One ultimate goal of vesicant research is to identify potential effective medical countermeasures to alkylation injury of the skin and to identify biomarkers for different stages of vesicant injury and wound repair. This would provide the methodology to screen large numbers of novel compounds quickly and act as a qualitative tool to determine their potential effectiveness in alleviating damage or enhancing wound repair. One approach is to identify biomarkers that correlate to histological improvements after application of a novel countermeasure.
Microarray technology is a useful tool which provides an indication of the changing gene expression between control samples and those exposed to toxicants. In general, transcriptional analysis can generate proxies that monitor critical events associated with post-exposure changes at the cellular level; the information from transcriptional signatures can lead to development of hypotheses associated with modes of action (Androulakis, 2005
). A majority of transcriptional studies to date focused on terminal events at a very coarse temporal resolution, loosely characterized as before vs. after, healthy vs. unhealthy, treated vs. vehicle, in an effort to identify clear and distinguishing variations between terminal states (Androulakis et al., 2007
). However, in order to understand the progression of a phenotypic response towards a terminal state or, more importantly, the identification of “intervention” strategies, the time-evolution of the transcriptional phenomenon is critical. Additional detailed time-course experiments provide “kinetic” information on the critical points and rates associated with the post-exposure response, and can provide insight into possible interventions that alter the progression of the response. Therefore, high(er) temporal resolution transcriptional studies enable improved characterization of the cellular processes as they evolve over time thus enabling the identification of the state changes and points of intervention associated with the progress of the response. Furthermore, time-course measurements will critically assist the development of quantitative and predictive state progression models. Therefore, the present study focused on longer time periods (24, 72, and 168 h) than previously reported, in order to allow enough time for wound repair to commence.
Initial analysis used the methodology of the Gene Ontology (GO) Consortium in combination with KEGG pathway analysis. Common gene pathways for the SM-treated time-points included cytokine–cytokine receptor interaction and Jak-STAT signaling. Both of these pathways correspond to persistent inflammation, which was observed histologically as well as in the gene chip analysis (data not shown). It will be interesting to see whether the inflammatory cell population follows a defined progression from neutrophils to macrophages to mast cells over time as is true for the incisional wound model (Stramer et al., 2007
). In addition to inflammatory pathways, two novel repair pathways became prominent 72 h post-treatment. These were cell communication and the Hedgehog signaling pathway, the latter of which is a pathway which plays a critical role in the regulation of the development of several tissues and organs (Ma et al., 2008
). By 168 h post-treatment several additional pathways are activated including cell adhesion molecules, neuroactive ligand–receptor interactions, glycosphingolipid biosynthesis, and complement and coagulation cascades. In a broad sense, these may all be considered repair pathways. It is fully expected that additional repair pathways will turn on even later, which would require further experiments that carry the model out at least a month or more. The data were analyzed a slightly different way, sorting according to the top biological functions, and similar results occurred. The biological functions included inflammation, immunological activation, cell movement, and cell death, also supporting the notion that cells are actively dividing and migrating in an attempt to heal the wound. These data are consistent with the belief that inflammation is persistent and a long-term process in the MEVM. In fact, genes involved in the inflammatory pathway topped the list in every type of analysis performed in this study. The most significant individual mRNA increase was for CXCL2 (also called MIP-2, macrophage inflammatory protein-2) and increased by a tremendous 500 fold by 168 h post-treatment (). This protein is involved with neutrophil recruitment and other inflammatory processes, so CXCL2 upregulation was not unexpected. Other inflammatory genes significantly upregulated after sulfur mustard exposure included CCL2, CCR1, Il1b, Il6, and PTGS2. All of these increased dramatically by 24 h post-SM exposure, demonstrating the value of analyzing early time points as well as the later ones. Several of these cytokines and Vcam1 were chosen for quantitation of their mRNA for the various time points (). In every case the mRNA validated the general trends of the microarray data. In order to validate that mRNA is translated into protein, interleukin 1 beta was quantitated and agreed with the microarray and mRNA data (). The major gene pathways affected by sulfur mustard were predominantly inflammatory, apoptosis, and stress-related pathways (–). These all reflect the severity of the wound caused by sulfur mustard exposure. Testing an inhibitor of matrix metalloproteinase activity demonstrated that subtle, but specific changes do occur in the gene profiles when microarray analysis is performed (). Since these changes are more quantitative than the histological changes which are more qualitative (evaluating amount of edema, number of invading inflammatory cells, and severity of necrosis), these differences in microarray patterns may correlate to predictable changes in SM exposed skin and lead to the identification of biomarkers for specific stages of injury. The pathway changes observed in the inhibitor I microarrays involved the metabolism, degradation, and biosynthesis of many molecules suggesting that the wound healing response was slightly accelerated using MMP-2/9 inhibitor I, although no gross changes in inflammation, or inflammatory genes were noted.
Taken together, the microarray data provide detailed information regarding the genes that become activated in mouse skin following exposure to sulfur mustard. Many of these genes reflect biologic processes. In general, a host of cytokines and mediators of inflammation are released and activated early (within 24 h) after SM exposure. These include genes in the NFKb pathway and the p38 MAP kinase pathway. Hyperproliferation and cell division are also activated early as seen by a number of genes under control of the p53 signaling pathway. Apoptosis and oxidative stress genes also turn on early and remain on throughout all the time points observed. When this methodology was applied to the MMP-2/MMP-9 inhibitor I data, there were numerous repair process genes that became activated when compared to SM-alone treated samples. This demonstrates the sensitivity of the assay since we did not observe any quantitative histological differences between SM-alone and MMP inhibitor I treated samples. If these results can be replicated and the same genes that were activated in this system identified, there is potential for use of microarrays as markers of repair.
Although the data were analyzed in several different ways, the major results were similar, depending on the analysis employed. However, there were subtle differences between the various analyses. Regardless of the different results, all the methods were valid and useful in analyzing gene expression at different time periods post-exposure. Higher time resolution gene expression experiments, partly performed in this study, beyond the standard dose–response analysis, will significantly boost the ability to cross over from the present, mostly, descriptive nature of genomics towards a more useful mathematical model-based analysis that identifies specific phases of wound progression and repair in order to developed targeted medical interventions. Microarray analysis shows strong potential for advancing the knowledge of the MEVM system and for use as a prescreening tool to predict the general effectiveness of medical countermeasures to vesicant injury.