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 Keap1Nfr2 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 Ergosterol 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 TGFb1 with a physiological appropriate concentration of SF, followed by dialysis for 4 h to simulate SF pharmacokinetics, results in enhanced Smadmediated transcription. As TGFb1/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 TGFb 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 TGFb1, 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 signaling 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 Ginsenoside-F5 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.