Chapter 3 Metabolism, kinetics and genetic variation Observations in humans is about 17 min in plasma and about 3 amount of urinary N-acetylcystei ne min in cells (Xu &Thornalley, 2000a). –ITC might have been underestimated, The 05H conjugates undergo fur- because these conjugates are unsta- Isothiocyanates ther enzymatic modification, in the ble and readily dissociate to isothio- Metabolism and disposition GSH portion, to give rise sequentially cyanates (Conaway et al., 2001). The The –N=C=S group of most isothio- to the cysteinyiglycine, cysteine and N- metabolic disposition of other isothio- cyanates is electrophilic and can react acetylcysteine conjugates, which are cyanates, including allyl-ITC and cul- readily with various nucleophiles, excreted in urine (Brüsewitz et al., foraphane, are similar in humans, as including thiols. Studies in both 1977), as shown in Figure 10. Urinary discussed below. humans and experimental animals excretion of N-acetylcysteine conju- have established that isothiocyanates gates is the principal route of disposi- Measurement of isothiocyanates and are metabolized in vivo to various tion of ingested isothiocyanates. For isothiocyanate metabolites dithiocarbamates principally by the example, when benzyl-ITC was admin- The cyclocondensation assay is a mercapturic acid pathway. An initial istered orally to six men, 54% of a dose highly sensitive quantitative method for conjugation with glutathione (aSH, the of 14.4 mg was recovered in the urine measuring isothiocyanates and their most abundant cellular thiol), which as N-acetyl-S-(N- ben zylthiocarba- metabolites (referred to collectively takes place spontaneously but is fur- moyl)-L-cysteine (benzyl-ITC--N-ace- here as isothiocyanate equivalent) ther promoted by glutathione S-trans- tylcysteine). This compound was (Zhang et al., 1996) and is a valuable ferase (GST), gives rise to the corre- excreted rapidly: maximum excretion tool for studying dietary consumption sponding conjugates. occurred within 2-6 h, and the com- of isothiocyanates and their metabo- In the spontaneous reaction, isoth- pound was essentially undetectable lism. The assay is based on the almost iocyanates react reversibly with cys- 10-12 h after dosing (Mennicke et al., universal ability of isothiocyanates to teinyl thiols, forming dithiocarbamates: 1988). Watercress is rich in gluconas- react quantitatively with 1,2-ben- zenodithiol, a vicinal dithiol, to give rise to a five-membered cyclic product and a free amine (Zhang et 1992a), as cystine residue—SH + RN=C=s - cystine residue -S—C —NHR al., 11 shown in Figure 11. Reactive isothio- s cyanates are converted to the same cyclic product, 1 ,3-benzodith iole-2- The equilibrium position and the chem- turtiin (constituting more than 30% of thione, and conversion is complete in ical relaxation time for equilibrium have total glucosinolates), the precursor of the presence of excess 1,2-ben- been estimated for phenethylisothio- phenethyl-ITC. When four volunteers 7enedithiol. The resultant 1,3-bon- cyanate (ITC) reacting with cysteinyl ate 30 g of watercress containing 21.6 zodithiole-2-thione can be measured residues in blood plasma (cysteinyl mg of gluconasturtiin, an average of accurately in amounts as low as a few thiol concentration, about 500 pmol/l) 47% of the amount was recovered as picomoles, with a simple high-perfor- and cells (cysteinyl thiol concentration, the N-acetylcysteine conjugate in 24-h mance liquid chromatography (HPLC) 5 mmol/l). The equilibrium constant, /Ç urine (Chung etaL, 1992). The amount procedure. Conjugation products of was 730 per mol. At equilibrium, about of phenethyl-ITC–N-acetylcysteine in isothiocyanates with thiols (dithiocar- 27% of phenethyl-ITC is bound to urine is probably greater when bamates), including N-acetyl-cys- plasma thiols in blood, and about 88% phenethyl-ITC is administered, because teine–isothiocyanate, also react quan- is bound to thiols in cells. For a plasma the my rosin ase-catalysed conversion titatively with 1 ,2-benzenedithiol, giving concentration of 5 pmol/l of phenethyl- of gluconasturtiin to phenethyl-ITC rise to the same cyclic product. Thus, ITC, the equilibrium relaxation time for may be incomplete after ingestion of this assay allows detection of the total formation of adducts with protein thiols watercress. In these studies, the amount of isothiocyanates and their 25 IARC Handbooks of Cancer Prevention Volume 9 Cruciferous vegetables, isothiocyanates and indoles Glu-Cys-Gly Glu-Cys-Gly Cys-Gly SH GST I GGT S + R—N C=S R—NH—C=S R—NH—C=S lsothiocyanate Dithiocarbamate CG NAC Cys S s R—NH—C---S Mercapturic acid Figure 10 Isothiocyanates are conjugated to glutathione by glutathione S-transferase (GST), metabolized sequentially by y-glutamyl transpeptidase (GGT), cysteinyiglycinase (CG) and N-acetyltransferase (AT) to form, ultimately, mercapturic acid. NAC, N-acetylcysteine R N=C=S R-NH2 Isothiocyanate SH + LJL>=S 1 ,3-Benzodithiole-2-thione 1 ,2-Benzenedithiol S / R—NH--C R—NH2 + R1—SH S—R, Dithiocarbamate Figure 11 Cyclocondensation reaction of isothiocyanate and dithiocarbatrate with 1,2-ben7enedithiol thiol conjugates. The assay has isothiocyanates. Even though individ- Use of the cyclocondensation allowed quantitative assessment of ual isothiocyanates and isothiocyanate assay is nevertheless subject to some total isothiocyanates in cruciferous metabolites can be quantified sensi- caveats. Although there are no endo- vegetables and isothiocyanate equiva- tively by other methods, including genous sources of urinary isothio- lents in human fluids, including blood HPLC-mass spectrometry (Ji & cyanates or dithiocarbamates in humans and urine. Because it reveals both Morris, 2003; Vermeulen et al., 2003), (Shapiro et cL, 1998), non-dietary isothiocyanates and their thiol metabo- such analyses might not provide accu- sources may exist. The contributions of lites, the total urinary concentrations of rate values, since the metabolites are cigarette smoke (possibly due to the these compounds are not affected by unstable and can dissociate to isothio- presence of carbon disulfide, which is dissociation of thiol conjugates to cyanates. also detected in the assay), agricul- 26 Metabolism, kinetics and genetic variation tural chemicals, chemicals used in the equivalent was recovered in urine rect conclusions about long-term rubber industry and medical com- within 10h (Shapiro of al., 1998). Thus, dietary intake of isothiocyanates. pounds such as disulfiram and anti- isothiocyanates are rapidly and effi- Cellular uptake of isothiocyanates septics, should be considered (Zhang ciently absorbed, rapidly cleared from is enhanced by GST isozymes (Zhang, etaL, 1996; Shapiro of al., 1998; Ye of blood and eliminated almost exclu- 2001); however, export of intracellular aL, 2002). Shapiro of al. (1998) found sively in urine. isothiocyanate—GSH conjugates is that when urine samples were stored The molecular basis for the rapid also highly efficient without GST at —20 °C, the cyclocondensation reac- metabolic disposition of isothio- involvement (Zhang & Calloway, 2002). tivity remained stable, but 35% of the cyanates in humans was elucidated by While GSTs are likely to be important dithiocarbamate content was lost in studying cultured human cells with the in isothiocyanate metabolism, current samples stored for 18 months at the cyclocondensation assay (Zhang & understanding does not allow a predic- same temperature. Talalay, 1998; Zhang, 2000, 2001; tion of the consequences of GST poly- Zhang & Callaway, 2002). Exposure of morphism on exposure of tissues or Measuring metabolic disposition of cells to an isothiocyanate led to rapid the rates and extent of urinary excre- isothiocyanates accumulation of the compound tion of isothiocyanates and isothio- Studies on dietary intake of isothio- through conjugation with cellular thiols. cyanate conjugates. cyanates are still extremely limited, f3SH, the most abundant intracellular and the information below was thiol, is the major driving force for Bioavailability of isothiocyanates obtained from studies of a small num- isothiocyanate accumulation, and cel- from cru ciferous vegetables ber of volunteers. Ye et al. (2002) deter- lular GST enhances isothiocyanate In a study designed to compare the mined the metabolic disposition of accumulation by promoting conjuga- bioavai ability of isothiocyanate in isothiocyanates from broccoli sprouts tion reactions. Peak intracellular isoth- fresh and steamed broccoli, 12 men by giving four volunteers a single serv- iocyanate accumulation was achieved were asked to eat 200 g of fresh or ing of a myrosinase-treated extract of within 0.5-3 h after the beginning of steamed broccoli, and urine samples 3-day-old broccoli sprouts containing exposure to isothiocyanate, reaching were collected during the subsequent 200 pmol total isothiocyanate. The 100-200 times the extracellular isoth- 24 h. The total content of isothio- isothiocyanate composition of the iocyanate concentration. Intracell ularly cyanate in fresh and steamed broccoli preparation was 77.2% sulforaphane accumulated isothiocyanate and con- after treatment with myrosinase was or iberin and 22.8% iberin (aliphatic jugates were also rapidly exported by virtually identical (1.1 and 1.0 pmol/g isothiocyanates with closely related membrane transporters, including the wet weight); however, the average 24- chemical structures). The isothio- multidrug resistance-associated pro- h urinary excretion of isothiocyanate cyanates were absorbed rapidly and