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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 2, 2004 as doi:10.1096/fj.04-1877fje. |
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Department of Health Sciences, University of Genoa, Genoa, Italy; and
* National Cancer Institute, National Institutes of Health, Rockville, Maryland, USA
2Correspondence: Department of Health Sciences, University of Genoa, Via A. Pastore 1, I-16132 Genoa, Italy. E-mail: sdf{at}unige.it
SPECIFIC AIMS
We have previously shown that sunlight-mimicking light induces genotoxic damage not only in skin but also in lungs, bone marrow, and peripheral blood of hairless mice. Moreover, light and environmental tobacco smoke (ETS) acted synergistically in the respiratory tract. The aim of the present study was to clarify the mechanisms involved at the transcriptional level. The expression of 746 toxicologically relevant genes (597 "Expression" and 149 "Stress" cDNA arrays) was evaluated in skin and lungs of SKH-1 hairless mice exposed for 28 days to light and/or ETS (Fig. 1
).
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PRINCIPAL FINDINGS
1. Exposure of mice to light up-regulates two genes in lungs and a variety of genes in skin
One group of mice was exposed, 5 days a week, 9 h a day, to the light emitted by halogen quartz bulbs covered with an UV-C blocking filter in order to mimic solar radiation wavelengths. The illuminance level was 10,000 lx. Exposure of mice to the light resulted in the overexpression of two genes in the lungs (i.e., glutathione S-transferase (GST) Pi 1 and catalase 1). The expression of these genes was increased more than two-fold as compared with unexposed mice.
Fifty-five genes (7.4%) were overexpressed in the skin of light-exposed mice. They included the genes encoding for 6 oxidative stress-related activities, among which catalase 1 and cyclooxygenase (COX) 2; 6 stress-response genes, among which p53 and its effector p21; 2 phase I metabolic activities (CYP2E1 and CYP4B1); 4 phase II activities, among which 3 glutathione S-transferase (GST) isoenzymes; 9 DNA or protein repair activities, 3 of which repairing UV-induced DNA damage and 2 repairing oxidative DNA damage; 4 proapoptotic activities; 6 stimulators of cell replication, including PCNA and 5 cyclins; oncogene products, including H-ras and a ras-related protein; 5 cytoskeleton proteins, among which 3 cytokeratins; the matrix metalloproteinase MMP2 and the MMP14 precursor; integrin ß 1; 2 transcription factors and 1 cell receptor; 4 proinflammatory activities; 1 immunosuppressive factor (interleukin 10).
2. Exposure of mice to smoke up-regulates a variety of genes in both skin and lungs
A whole-body exposure of mice to ETS was achieved in a smoking machine by burning 5 Kentucky 2R1 reference cigarettes at one time, 6 h a day, 5 days a week. This accounted for a cumulative exposure to the smoke generated by 120 cigarettes/day, with an average total particulate matter of 83 mg/m3 and a CO concentration of 612 ppm. Exposure to ETS resulted in the overexpression of 14 genes (1.9%) in skin and 24 genes (3.2%) in lungs. In particular, in skin the ETS-up-regulated genes included 5 metabolic activities located either in the endoplasmic reticulum (CYP1A1 and GST), or in the cytosol (epoxide hydrolase and a GSH peroxidase precursor), or in the cell membrane (multidrug resistance [MDR] protein 1); 1 stress response activity (MAPK kinase 4); 3 DNA repair activities; 2 activities removing damaged proteins; 1 proapoptotic factor; 2 negative regulators of the cell cycle (GADD45 and GADD153). In lungs, the ETS-regulated genes encoded for 5 metabolic activities located either in the endoplasmic reticulum (among which CYP1A1) or in the mitochondria (thiosulfate sulfurtransferase) or in the cell membrane (MDR protein 1); 3 oxidative stress activities, among which a SOD 2 precursor and catalase 1; 3 stress response activities, among which were MAPK kinase 4 and plasminogen activator inhibitor; 6 factors involved in repair and removal of damaged proteins; 1 proapoptotic activity (caspase-7); 1 positive regulator of the cell cycle; 4 factors involved in the immune response. ETS also up-regulated the trypsin-chymotrypsin related serine protease gene and at the same time down-regulated the 
-1-antitrypsin 1-2 precursor gene.
3. Light increases smoke-induced gene expression in lungs, while smoke attenuates light-induced gene expression in the skin of mice exposed to both agents
Exposure of mice to daily cycles of light and ETS resulted in the up-regulation of 46 genes (6.2%) in skin and 29 genes (3.9%) in lungs, as compared with unexposed mice. The induced genes were generally the same that were individually up-regulated by light and/or ETS in both skin and lungs. However, the light tended to increase ETS-induced gene expression in lungs, while ETS tended to attenuate light-induced gene expression in skin.
4. Administration of sulindac attenuates the light- and ETS-related gene overexpression in mouse skin and lungs
One group of mice exposed to daily cycles of ETS and light received sulindac in drinking water (150 ppm), starting 3 days before the first exposure and continuing until the end of the experiment. Administration of this nonsteroidal anti-inflammatory drug (NSAID) caused a considerable decrease of overexpressed genes in skin and lungs. In fact, compared with untreated mice, only 22 genes (3.0%) were overexpressed in skin and 11 genes (1.5%) overexpressed in the lung of sulindac-treated mice exposed to both ETS and light. In mouse skin, sulindac was quite efficient in inhibiting the light-induced overexpression of COX 2 and related oxidative stress response genes, especially of two SOD precursors. In the lungs, sulindac down-regulated a variety of ETS-induced genes involved in oxidative stress response, removal of damaged proteins, inflammation, and the immune response.
CONCLUSIONS AND SIGNIFICANCE
Exposure of mice to light and/or ETS produced remarkable alterations of gene expression in skin and lungs. Both agents, either individually or in combination, appear to be capable of inducing a systemic damage not only at the genomic level, as previously documented, but also at the transcriptional level. The most striking result was that exposure to light caused per se overexpression of GST Pi and catalase 1 in mouse lungs. GST Pi is involved in the protection against electrophiles, including lipid peroxidation products, and reactive oxygen species (ROS), such as H2O2. H2O2 is the substrate for catalase, a vital component of the defense machinery of the lung against oxygen toxicity. These data support the hypothesis that UV-containing light induces in skin the generation of ROS, lipid peroxidation products, and other derivatives that may sufficiently be long-lived to travel at a distance from the skin. In this perspective, it is noteworthy that a variety of genes were overexpressed in the skin of light-exposed mice, among which the COX 2 gene and other genes involved in the response to oxidative stress. This supports the role of the COX pathway as a mediator of UV effects.
The whole-body exposure of mice to ETS increased the expression of many genes belonging to a variety of functional categories in both skin and lungs. Among others, ETS stimulated some genes involved in various metabolic functions, including MDR protein 1. This suggests for the first time that MDR 1 may provide a defense mechanism against cigarette smoke by extruding its toxic components outside skin and lung cells. In general, ETS attenuated the light-induced overexpression of genes in skin, with particular reference to cell cycle regulators, which were stimulated by light while being inhibited by ETS. On the other hand, the light tended to enhance the ETS-induced overexpression of genes in lungs, mainly due to impairment by light of pathways detoxifying ETS components. This conclusion is consistent with our previous finding that exposure of mice to light significantly increases ETS-related genomic alterations in lungs.
Sulindac exerted important protective effects toward ETS-induced overexpression of genes in lungs and light-related overexpression of genes in skin, including COX 2. COX inhibition represents the main mechanism of NSAIDs. Our results suggest that sulindac, which so far has mainly be proposed as a chemopreventive agent in colon carcinogenesis, has the potential capability to attenuate light-induced alterations in skin as well as smoke-induced alterations in lungs.
The results reported here provide new mechanistic insights regarding the genomic and transcriptional alterations produced in different organs by light and smoke, and portray the role of both genotoxic and epigenetic events in the pathogenesis of related diseases. The reported data bear relevance in light-induced skin carcinogenesis and smoke-induced lung carcinogenesis, and indicate that exposure to light may increase smoke-related damage in the lungs. The finding that the light induces genomic and transcriptional alterations in both skin and respiratory tract raises the suspicion that UV-containing light may be carcinogenic not only to the skin but also to distant organs. The patterns of transcriptional data elucidate the mechanisms involved in the pathogenesis of smoke- or light-related chronic degenerative diseases other than cancer. For instance, the finding that UV-containing light induces apoptosis in skin cells and at the same time favors their proliferation, along with the increased expression of MMP and cytokeratin genes, may be related to the pathogenesis of cutaneous photoaging. The ETS-related overexpression in lungs of the plasminogen activator inhibitor, having a prothrombotic activity, contributes to explaining the increased risk of stroke in smokers. Of particular relevance to the pathogenesis of smoke-related emphysema is the simultaneous up-regulation in the lung of the proteolytic activity due to trypsin-chymotrypsin related serine protease and down-regulation of its inhibitor -1-antitrypsin 1-2 precursor.
FOOTNOTES
1 Permanent address: National Center of Oncology, Sofia-1756, Bulgaria 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1877fje;
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