To our knowledge, this is the first dietary intervention study to analyse global gene expression profiles within a target tissue before and after a 12 month intervention, and to stratify gene expression profiles by genotype. While we do not observe any consistent changes in plasma PSA levels over the 12 month period of the intervention, we were able to quantify extensive changes in gene expression. We find little evidence to support potential mechanisms derived from animal and cell models to explain the observational data that consuming broccoli may reduce risk of cancer, but considerable evidence for the perturbation of several signalling pathways that are associated with carcinogenesis and inflammation (). It is possible that the net effect of perturbation of these pathways may reduce the risk of cell proliferation, and maintain cell and tissue homoeostasis. However, whilst quantification of gene expression and pathway analyses provides information concerning which pathways may be modified by time or diet, it can provide little information about the precise nature of how these pathways are perturbed. This requires further analysis of mRNA and protein turnover, and post
translational protein modifications such as phosphorylation, associated with components of the signal transduction pathway and downstream targets. Furthermore, the effect of pathway perturbation may depend upon the precise cell type, with potentially differential effects occurring in epithelial and stromal cells. Despite these reservations, it is of considerable interest that broccoli intervention is associated with perturbation of TGFβ1, EGF and insulin signalling, each of which has been associated with prostate carcinogenesis 
, in addition to carcinogenesis at other sites 
, and inflammation associated with myocardial infarction 
. It is noteworthy that broccoli consumption was also associated with alterations in mRNA processing, and this is being further explored.
It is likely that the major bioactive products derived from broccoli are the isothiocyanates, sulforaphane and iberin. These have been shown to have a multitude of biological activities in cell models consistent with anticarcinogenic activity 
. However, these studies largely involve exposing cells to concentrations of SF and IB far in excess of those which occur transiently in the plasma after broccoli consumption, and are mediated by the intracellular activity of the ITCs by, for example, perturbing intracellular redox status, depletion of glutathione and perturbation of the Keap1-Nfr2 complex. We question whether these processes would occur in vivo
, as any of the ITCs entering cells would immediately be inactivated through conjugation with glutathione that would be present in relatively high concentration. Thus, we explored whether the biological activity of ITCs may be mediated through their chemical interaction with signalling peptides within the extracellular environment of the plasma, which has a low glutathione concentration. We demonstrated that ITCs readily form thioureas with signalling proteins in the plasma through covalently bonding with the N-terminal residue. It is likely that ITCs chemically react with other plasma proteins and a global analysis of plasma protein modifications by ITCs is warranted. It is also possible that other types of chemical modification of plasma proteins by ITCs may occur, such as covalent bonding through cysteine and lysine residues 
Previous studies have shown that isothiocyanate-derived thioureas modify the physicochemical and enzymatic properties of the parental proteins 
. Thus, it is possible that the perturbation of signalling pathways in the prostate is mediated by protein modifications that occur in the extracellular environment. We provide further evidence for this hypothesis by demonstrating that pre incubation of TGFβ1 with a physiological appropriate concentration of SF (2 µM for 30 minutes), followed by dialysis for 4 h to simulate SF pharmacokinetics, results in enhanced Smad-mediated transcription. As TGFβ1/Smad-mediated transcription inhibits cell proliferation in non-transformed cells 
, the enhancement of Smad-mediated transformation by SF would be consistent with the anticarcinogenic activity of broccoli, in addition to reduced risk of myocardial infarction 
. In certain circumstances, enhancement of TGFβ signalling has been associated with tumour progression within already initiated cells, although the precise pathways by which this is mediated have not been fully resolved 
. To what extent a broccoli-rich diet may influence these processes requires further studies. However, we consider it likely that it is the net effect of changes in several pathways, as opposed to just TGFβ1, which may underlie the observed reduction in both cancer and myocardial infarction through broccoli/crucifer consumption.
A previous study has demonstrated that isothiocyanates can inhibit EGF signalling, but without a mechanistic explanation 
. In the current study, we show that SF will bind to the EGF ligand, and this may underlie our results and those reported previously 
. Moreover, chemical modification of signalling proteins by ITCs may be complemented by modification of receptor proteins, as has previously been shown for the TRPA1 receptor 
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
null genotype with enhanced risk of prostate cancer 
enzyme activity catalyses both the formation and the cleavage of SF – glutathione conjugates 
. We suggest that following transport into the plasma from enterocytes, GSTM1
activity (originating either from hepatic cell turnover 
or leakage from peripheral lymphocytes 
) catalyses the cleavage of the SF-glutathione conjugate within the low glutathione environment of the plasma 
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 (). 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
, 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 complement histological examination of needle biopsies and measurement 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 derivatives, could also chemically interact with signalling peptides in the plasma, in a similar manner to the suggested mechanism of action of isothiocyanates.
In conclusion, we consider that our study has provided a mechanistic basis for the reduction in risk of prostate cancer through broccoli consumption, as suggested by epidemiological studies. Further studies with larger cohorts, combined with the assessment of clinical endpoints, are warranted.