Sulforaphane (SFN), a compound found at high levels in broccoli and broccoli sprouts, is a potent inducer of phase 2 detoxification enzymes and inhibits tumorigenesis in animal models. SFN also has a marked effect on cell cycle checkpoint controls and cell survival and/or apoptosis in various cancer cells, through mechanisms that are poorly understood. We tested the hypothesis that SFN acts as an inhibitor of histone deacetylase (HDAC). In human embryonic kidney 293 cells, SFN dose-dependently increased the activity of a beta-catenin-responsive reporter (TOPflash), without altering beta-catenin or HDAC protein levels. Cytoplasmic and nuclear extracts from these cells had diminished HDAC activity, and both global and localized histone acetylation was increased, compared with untreated controls. Studies with SFN and with media from SFN-treated cells indicated that the parent compound was not responsible for the inhibition of HDAC, and this was confirmed using an inhibitor of glutathione S-transferase, which blocked the first step in the metabolism of SFN, via the mercapturic acid pathway. Whereas SFN and its glutathione conjugate (SFN-GSH) had little or no effect, the two major metabolites SFN-cysteine and SFN-N-acetylcysteine were effective HDAC inhibitors in vitro. Finally, several of these findings were recapitulated in HCT116 human colorectal cancer cells: SFN dose-dependently increased TOPflash reporter activity and inhibited HDAC activity, there was an increase in acetylated histones and in p21(Cip1/Waf1), and chromatin immunoprecipitation assays revealed an increase in acetylated histones bound to the P21 promoter. Collectively, these findings suggest that SFN may be effective as a tumor-suppressing agent and as a chemotherapeutic agent, alone or in combination with other HDAC inhibitors currently undergoing clinical trials.

Isothiocyanates (ITCs) are a group of naturally occurring compounds that occur as thioglucoside conjugates, termed glucosinolates, in plants and cruciferous vegetables such as watercress, Brussels sprouts, broccoli, cabbage, kai choi, kale, horseradish, radish and turnip. ITCs inhibit the development of tumors in many of the experimental models investigated, and are being investigated as possible chemopreventive agents for specific human cancers. The goal of this review is to provide a mechanistic understanding for the biological activities of ITCs and to relate the metabolism of ITCs to their action as chemopreventive agents. In vivo animal studies have been conducted to address issues of tissue disposition, pharmacokinetics, and metabolism of ITCs. Methods for analysis of ITCs and their metabolites in urine and plasma have been developed. The metabolism of several naturally occurring ITCs as constituents of foodstuffs or as drugs has also been investigated in human studies. Finally, based on recent epidemiological studies, the role of dietary consumption of vegetables containing ITCs in prevention of human cancers and human cancer susceptibility is discussed.

The cancer-chemopreventive effects of broccoli may be attributed, in part, to isothiocyanates (ITCs), hydrolysis products of glucosinolates. Glucosinolates are hydrolyzed to their respective ITCs by the enzyme myrosinase, which is inactivated by heat. In this study, the metabolic fate of glucosinolates after ingestion of steamed and fresh broccoli was compared in 12 male subjects in a crossover design. During each 48-hour baseline period, no foods containing glucosinolates or ITCs were allowed. The subjects then consumed 200 g of fresh or steamed broccoli; all other dietary sources of ITCs were excluded. Blood and urine samples were collected during the 24-hour period after broccoli consumption. Total ITC equivalents in broccoli and total ITC equivalents in plasma and urine were assayed by high-performance liquid chromatography as the cyclocondensation product of 1,2-benzenedithiol. The content of ITCs in fresh and steamed broccoli after myrosinase treatment was found to be virtually identical (1.1 vs. 1.0 micromol/g wet wt). The average 24-hour urinary excretion of ITC equivalents amounted to 32.3 +/- 12.7% and 10.2 +/- 5.9% of the amounts ingested for fresh and steamed broccoli, respectively. Approximately 40% of total ITC equivalents in urine, 25.8 +/- 13.9 and 6.9 +/- 2.5 micromol for fresh and steamed broccoli, respectively, occurred as the N-acetyl-L-cysteine conjugate of sulforaphane (SFN-NAC). Total ITC metabolites in plasma peaked between 0 and 8 hours, whereas urinary excretion of total ITC equivalents and SFN-NAC occurred primarily between 2 and 12 hours. Results of this study indicate that the bioavailability of ITCs from fresh broccoli is approximately three times greater than that from cooked broccoli, in which myrosinase is inactivated. Considering the cancer-chemopreventive potential of ITCs, cooking broccoli may markedly reduce its beneficial effects on health.

Exocyclic adducts are unique DNA modifications resulting from bindin at two sites of bases that normally are involved in hydrogen-boning for maintaining the doublehelical structure of DNA. These adducts have been shown to be formed in rodents upon exposure to carcinogens. Using a sensitive 32P-postlabeling method combined with high performance liquid chromatography, we obtained evidence that 1,N2-propanodeoxyguanosine adducts of acrolein (AdG) and crotonaldehyde (CdG) are present in the liver DNA of humans and rodents without carcinogen treatment. (22). In this study, we used this method to show that AdG and CdG (Fig. 1) are present in the liver DNA of rodents and humans and that the in vivo formation of these adducts appears to be stereoselective. MATERIALS AND METHODS DNA Isolation and Hydrolysis and Collection of AdductFractions by HPLC. Male A/J mice (25-30 g) and Fischer rats (200-230 g) were purchased from Charles River Breeding Laboratories and housed in polycarbonate cages (250C ± 2TC, 50 ± 10% relative humidity, and 12-hr light/dark cycle). These animals were fed the modified AIN-76A diet and tap water ad libitum. After 3 weeks of acclimatization, all animals were sacrificed. Livers were quickly removed, minced, and frozen at -800C until DNA isolation. DNA was isolated by a modified Marmur's procedure (23). DNA samples were stored at -800C until analysis.DNA (105-300 pg) was obtained from livers ofeach offour mice and four rats and from each of five humans (two males of ages 37 and 94 years; three females of ages ranging from 50 to 60 years). Human liver DNA was isolated from autopsy samples and was provided by Regina Santella (Columbia University). The DNA was enzymatically hydrolyzed to 3'-monophosphates (22). The enzyme digest was filtered through a 0.2-gm Acrodisc Mini Spike syringe filter (Gelman) and was analyzed by HPLC system 1. Fractions corresponding to AdG 3'-monophosphate and CdG 3'-monophosphate were collected according to the retention times of the synthetic standards (22). To ensure that the assay was free of contamination, blank samples were obtained after injecting 300 p4 of water prior to the collection of fractions of DNA hydrolysates from each species. The fractions from the Abbreviations: enals, a,jf-unsaturated aldehydes; AdG, acroleinderived 1,N2-propanodeoxyguanosine; CdG, crotonaldehydederived 1,N2-propanodeoxyguanosine.*To whom reprint requests should be addressed. 7491The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

A number of promutagenic exocyclic DNA adducts have recently been detected in both humans and rodents without carcinogen treatment. These observations raised questions about their origins and potential significance in carcinogenesis. In this commentary, we present our views pertaining to the in vivo sources of these cyclic adducts, specifically the cyclic propano and etheno adducts. The basis for our discussion comes mainly from the information generated through a span of more than a decade from several laboratories, including ours. This commentary summarizes the data from the chemical and biochemical studies that provide support for the hypothesis that lipid peroxidation is involved in the endogenous formation of these exocyclic adducts.