorphonuclear leukocyte counts caused by exposures to particulate matter (PM) with a mass median aerodynamic diameter ≤ 10 µm (PM10).
However, most previous studies evaluated only downstream markers for systemic inflammatory responses. Direct human evidence is still lacking that shows particulates can induce systemic inflammation, although previous human studies and animal experiments did generate data, suggesting the involvement of inflammatory responses in particulate-mediated acute cardiac events. If particulate-mediated systemic inflammation were responsible for the observed adverse effects on the cardiovascular system, we would expect to see corresponding changes in mRNA expression for particulate-mediated systemic inflammation. The study described in this article addresses this mechanistic gap by investigating the systemic inflammatory response to welding-fume exposure using cDNA microarray technology on whole-blood total RNA. Blood samples were collected from welders and nonwelding controls before and after the work shift. We hypothesized that welding-fume exposure would be associated with systemic inflammation, as indicated by the findings that genes involved in systemic inflammation have significantly altered expressions. Furthermore, previous epidemiologic studies have shown that cigarette smoking significantly affects CRP, fibrinogen, and WBC levels (Frohlich et al. 2003; Smith et al. 2003). Therefore, we also hypothesized that smoking status would significantly affect the association between welding fume and the various systemic inflammatory gene expressions.
Microarray technology provides a format for the simultaneous measurement of the expression of thousands of genes in a single experimental assay and quickly becomes one of most the powerful and versatile tools for genomics and biomedical research (Murphy 2002). Peripheral blood is an essential tissue type for biomedical and clinical research because of its critical roles in immune response and metabolism. Furthermore, considering the simplicity and ease of collection, peripheral blood is also essential for discovery of biomarkers of hematologic diseases and surrogate markers of a wide range of nonhematologic disorders. Thus, applying microarray technology on peripheral blood may provide new insights of variations in global gene expression specifically associated with states of normal and disease and has the potential of applying the technology in disease detection and diagnosis. However, with the challenges unique to the blood sample, including complex composition of heterogeneous cell types and ex vivo changes of expression profiles induced by different handling and processing methods, it is difficult to apply microarray technology on whole-blood total RNA, and there are few previous publications of such research. To this end, this study is also an exploratory research with the purpose of developing proper methods for applying microarray technology on whole-blood total RNA.
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