Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
ReviewCancer-preventive isothiocyanates: measurement of human exposure and mechanism of action
Introduction
Isothiocyanates (ITCs) are a family of compounds derived almost exclusively from plants, although marine sponges and fungi also have been reported to produce a few ITCs [1]. They are synthesized and stored as glucosinolates (β-thioglucoside N-hydroxysulfates, GS) in plants and are released when damage to plant tissue occurs. The conversion is catalyzed by myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1), an enzyme that coexists with but is physically separated from GS in normal plants (Fig. 1). However, it is also known that under certain conditions hydrolysis of GS may lead to non-ITC products, including thiocyanates, nitriles, epithionitriles, indoles, and oxazolidine-2-thiones. Extensive information on myrosinase, GS hydrolysis, and product formation is available elsewhere [1], [2], [3], [4], [5], [6]. Moreover, GS that escape the plant myrosinase may be hydrolyzed in the intestinal tract, as the microflora are known to possess myrosinase activity [6], [7], [8]. Although at least 120 different GS have been identified in various plants [1], the majority of which likely produce ITCs, only a small number of ITCs may be commonly consumed by humans. Cruciferous vegetables are the principle dietary source of ITCs, but the types of crucifers frequently consumed by humans are limited. Examples of popular crucifers that are particularly rich in certain ITCs include mustard and horseradish—allyl-ITC (AITC) [9], watercress—phenethyl-ITC (PEITC) [10], dikon—dehydroerucin [1], [11], and broccoli and broccoli sprouts—sulforaphane (SF) [12], [13] (see Fig. 2 for chemical structure).
To date, the most important known biological activity of ITCs is their ability to inhibit cancer development. There is convincing evidence that certain natural ITCs, such as AITC, benzyl ITC (BITC), PEITC and SF, as well as a number of synthetic analogs, are effective inhibitors of chemically induced tumors in one or more organ sites of rodents, including the bladder, colon, esophagus, mammary glands, pancreas, and stomach. Numerous studies have been performed in this area, and many comprehensive review articles have been written [14], [15], [16], [17], [18]. Moreover, in agreement with the rodent results, several recent epidemiological studies already show that dietary intake of ITCs is inversely correlated with cancer risk of several organ sites, as discussed in Section 4. These findings have generated much enthusiasm about ITCs as potential cancer-preventive agents in humans. Much of the molecular basis of the cancer-preventive activity of these compounds has been learned, as described in Section 5, which indicates that ITCs not only inhibit the development of cancer cells, but also eliminate established cancer cells. For example, both AITC and SF significantly inhibited the growth of PC-3 human prostate cancer cell xenografts in mice [19], [20].
All ITCs are characterized by the presence of an NCS group, whose central carbon often is highly electrophilic. The biological activities of ITCs, perhaps their toxic effects as well, may be primarily mediated through the reaction of this carbon atom with cellular nucleophilic targets. In contrast, GS are not electrophilic, and there is no clear evidence that intact GS are cancer-preventive. It is believed that the side chains of ITCs may play secondary roles, e.g., affecting the electrophilicity of the NCS group, altering the steric hindrance to the reactive carbon atom, and controlling the lipophilicity of the molecule. The NCS group also governs the metabolism of ITCs. ITCs are metabolized in vivo principally by the mercapturic acid pathway: an initial conjugation through the NCS group with glutathione (GSH), which takes place spontaneously but is further promoted by glutathione transferases (GST) [21], [22], [23], gives rise to the corresponding conjugates. The GSH conjugates then undergo further enzymatic modifications (modifications of the GSH portion) to form sequentially the cysteinylglycine-, cysteine-, and N-acetylcysteine (NAC)-conjugates, which are excreted in urine [24] (Fig. 3).
Section snippets
The cyclocondensation assay
Recent development of a highly sensitive and quantitative method, namely the cyclocondensation assay, for measuring ITCs and their mercapturic acid pathway metabolites [25] (Fig. 4), has provided a valuable tool to better understand human consumption of dietary ITCs, their metabolism and disposal in vivo, and cell and tissue exposure to these compounds. This assay was developed based on the discovery of the almost universal ability of the central carbon of ITCs to undergo successive
Measuring human uptake of dietary ITCs
In agreement with the observation that ITCs are readily absorbed, principally metabolized in vivo through the mercapturic acid pathway, and excreted in urine as NAC–ITCs, many studies have shown that dietary ITC intake can be measured by detection of its NAC conjugate in the urine. For example, when BITC was administered orally to humans, 54% of the dose (14.4 mg) was recovered in the urine as NAC–BITC. NAC–BITC was excreted rapidly; maximum excretion occurred 2–6 h after and was essentially
Evidence of cancer-preventive activity of ITCs in humans
A few studies on the relationship between exposure to ITCs and human cancer risk have been reported, although there have been no studies on the effect of dietary ITCs on human bladder cancer.
London et al. [45] conducted a case–control study in which a cohort of 18,244 men in Shanghai, China was followed from 1986 to 1997. Each participant provided a single-void urine sample at the start of the study. Of the 232 individuals that subsequently developed lung cancer and 710 matched controls, 89.7
The molecular basis of cancer-preventive effects of ITCs
It has been well established that ITCs can inhibit cancer development through multiple mechanisms, including: (i) protecting DNA by modulating carcinogen-metabolizing enzymes; (ii) reducing oxidative stress by elevating and maintaining cellular antioxidants; (iii) inhibiting cell proliferation, thereby retarding or eliminating clonal expansion of initiated, transformed, and/or neoplastic cells. Other effects, including anti-inflammation, anti-infection, and perhaps induction of differentiation
Concluding remarks
ITCs, many of which are commonly present in the human diet and are derived from cruciferous vegetables, are highly promising cancer-preventive agents. Analytical methods have been developed to allow assessment of human exposure to dietary ITCs, and to understand the bioavailability of these compounds from dietary sources. There is epidemiological evidence that dietary ITCs may indeed provide protection against human cancers. ITCs possess multi-faceted cancer-preventive mechanisms, capable of
Acknowledgement
I would like to thank my coworkers Jun Li, Joseph D. Paonessa, and Li Tang for critical reading of this manuscript. This work was supported in part by National Cancer Institute grants CA 80962 and CA100623.
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