Quantitation of Human Uptake of the Anticarcinogen Phenethyl Isothiocyanate After a Watercress Meaf1
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vol. 1, 383-388, July/August 1992 Cancer Epidemiology, Biomarkers & Prevention 383 Quantitation of Human Uptake of the Anticarcinogen Phenethyl Isothiocyanate after a Watercress MeaF1 F-L. Chung,2 M. A. Morse, K. I. Eklind, and J. Lewis humans (2). However, as stated in a neport by the Na- Division of Chemical Carcinogenesis, American Health Foundation, tional Academy of Sciences Committee on Diet, Nutni- Valhalla, New York 10595 [F-L. C., M. A. M., K. I. E.], and Department tion, and Cancer, #{176}becausethe indices of vitamin A intake of Food Molecular Biochemistry, AFRC Institute of Food Research, in these studies were derived from foods that also contain Norwich Research Park, Colney Lane, Norwich, NR4 7UA, England El. L.} other natural inhibitors of carcinogenesis, it is also pos- sible that dietary constituents other than preformed vi- tamin A or (3-carotene are relevant risk-reducing factors” Abstrad (2). Our previous studies showed that phenethyl A more recent population-based study of diet and isothiocyanate (PEITC), a cruciferous vegetable lung cancen suggested other ingredients in cruciferous constituent, inhibited the lung tumorigenesis induced vegetables appear to have a stronger inverse association by a potent tobacco-specific carcinogenic nitrosamine with risk than /9-carotene (3). Our previous studies in animals. These results implicate dietary PEITC as a showed that PEITC3 and indoles, both of crucifenous risk-reducing fador of lung cancers induced by origin, inhibited tobacco-specific NNK-induced lung can- smoking. To define the effed of dietary PEITC on cinogenesis in laboratory animals (4, 5). NNK is a nic- human cancers, a method of measuring its uptake is otine-denived nitrosamine which is believed to be in- needed. Since watercress is rich in gluconasturtiin, a volved in the development of lung cancer in smokers glucosinolate precursor of PEITC, it was chosen to be because of its carcinogenic potency and organ specificity the source of PEITC. Four individuals were asked to eat for the induction of lung tumors in laboratory animals (6, watercress as part of a breakfast meal, and 24-h urine 7). NNK-tneated rats fed diet containing PEITC developed samples were colleded. A urinary metabolite was significantly fewer lung tumors than NNK-treated rats fed found, and its identity was confirmed as the N- a control diet (4). More recently, we have demonstrated acetylcysteine conjugate of PEITC by comparison with that pretreatment with PEITC and other structurally ne- the synthetic standard using nuclear magnetic lated aromatic isothiocyanates effectively blocks the lung resonance and mass spedrometry. A dose-dependent tumorigenic activity of NNK in A/i mice (8, 9). The excretion of this conjugate was observed. These results anticarcinogenic potential of PEITC has been also dern- clearly showed that PEITC was released in the human onstnated in many other studies. Wattenbeng showed body upon ingestion of watercress and suggest that the that pretreatment with PEITC inhibited the mammary N-acetylcysteine conjugate of PEITC may be a useful and lung tumorigenesis of 7,1 2-dimethylbenz(a)- marker for quantitating human exposure to this anticarcinogen as a tool for epidemiological anthnacene (10). Feeding PEITC in the diet also inhibited investigations. the formation of esophageal tumors in rats treated with N-nitrosobenzylmethylamine (1 1). Therefore, it appeans that PEITC possesses a broad spectrum of anticarcino- Introdudion genic activity. Since some of the carcinogens examined Despite the well-established inverse relationship be- in these studies may be involved in the etiology of human tween vegetable consumption and the incidence of var- cancers, it is possible that dietary exposure to PEITC may ious cancers in humans, the exact nature of the ingredi- be beneficial to humans in the reduction of risk of certain ents which might be responsible for the protection is still cancers, such as tobacco-related lung cancer. To assess not well defined. Fibers found in many vegetables have the potential role of dietary PEITC in human cancers, a been identified as a risk-lowering factor for certain types method for measuring its uptake is needed. Watencness of cancer such as colon cancer (1). However, relatively is rich in gluconasturtlin, a glucosinolate of PEITC, which few experimental data regarding the protection against releases PEITC upon hydrolysis by myrosinase, an en- lung cancers by vegetable ingredients are available. Vi- zyme activated by chopping on chewing this vegetable. tamin A and its precursor, /3-carotene, have been identi- We chose to use watercress as a dietary source for PEITC. fied as constituents which may possibly contribute to the This study describes the development of an assay for the protective effects of vegetables against lung cancer in quantitation of dietary uptake of PEITC in humans after a watercness meal. Received 1/10/92. 1 Supported by Grant CA46535 from the National Cancer Institute. 2 To whom requests for reprints should be addressed, at Division of 3 The abbreviations used are: PEITC, phenethyl isothiocyanate; NNK, 4- Chemical Carcinogenesis, American Health Foundation, 1 Dana Road, (methylnitrosamino)-1-(3-pyridyl)-1 -butanone; HPLC, high-performance Valhalla, NY 10595. liquid chromatography. Downloaded from cebp.aacrjournals.org on October 3, 2021. © 1992 American Association for Cancer Research. 384 Marker for Uptake of Anticarcinogen Phenethyl lsothiocyanate Materials and Methods sS PEITC was purchased from Aldrich Chemical Co. (Mil- HH C-S “‘““C’s waukee, WI). The conjugates of PEITC were prepared as previously described (12). Gluconasturtiin was synthe- sized by a published method (13). Myrosinase was iso- :1 HO COOH lated from mustard seeds according to a published pro- cedure (14). Watercress was obtained from a local gro- cery store. HH S HH Mouse Study. Seven-week-old female A/i mice, three in each group, were fed AIN-76 diet containing gluconas- O-”I\ C-.C-NH-C-S-C-C-COOHII ii - I’ll” I’ll turtiin (10 or 50 zmol/g diet) and supplemented with 2 NH--CH3 myrosinase (5 or 25 mg/g diet, respectively). After 24 h 0 on this diet, mice were fed AIN-76 diet for an additional 48 h. Seventy-two-h urine samples were collected at 24- h intervals during the experiment and stored in a dry ice Control Urine bath until analysis of PEITC metabolites. The collected urine samples were processed by adding an equal vol- ume of methanol to the pooled urine of each group to precipitate solids. Filtered samples were concentrated in vacuo to obtain final samples for the analysis by reverse- phase HPLC as described previously (12). Human Study. Four volunteers (two males and two fe- males) ingested 57 g (Experiment 1) or 30 g (Experiment 2) of watercress at a breakfast with croissants on bagels = and cream cheese. All participants were told to avoid crucifenous vegetables and mustard in the diet during the 0 experiment. For the control diet, all participants ate the ‘4 same food except watercress 2 days prior to the expeni- .0 mental diet. Subject 3 of Experiment 1 did not participate in Experiment 2 and was replaced by another volunteer of the same sex. Urine samples were collected at 2, 4, 8, and 24 h after breakfast and were stored frozen until analysis. Upon thawing, samples were adjusted to pH 3 with phosphoric acid. Three 2-mI aliquots of each urine sample were placed in 7-mI vials. Two g of ammonium sulfate and 3 ml of ethyl acetate were added to each vial. 1 Each vial was vortexed for 20 s and centrifuged to sepa- nate the ethyl acetate layer. This extraction was carried out twice. The extract was reconstituted in 0.5 ml of 2 HPLC mobile phase and analyzed by reverse-phase HPLC as described previously (12). This method resulted in quantitative recovery of the N-acetylcysteine conju- 10 20 30 40 50 60 gate of PEITC. A significant amount of N-acetylcysteine Time (mm) conjugate was excreted in the urine; therefore, it was isolated in sufficient quantity by repetitive HPLC runs. Fig. 1. Reverse-phase HPL.C chromatogram obtained from analysis of The identity of this urinary metabolite as the N-acetyl- urine from mice fed diet containing gluconasturtiin and myrosinase. Peaks cysteine of PEITC was confirmed by comparing the 360 1 and 2 are identical to those found in the urine of mice treated with PEITC (12), and their structures are shown. MHz proton nuclear magnetic resonance and mass spec- tra with those obtained from the synthetic standard. Analysis of Gluconasturtiin Content of Watercress. The watercress was freeze-dried in New York; approximately pynidinium acetate solution, the column (containing the 300 g were sealed in a polyethylene bag containing CaCI2 intact glucosinolates) was treated with sulfatase, and the and sent to Norwich. The analysis of gluconasturtiin desulphoglucosinolates were eluted with water. HPLC content of th freeze-dried watercress was performed analysis was carried out using the method of Spinks et following a published method of Heaney and Fenwick al. (16). (1 5). Briefly, the freeze-dried watercress was ground to a fine powder and extracted for 1 5 mm in 100 ml 70% methanol to inactivate myrosinase. Two ml (in duplicate) Results of the extract was added to a solution of glucotropaeolin As a prelude to the human study, we first examined (internal standard), and 100 l of 1:1 (v/v) mixture of whether A/i mice fed gluconasturtimn diet supplemented barium acetate (0.5 M) and lead acetate (0.5 M) in solution with myrosinase would excrete these metabolites in the was added. The mixture was centrifuged, and the super- urine. As shown in Fig. 1, two major metabolites, identical natant was applied to a microcolumn of DEAE Sephadex to the conjugates extracted in the urine of mice treated A25 (15).