New Biomarkers for Monitoring the Levels of Isothiocyanates in Humans

Abstract

Isothiocyanates (ITCs) found in cruciferous vegetables have demonstrated cancer preventive activity in animals, and increased dietary intake of ITCs has been shown to be associated with a reduced cancer risk in humans. ITCs exert their cancer chemopreventive action by multiple mechanisms, for example, by modulating the activities of phase I and phase II drug metabolism enzymes, by inhibiting the cell cycle and histone deacetylase, and by causing apoptotic cell death. In cells, protein adducts account for most of total cellular ITC uptake at 4 h after treatment. The time course of this protein binding correlates well with the inhibition of proliferation and the induction of apoptosis. Animal studies have shown that glutathione conjugates are the major products of ITCs. The major urinary excretion products of ITCs in human are N-acetyl cysteine conjugates. Urinary metabolites might provide the exposure history of the last 24 h, if the urine of the full next day is collected. However, this is not feasible in large epidemiological studies. Furthermore, the mercapturic acids of ITC are not stable. Therefore, stable biomarkers are needed that reflect a larger time span of the ITC exposure history. We developed a method to determine stable (not cysteine adducts) reaction products of ITCs with albumin and hemoglobin in humans and mice. We reacted albumin with the ITCs: benzyl isothiocyanate (BITC), phenylethyl isothiocyanate (PEITC), sulforaphane (SFN), and allyl isothiocyanate (AITC). After enzymatic digestion, we found one major product with lysine using LC-MS/MS. The identity of the adducts was confirmed by comparing the analyses with synthetic standards: N(6)-[(benzylamino)carbonothioyl]lysine (BITC-Lys), N(6)-{[(2-phenylethyl)amino]carbonothioyl}lysine (PEITC-Lys), N(6)-({[3-(methylsulfinyl)propyl]amino}carbonothioyl)lysine (SFN-Lys), and N(6)-[(allylamino]carbonothioyl]lysine (AITC-Lys). The adduct levels were quantified by isotope dilution mass spectrometry using the corresponding new ITC-[(13)C(6)(15)N(2)]lysines as internal standards. The applicability of the method was tested for biological samples obtained from different experiments. In humans consuming garden cress, watercress, and broccoli and/or in mice exposed chronically to N-acetyl-S-{[(2-phenylethyl)amino]carbonothioyl}-l-cysteine, albumin and hemoglobin adducts were found. BITC-Lys, PEITC-Lys, and SFN-Lys released after enzymatic digestion of the proteins were quantified with LC-MS/MS. This new method will enable quantification of ITC adducts in blood proteins from large prospective studies about diet and cancer. Protein adducts are involved in the chemopreventive effects of ITCs. Therefore, blood protein adducts are a potential surrogate marker for the effects of ITCs at the cellular level. This new technique will improve the assessment of ITC exposure and the power of studies on the relationship between ITC intake and cancer.