Chemoprotective glucosinolates and isothiocyanates of broccoli sprouts: metabolism and excretion in humans.

Broccoli sprouts are a rich source of glucosinolates and isothiocyanates that induce phase 2 detoxication enzymes, boost antioxidant status, and protect animals against chemically induced cancer. Glucosinolates are hydrolyzed by myrosinase (an enzyme found in plants and bowel microflora) to form isothiocyanates. In vivo, isothiocyanates are conjugated with glutathione and then sequentially metabolized to mercapturic acids. These metabolites are collectively designated dithiocarbamates. We studied the disposition of broccoli sprout glucosinolates and isothiocyanates in healthy volunteers. Broccoli sprouts were grown, processed, and analyzed for (a) inducer potency; (b) glucosinolate and isothiocyanate concentrations; (c) glucosinolate profiles; and (d) myrosinase activity. Dosing preparations included uncooked fresh sprouts (with active myrosinase) as well as homogenates of boiled sprouts that were devoid of myrosinase activity and contained either glucosinolates only or isothiocyanates only. In a crossover study, urinary dithiocarbamate excretion increased sharply after administration of broccoli sprout glucosinolates or isothiocyanates. Cumulative excretion of dithiocarbamates following 111-micromol doses of isothiocyanates was greater than that after glucosinolates (88.9 +/- 5.5 and 13.1 +/- 1.9 micromol, respectively; P < 0.0003). In subjects fed four repeated 50-micromol doses of isothiocyanates, the intra- and intersubject variation in dithiocarbamate excretion was very small (coefficient of variation, 9%), and after escalating doses, excretion was linear over a 25- to 200-micromol dose range. Dithiocarbamate excretion was higher when intact sprouts were chewed thoroughly rather than swallowed whole (42.4 +/- 7.5 and 28.8 +/- 2.6 micromol; P = 0.049). These studies indicate that isothiocyanates are about six times more bioavailable than glucosinolates, which must first be hydrolyzed. Thorough chewing of fresh sprouts exposes the glucosinolates to plant myrosinase and significantly increases dithiocarbamate excretion. These findings will assist in the design of dosing regimens for clinical studies of broccoli sprout efficacy.

Human metabolism and excretion of cancer chemoprotective glucosinolates and isothiocyanates of cruciferous vegetables.

Isothiocyanates and their naturally occurring glucosinolate precursors are widely consumed as part of a diet rich in cruciferous vegetables. When plant cells are damaged, glucosinolates are released and converted to isothiocyanates by the enzyme myrosinase. Many isothiocyanates inhibit the neoplastic effects of various carcinogens at a number of organ sites. Consequently, these agents are attracting attention as potential chemoprotectors against cancer. As a prerequisite to understanding the mechanism of the protective effects of these compounds, which is thought to involve the modulation of carcinogen metabolism by the induction of phase 2 detoxication enzymes and the inhibition of phase 1 carcinogen-activating enzymes, we examined the fate of ingested isothiocyanates and glucosinolates in humans. Recently developed novel methods for quantifying isothiocyanates (and glucosinolates after their quantitative conversion to isothiocyanates by purified myrosinase) and their urinary metabolites (largely dithiocarbamates) have made possible a detailed examination of the fates of isothiocyanates and glucosinolates of dietary crucifers. In a series of studies in normal volunteers, we made these findings. First, in nonsmokers, urinary dithiocarbamates were detected only after the consumption of cruciferous vegetables and condiments rich in isothiocyanates and/or glucosinolates. In sharp contrast, the consumption of noncrucifers (corn, tomatoes, green beans, and carrots) did not lead to the excretion of dithiocarbamates. Moreover, the quantities of dithiocarbamates excreted were related to the glucosinolate/isothiocyanate profiles of the cruciferous vegetables administered (kale, broccoli, green cabbage, and turnip roots). Second, eating prepared horseradish containing graded doses of isothiocyanates (12.3-74 micromol; mostly allyl isothiocyanate) led to a rapid excretion of proportionate amounts (42-44%) of urinary dithiocarbamates with first-order kinetics. The ingestion of broccoli in which myrosinase had been heat-inactivated also led to proportionate but low (10-20%) recoveries of urinary dithiocarbamates. Broccoli samples subsequently treated with myrosinase to produce the cognate isothiocyanates were much more completely (47%) converted to dithiocarbamates. Finally, when bowel microflora were reduced by mechanical cleansing and antibiotics, the conversion of glucosinolates became negligible. These results establish that humans convert substantial amounts of isothiocyanates and glucosinolates to urinary dithiocarbamates that can be easily quantified, thus paving the way for meaningful studies of phase 2 enzyme induction in humans.

Quantitative determination of isothiocyanates, dithiocarbamates, carbon disulfide, and related thiocarbonyl compounds by cyclocondensation with 1,2-benzenedithiol.

A recently developed UV spectroscopic method for quantitating isothiocyanates (R-N=C=S) at the nanomole level is based on the observation that the highly electrophilic central carbon atom of the -N=C=S group can undergo successive nucleophilic additions with reagents containing two sulfhydryl groups on adjacent carbon atoms to form a cyclic thiocarbonyl product and release the nitrogen atom as a primary amine (Y. Zhang, C.-G. Cho, G. H. Posner, and P. Talalay, Anal. Biochem. 205, 100-107, 1992). The assay utilizes 1, 2-benzenedithiol as the vicinal dithiol reagent and measures the reaction product, 1,3-benzodithiole-2-thione, spectroscopically (am of 23,000 M-1 cm-1 at lambdamax = 365 nm). This paper reports a dramatic improvement in the analytical sensitivity of this method. By separating the cyclocondensation product by a simple isocratic HPLC method and using an automatic integrator, the sensitivity of detection has been lowered to a few picomoles of isothiocyanate. Furthermore, we now find that the chemical specificity of the cyclocondensation reaction is not restricted to isothiocyanates, but includes dithiocarbamates, and related thiocarbonyl compounds such as carbon disulfide, certain substituted thiourea derivatives, and xanthates. The availability of such analytical methods is important not only because isothiocyanates (and their glucosinolate precursors) are present in edible plants and are consumed by humans in substantial quantities, but also because some dithiocarbamates are toxic, and are widely used in the rubber industry as vulcanization accelerators and in agriculture as fungicides, insecticides, and herbicides. The analysis of many isothiocyanates is complicated by their extreme volatility. This difficulty can be circumvented by converting isothiocyanates quantitatively into dithiocarbamates (by facile addition of a mercaptan such as N-acetylcysteine) and quantitating the nonvolatile dithiocarbamate by the cyclocondensation reaction.

Mercurials and dimercaptans: synergism in the induction of chemoprotective enzymes.

The induction of NAD(P)H:quinone reductase (EC 1.6.99.2; QR) in Hepa 1c1c7 murine hepatoma cells provides a versatile quantitative model for measuring the potencies of inducers for Phase 2 detoxication enzymes. Since many inducers of these enzymes also protect animals and their cells against the toxic and neoplastic effects of carcinogens, understanding the mechanisms of induction of Phase 2 enzymes is important. Both HgCl2 and 2,3-dimercaptopropanol (BAL) are inducers of QR in these cells, and paradoxically BAL (which is about 30 times less potent than HgCl2) enhances the inducer potency of HgCl2 substantially. This synergism depends on the presence of two thiol groups on adjacent carbon atoms. Since nonchelated mercury(II)-thiol compounds did not show synergism, the formation of very high affinity bidentate chelates appears to be essential for such synergism. A major mechanism for the augmentation of the inducer potency of mercury(II) by BAL is the more rapid cellular uptake and the accumulation of higher intracellular concentrations of mercury. It is also possible that BAL-mercury chelates are intrinsically more potent as inducers. Although equimolar mixtures of BAL and HgCl2 and the synthetic chelate isolated from such mixtures were more potent inducers than HgCl2 alone, the presence of excess BAL increased this inducer synergism even further. By chromatography we showed the reversible formation of higher order complexes between BAL and mercury(II). Such complexes are transported into cells more efficiently and appear to be more potent than free HgCl2 or the chelate obtained from equimolar mixtures of BAL and HgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)