BAT Science - Inflammation and oxidative stress

Cigarette smoke is a complex and dynamic mixture of more than 5,000 individual chemical constituents [1] and there is strong evidence linking cigarette smoking with a number of disease processes. Cigarette smoke is well known to cause cellular oxidative stress, which is a factor in many smoking-related diseases [2-3,7].

Oxidative stress occurs when an imbalance between excessive generation of oxidants (free radicals) and/or insufficient antioxidant defence mechanisms results in cell, tissue or organ injury. This can initiate numerous pathological processes and contribute to the development of various diseases including atherosclerosis, cancer and chronic obstructive pulmonary disease (COPD) [ 4 ].

Free radicals include reactive oxygen (ROS) and reactive nitrogen species (RNS) and are formed naturally during a variety of biochemical reactions and cellular functions, for example during mitochondrial metabolism. Under normal circumstances, free radicals are neutralised by the cells’ antioxidant defence mechanisms. Oxidative stress can however result in an imbalance in the formation and degradation of pro-oxidant free radicals, leading to DNA damage, mitochondrial dysfunction, membrane damage and lipid peroxidation, protein oxidation and apoptosis [ 5-6 ]. Both in vivo and in vitro studies have demonstrated that chemical constituents in cigarette smoke have the potential to generate ROS and induce oxidative stress by increasing the pro oxidant burden and/or decreasing antioxidant protection. Cigarette smoke-derived ROS cause oxidative damage to cellular components and activate numerous signal pathways that modulate cellular responses and may ultimately lead to pathological changes in cell function [ 7 ].

Direct measurement of ROS production

The induction of the production of reactive oxygen species (ROS) by intracellular enzymes in response to cigarette smoke is a key factor in the oxidative stress associated with smoke-related diseases. Using specific dyes to monitor ROS levels, including dichlorofluorescein (DCF) and dihydroethidium (DHE), we are developing tools with the ability to monitor the intracellular production of ROS in cell types relevant to each of the three main smoking-related diseases. The example below shows the detection of ROS by DCF in the lung epithelial (NCI-H292) cell line in response to exposure to cigarette smoke extracts.

            

NCI-H292 cells above loaded with the ROS-sensitive dye, DCF (dichlorofluorescein). Images are (left) of control cells exposed to 1 % DMSO and (right) of cells exposed to 100 mg/ml TPM for 1 hour.

Monitoring intracellular antioxidant status

As well as measuring directly the production of ROS, we are monitoring changes in oxidative stress indirectly by examining changes in the levels of the antioxidant, reduced glutathione, in cells. This is being carried out using the luminescent GSH-glo assay and our initial data demonstrate the use of such an assay in monitoring antioxidant levels as well as in determining cellular mechanisms underlying the response to cigarette smoke [ 8-9 ].

Monitoring oxidative stress-induced transcriptional regulation

The study of transcription factors related to cigarette smoke exposure via oxidative stress, such as nuclear transcription factor κB (NF-κB) [ 10-11 ], has gained considerable interest of the field of free radical biology. NF-κB is a transcription factor that resides in an inactive state in the cytoplasm and which is activated and translocates to the nucleus in response to oxidative stress. The activated NF-κB regulates various cellular processes and stimulates pro inflammatory responses. These include switching on the expression and secretion of a number of chemokines such as interleukins 6 and 8 (IL 6 and IL 8) which mediate pathological inflammatory processes and are implicated in numerous human diseases [11]. We are currently using powerful and integrated immunocytochemical and protein profiling approaches to monitor NF-κB activation and protein secretion in response to cigarette smoke extracts in both lung epithelial (H292) and endothelial (HUVEC) cells.

        

The above picture shows H292 lung epithelial cells immunostained with an anti-pNF-kB antibody (red fluorescence). The blue fluorescence is the DAPI-stained nuclei. In response to TPM, NF-kB levels were increased and the transcription factor accumulated in the nucleus.

Monocyte function

As described in the pages on cardiovascular disease, monocytes/macrophages play a key role in the development and progression of atherosclerosis, and are also involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). We are currently utilising the monocyte cell line, THP-1, to monitor the release of chemokines from these cells in response to cigarette smoke using the MesoScale Discovery (MSD®) protein profiling platform. We are also developing external collaborations to enable us to examine numerous facets of monocyte/macrophage function in relation to cardiovascular disease, including lipid uptake and metabolism and the expression and secretion of inflammatory markers and matrix metalloproteinases.

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7. Halliwell, B., Poulsen, H.E. (2006). Cigarette smoke and oxidative stress. Springer Press, ISBN 3-540-31410-31415.
8. Tai, T., Carr, T., Oke, O., Faux, S., Gaca, M. (2008). Detection of cigarette smoke induced reactive oxygen species (ROS) using a CM-H2DCFDA fluorescence probe. Poster presented at Society for Free Radical Biology and Medicine Conference, Indianapolis, November 2008. Link to poster PDF. 
9. Taylor, M., Carr, T., Cockcroft, N., Fearon, I.M. (2009). Changes in reduced glutathione levels in response to cigarette smoke extracts: potential role of ROS production at mitochondrial complex I. Poster presented at Society for Free Radical Biology and Medicine Conference, San Francisco, November 2009. Link to poster PDF. 
10. Li, X., Stark, G.R. (2002). NFkappaB-dependent signaling pathways. Experimental Hematology. 30: 285 296.
11. Ahn, K.S., Aggarwal, B.B. (2005). Transcription factor NF-κB: a sensor for smoke and stress signals. Annals of the New York Academy of Sciences. 1056: 218-233.