Enhancement of Glutathione S-Transferase Activity of the Esophagus by Phenols, Lactones, and Benzyl Isothiocyanate1

The effects of feeding p-methoxyphenol, benzyl isothiocyanate, coumarin, a-angelicalactone, 2-fert-butyl-4-hydroxyanisole, and 3-ferf-butyl-4-hydroxyanisole on the glutathione Stransferase activity and sulfhydryl levels of esophagus and small bowel mucosa of ICR/Ha mice have been investigated. p-Methoxyphenol, benzyl isothiocyanate, coumarin, and 2-tertbutyl-4-hydroxyanisole increased glutathione S-transferase ac tivity of the esophagus by 68 to 135%, a-angelicalactone was less effective, and 3-fert-butyl-4-hydroxyanisole had only a small enhancing capacity. All six compounds increased the sulfhydryl levels of the esophagus. The ranking order and magnitude of the enhancing effects of the six compounds on glutathione S-transferase activity are similar for esophagus and forestomach (previously published) but differ from that of the small bowel mucosa. Since esophageal cancer is an important cause of cancer deaths in many parts of the world, information as to factors which can enhance protective systems of this organ may be of value. INTRODUCTION GSH3 S-transferase has been studied extensively as a major detoxification system that catalyzes the reaction of a wide variety of electrophiles to GSH (2, 8). Since the reactive ulti mate carcinogenic forms of chemical carcinogens are electro philes, GSH S-transferase takes on considerable importance as a mechanism for carcinogen detoxification. Enhancement of the activity of this system potentially could increase the capacity of the organism to withstand the neoplastic effects of chemical carcinogens (1, 16). Experiments have been carried out aimed at determining if there is a relationship between increased GSH S-transferase activity in a target organ of chem ical carcinogenesis and response to the carcinogen. For this purpose, the forestomach of the mouse was used. Members of several classes of inhibitors of BP-induced neoplasia of the mouse forestomach were studied for their effects on the GSH S-transferase activity in that structure. Five of the compounds increased the GSH S-transferase activity of the forestomach 78 to 182%. The 5 compounds were p-methoxyphenol, 2-BHA, coumarin, a-angelicalactone, and benzyl isothiocyanate. All 5 inhibited BP-induced neoplasia of the forestomach. These data 1 Supported by USPHS Grants CA-14146 and CA-09599 from the National Cancer Institute. 2 Present address: Institute of Cancer, Chinese Academy of Medical Sciences, Beijing, People's Republic of China. 3 The abbreviations used are: GSH, reduced glutathione: BP, benzo(a)pyrerie; BHA, fert-butyl-4-hydroxyanisole; 3-BHA, 3-fert-butyl-4-hydroxyanisole [the ma jor isomer occurring in the commercial mixture of 2(3)-(erf-butyl-4-hydroxyanisole]; 2-BHA, 2-fert-butyl-4-hydroxyanisole (the minor isomer found in the com mercial mixture). Received July 27, 1981 ; accepted December 15, 1981. indicate that enhancement of the GSH S-transferase activity by about 75% or greater is associated with a reduced carcino genic response of the forestomach to BP (14). The data also suggest that the capacity to enhance GSH S-transferase activ ity might be used as a method of identifying compounds or natural products likely to inhibit BP or other carcinogens de toxified in a similar manner (18). The forestomach of the mouse is lined by a stratified squamous epithelium without other structures, such as glands, being present. The epithelium is similar to that of the esopha gus. However, their milieu is not the same. In addition, epithelia with similar morphology do not necessarily have the same biochemical composition. The esophagus is a particularly in teresting organ in that in several areas of the world, including parts of China, esophageal cancer is a leading cause of cancer deaths (3, 10, 13, 15, 19). In the present investigation, the effects on esophageal GSH S-transferase activity and acidsoluble sulfhydryl levels of compounds previously shown to cause increases in both of these entities in the forestomach have been studied. Parallel studies of inhibition of carcinogen esis have not as yet been undertaken. MATERIALS AND METHODS Animal Experiments. Female ICR/Ha mice from the Madison, Wis., facility (HarÃ-an Sprague-Dawley, Indianapolis, Ind.) were used in all experiments. Mice were randomized by weight at 7 weeks of age. At that time, they were divided into groups of 5 animals each and placed on experimental diets. Two basic diets were used. One was a semipurified diet consisting of 27% vitamin-free casein, 59% starch, 10% corn oil, 4% salt mix (U.S.P. XIV), and a complete mixture of vitamins (Teklad Inc., Madison, Wis.). The other was powdered Purina rat chow (Ralston Purina Company, St. Louis, Mo.). To these diets were added the test compounds. The diets were fed for 9 days, at which time the experiment was terminated. Mice were killed at 9 a.m. The esophagus and proximal half of the small bowel were removed. The small bowel was opened, washed, and the mucosa was scraped off gently. In some experiments, the test compounds dissolved in cottonseed oil were given by P.O. intubation once a day for 3 days, the last administration being 24 hr prior to killing the mice. Preparation of Homogenates and Cytosol. All steps were done at 0-4°. The tissues to be studied were homogenized in 0.1 M phosphate buffer (pH 7.5). The homogenate was centrifuged at 100,000 x g for 1 hr. The supernatant was used for the assay of cytosolic GSH Stransferase activity. Determinations of GSH S-Transferase Activity and Acid-soluble Sulfhydryl Levels. The activity of cytosol GSH S-transferase was determined spectrophotometrically at 30°with 1-chloro-2,4-dinitrobenzene as substrate, according to the procedure of Habig et al. (7). The reaction mixture (1 ml) contained 100 jumol phosphate buffer (pH 6.5), 5 fimol GSH, and 1 ftmol 1-chloro-2,4-dinitrobenzene. The reaction was started by addition of cytosol. There are multiple GSH S-transferases that have been identified in the cytosol of tissues. However, the esophagus has not been investigated in this regard. The known multiple forms of GSH S-transferase have low substrate specificities which