Tobacco Documents Online

Txr Couxcir. FOR TOBACCO RrsraRcg-U.S.A., INC. MEiWR1ITIDLM TO: Dr. S.C. Scar¢ners and Staff FFdaM: D.H. Ford Septenber 17, 1984 RE: Site visit with Dr. Y. OsaO; Medical Foundation of Buffalo, NY, Sept. 13, 1984. Grant No. 1643 entitled "Aromatase inhibitors in cigarette smoke and tobaceo" OVERVIEW: Dr. Osawa is head of the Endocrine Biochemistry Dept. of a small privately funded research institute located adjacent to the Roswell Park Foundation, with which it maintains a working affiliation which.permits the use of the library facilities as well as to undertake collaborative investigations. Further, Dr. Osawa maintains an affiliation with the State University of N.Y. at Buffalo. This is essentially a basic research institute with a major interest in honnone investi- gations. Dr. Osawa shares a large laboratory facility which is well equipped for biochemical and endocrine research. He has recently been joined by a Dr. Tochigi from Japan. Dr. Tochigi is an expext on steroid metabolism and synthesis during pregnancy. His interest at the Foundation is directed toward understanding the effect of cigarette smoke constituents on placental enzyme activities. The orienta- tion of this group appears sanewhat crniparable to that of the Worcester Foundation. Dr. Osawa's current CTR-supported work is based on observations that a sub- stance which can be extracted from tobacco smoke concentrates,. cigarette tobacco or uncured tobacco leaves will inhibit the activity.of aromatase, an enzyme which converts adrogenic hormone to estrogen. In his in vitro test.hiodel system, Dr. Osawa uses hunan placental aromatase, which can Be obtained in large quantities. It is also a good test system, because it is similar to the rat ovarian aranatase, which he will be testing in-his in vivo studies, wherein he hopes to use the inhibitor to block or induce regression of estrogen-dependent breast cancer in Buffalo rats. SUTO1ARY OF RECENT RESSUI,TS : STUDIES WITH CIGARETiE SNY3KE: 1. Aqueous and organic extracts of smoke concentrates contained a material which inhibited the activity of placental aromatase (synthesis of estrogen from andrQ- gens) in a dose dependent manner. 50% inhibition was achieved by a 0.25 cigarette equivalent with the aqueous extract and a 0.07 cigarette equivalent with the organic extract. 2. The inhibition appeared to be campetitive rather than inhibitory. 3. A 15-25% difference in inhibition rate was observed with ccamexcial cigarettes. Menthol had no effect. He is now and will in the future be using reference cigarettes obtained from Kentucky. 4. Nicotine showed a 50% inhibition of aromatase only with very high con- centrations (7.5 mg., equivalent to the smoke from 7 cigarettes). Thus, nicotine is not considered to be a major armatase inhibitor. 5. The ether soluble basic fraction of smoke contained a major inhibitor; 36r g induced a 50% inhibition of aromatase. STUDIES WITH TOBACCO EXTRACTS: 1. Ethanol extracts of cnmiercial tobacco showed dose and time dependent 50% inactivation of homan placental aromatase (= to a 0.025 cigarette equivalent). NADPH was not needed as a co-factor for this activity. 2. The ether soluble fraction (possessed a characteristic tobacco arcma).. was the strongest aromatase inhibitor and was clearly dose dependent in its in- hibitory activity. 50% aromatase inactivation was achieved with a 0.08 cigarette equivalent.

3. Silica gel coltann chrcmatography of the acid fraction provided 4 peaks with antiarcznatase activity. 4. Green leaf (obtained fran Kentucky) contained an aranatase inhibitor in the ethanolic fraction, which possessed dose and time dependent activity. Osawa believes this is the first known case of there being a natural occurring aranatase inhibitor in plants. ORGANIC ACIDS: 93 organic acids have been identified as components of Turkish tobaceo leaves. They are mostly fatty acids, aranatic acids and terpinoid acids. To date, Dr. Osawa has assayed 8 aromatic and fatty acids for antiaranatase activity, but found none. Since the strongly acid fraction of tobacco leaves possessed potent inhibitory activity, Osawa will proceed to examine the terpinoid and other organic acids in tobacco leaf extracts. IN VIVO STUDIFS: These have already been initiated with female BUF/N rats, which have been injected with nitroscanethylurea (NMU), which causes an estrogen dependent breast cancer in this rat strain. He will be determining the incidence of breast cancer in untreated, NM[F-treated, and in NMU-treated-ovariectantzed rats. In other NMU-treated rats he will be determining to what degree the arcmatase inhibitor inhibits development of the estroQen dependent cancer. He also plans to deterntine the ef€ect of a6ainis=:xation o2" 6,8--brorioandros-cenedior_e, an anaarcma-c,ase which does not inhibit the estrous cycle (other natural arematase inhibitors which also block the estrous cycle will also be tested). QUESTIONS: 1. Does the armatase inhibitor in tobacco sictply inhibit the ovarian enzyme and thus estrogen synthesis, or does it also block the estrous cycle? The latter might imply an effect at the level of the CNS or pituitary. 2. Does the inhibitor also affect brain aramatase, which is believed to be some- what different from the ovarian or placental enzyme? 3. will the inhibitor work in vivo? 4. [Vhat is the biochemical nature of the inhibitor? 5. Can the inhibitor inhibit the production of estrogen dependent breast cancer, or cause its regression? CCNIIMENI: An exciting basic bio-molecular investigation of factors in smoke or whole tobacco which may interfere with a normal enzyme activity important in regu- lating steroid hormone biosynthesis. Since estrogen plays an important role in some cancers, in female reproduction and behavior, the results of the study could have a profound biologic significance. It should be most interesting to follow this study and observe its progress. At this time the investigation merits con- tinued CIR support in view of its potential significance in relation to breast cancer and female reproductive functions. D.H. Ford DHF:cf