Broccoli & Signalling Pathways Figure 6. LC-MS of TGFb1 incubated with and without SF. Extracted ion chromatograms (MS) of precursor masses representing the unmodified N-terminal peptide of TGFb1 (m/z 768.5) and the modified N-terminal peptide (m/z 877.2) A of m/z 768.2–769.2 from DMSO treated TGFb1, B of m/z 768.2–769.2 from SF treated TGFb1, C of m/z 876.7–877.7 DMSO treated TGFb1 and D of m/z 876.7–877.7 SF treated TGFb1. doi:10.1371/journal.pone.0002568.g006 proteins by ITCs may be complemented by modification of receptor proteins, as has previously been shown for the TRPA1 receptor [39,40]. Perturbation of signalling pathways is additionally determined by GSTM1 genotype. The interaction between diet and GSTM1 on gene expression may partially explain the contradictory results from those case control studies which lack dietary assessment and which have or have not associated the GSTM1 genotype with enhanced risk of prostate cancer [44–47]. GSTM1 enzyme activity catalyses both the formation and the cleavage of SF – glutathione conjugates [48]. We suggest that following transport into the plasma from enterocytes, GSTM1 activity (originating either from hepatic cell turnover [49] or leakage from peripheral lymphocytes [50]) catalyses the cleavage of the SF-glutathione conjugate within the low glutathione environment of the plasma [51] to determine the extent of free SF that is available for protein modification, as discussed above, and which is not excreted via mercapturic acid metabolism (Figure 1). Thus low levels of SF, as would be expected from normal dietary consumption of broccoli, may lead to subtle changes in cell signalling, which, over time, result in profound changes in gene expression. In this manner, consuming one portion of broccoli per week if one is GSTM1 positive, or more if one is GSTM1 [14], may contribute to a reduction in cancer risk. In addition to the insight this study provides to the effect of broccoli consumption on gene expression, we consider that our study may have broader implications. First, we demonstrate that routine prostate needle biopsies can be used for global gene expression analyses in addition to histological assessment, and that it is possible to monitor changes in expression with time. It is notable that men within both dietary arms of the study had significant changes in the androgen receptor pathway. It is possible that these changes in androgen signalling are associated with aging and independent of diets, or they may have been induced by a common component of both the broccoli-rich and pea-rich diet. To our knowledge there is no data on the rate of change on androgen signalling in men of this age with HGPIN. This observation suggests further study is warranted. Analysis of the rate of change of gene expression of men diagnosed with either HGPIN or localized prostate cancer through sequential biopsies may provide reliable biomarkers to measure the likelihood of both carcinogenesis and progression to aggressive cancer, and comple- ment histological examination of needle biopsies and measure- ment of plasma PSA levels. Secondly, stratification of global gene expression profiles by genotype has been informative, and this approach could be extended to other genes to dissect patterns of gene expression in prostate or other tissues. Lastly, it is conceivable that other dietary phytochemicals, such as polyphenolic deriva- tives, could also chemically interact with signalling peptides in the plasma, in a similar manner to the suggested mechanism of action of isothiocyanates. PLoS ONE | www.plosone.org 11 July 2008 | Volume 3 | Issue 7 | e2568