1)MASAKAZU HATANAKA, M.D., PH.D.,
National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205
Host Institution:
Professor Tsutomu Sugahara, Faculty of Medicine, Kyoto University, Kyoto, Japan Dates of Visit: September 10 to October 20, 1979
Summary of Activities:
Indian muntjac (Muntjacus muntjak) cells have a diploid chromosome number 6 in the female and 7 in the male, the lowest number yet described in a mammal, and have maintained diploidy after many passages in culture. We have successfully transformed the cells by a xenotropic murine sarcoma virus, 43-2 XV, and isolated morphologically transformed permanent lines that retain a stable diploid chromosome number (M. Hatanaka and R. Klein, “Tumorigenicity of Indian Muntjac Diploid Cells by the Proviral Integration of Sarcoma Gene of a Mouse Retrovirus” Journal of Experimental Medicine, 150: 1195-1201, 1979). These diploid clones of Indian muntjac permanent cells have physical, numerical and cytogenetic advantages to study and score systematically chromosomal aberrations (CA) and sister chromatid exchanges (SCE) induced by environmental mutagens and carcinogens.
Professor Sugahara in the Department of Experimental Radiology, Faculty of Medicine, Kyoto University, Kyoto, Japan has been interested in using our permanent lines of Indian muntjac for studies of carcinogenesis and mutagenesis. Thus, I worked with the staff of Dr. Sugahara using his facilities during my stay in Japan under the auspices of the U.S.-Japan Cooperative Cancer Research Program from September 10 to October 21, 1979.
Six alkylating agents, four N-nitroso-and two methane-sulfonate compounds were tested for the induction of chromosomal aberrations (CA) and sister chromatid exchanges on one of our clonal isolates, 12-1-58. The magnitudes of detecting sensitivity of CA and SCE on the Indian muntjac chromosomes after mutagenic exposures were found to be similar, implying the greater advantages by use of Indian muntjac cells for the screening of mutagens and carcinogens compared to other mammalian cells as an indicator. Scoring CA in the large and easily identifiable 6 chromosomes of Indian muntjac requires a minimal degree of skillfulness. These features may be exploitable in the standardization of CA screening and useful to regulatory institutions and hospitals. With certain carcinogens that fail to induce SCE, such as X-ray, we were able to rapidly and quantitatively detect CA of Indian muntjac chromosomes. These effects were much easier to detect than in other mammalian chromosomes.
Some of the results will be published in the Journal of Mutation Research by Peter Raff and Tsutomu Sugahara entitled “Sister Chromatid Exchanges, Chromosomal Aberrations and Cytotoxicity in Cultured Indian muntjac cells treated with Alkylating Agents.” We have also investigated chromosome changes in 60 clonal isolates of Indian muntjac, collaboration with Dr. Masao Sasaki. In preliminary studies we found two unusual translocations in two clonal isolates of Indian muntjac; (X/X, 3/3) in 12-1-60 T clone and (1p/X, 1q/3) in 12-(1)-01 clones. Both clones produce highly malignant tumors in nude mice (M. Hatanaka and R. Klein, Journal of Experimental Medicine, 150, 1195-1201, 1979). These unique translocations have never been found in the parental cells. The results reported here have been obtained under the auspices of the U.S.-Japan Cooperative Cancer Program during my stay in Japan. These informations may provide a useful basis for establishing a new sensitive method for CA screening of mutagens and carcinogens, and new insights for studies on chromosomal organization and chromosomal rearrangements.
2) NEIL ELLIOT SPINGARN,
American Health Foundation, Valhalla, New York 10595
Host Institution:
Dr. Takashi Sugimura, National Cancer Center Research Institute, Tokyo, Japan
Dates of Visit: October 3, 1979-December 13, 1979
Summary of Activities:
The purpose of my visit to Japan was to collaborate with Drs. Sugimura and Matsushima on isolation and identification of the principal mutagens from cooked beef. This is part of an ongoing project investigating the health effects of food cooking procedures and their postulated role in the etiology of human cancer, particularly colonic cancer. We succeeded in purifying and partially identifying one of the two mutagens present, and partially purified the second mutagen. A paper describing this work is currently in press. [Spingarn, N.E. et al, Cancer Letters, 9, 177-183 (1980). “Formation of Mutagens in Cooked Foods. III: Isolation of a Potent Mutagen from Beef.”]
This project was successful in achieving its objectives since by combining our individual techniques, both laboratories (ours and theirs) can now purify mutagens from food more effectively. A major step forward has resulted from isolation of this mutagen from beef. We plan to continue our very fruitful collaboration in the pursuit of mutagens/carcinogens in food. Visits to or from Japan may be desirable at a later date to continue this collaboration.
3) LYNNE A. HAROUN, B.A., M.P.H.,
Department of Biochemistry University of California, Berkeley, California
Host Institution:
Dr. Takashi Sugimura, Director, National Cancer Center Research Institute, Tokyo, Japan
Dates of Visit: November 1, 1979 to January 31, 1980
Summary of Activities:
a)The Testing of Polychlorinated Hydrocarbons in the Salmonella Assay: Activation by House Fly S9
Almost every member of the class of multiply chlorinated compounds that has been tested in experimental animals has been shown to be a carcinogen. This important class of industrial and agricultural compounds includes such widely used chemicals as the solvents, carbon tetrachloride and chloroform; and the pesticides, aldrin and DDT. However, most of these compounds are not detected as mutagens in the Salmonella/Ames test. Since most carcinogens are mutagens in the assay, we have been concerned with developing modifications of the Salmonella assay for the detection of these compounds. There are several possible explanations for the negative response in the Salmonella assay. These compounds have relatively low chemical and metabolic reactivity and the S9 mix (prepared from rat liver) routinely used in the assay may not metabolize these compounds to active intermediates, either because essential cofactors are missing, or the enzymes needed for activation are not present at sufficiently high levels. Another possibility is that many of these compounds are metabolized to free radicals whose half-lives are so short that they are reacting with other components of the reaction mixture before reaching the target DNA. Other mechanisms under consideration are that these compounds act indirectly through a common mechanism, possibly through the peroxidation of lipids and generation of free radicals or malonaldehyde.
My work in Japan was concerned with one aspect of this problem the use of S9 that would be able to better activate this class of compounds. The work involved the preparation and testing of an S9 fraction from domestic house flies. Insect microsomal fractions are able to catalyze several different reaction types, being similar in metabolic versatility to mammalian liver microsomes. Strains of house flies were obtained from Dr. T. Hiroyoshi at Osaka University that were resistant to some of the chlorinated pesticides. While the biochemical basis for resistance is not known it was thought that an S9 prepared from both resistant and wild-type strains would be a useful tool for looking at the metabolism of these compounds. The Salmonella test was used to monitor the generation of reactive mutagenic metabolites.
An S9 fraction from both wild-type and resistant strains of house fly larva was prepared and then used to screen a number of known mutagens in the Salmonella assay. It was necessary to first develop techniques for preparing a sterile fraction. Five compounds, benzo(a)pyrene, 7,12-dimethylbenzanthracene, acetylaminofluorene, tris (2,3-dibromopropyl)phosphate and sterigmatocystin were then screened in the assay. Four of the five compounds were activated by the fly larva S9. In these preliminary experiments it was shown that: (1) The larva S9 was able to metabolize known mutagens to their active metabolites; (2) There were differences in the metabolic activity of the S9 from wild-type and resistant strains and that these differences were compound dependent; and (3) The activity of larva S9 ranged from less active to more active than rat liver S9, depending on the compound being tested. Eleven chlorinated hydrocarbons1 representing a variety of structural types were screened in the Salmonella assay using the house fly S9. All eleven compounds were negative in the assay.
Further experiments with fly S9 fractions included: (1) The preparation of S9 fractions from PCB, DDT or aldrin induced fly larvae, and (2) The preparation of S9 fractions from induced and uninduced adult flies (reports in the literature suggest that enzyme levels in adult flies are higher than in larvae). However, negative results were obtained when the chlorinated hydrocarbons were tested with these S9 fractions.
Although the house fly S9 was not able to metabolize the polychlorinated hydrocarbons to intermediates mutagenic to Salmonella it was possible that some of the other compounds that are false negatives (carcinogens/nonmutagens) in Salmonella would be activated by the house fly S9. There are a few compounds that are negative in Salmonella when tested in the presence of rat liver S9, but positive when tested in the presence of an S9 fraction from a different species. I screened several compounds from different chemical classes in the Salmonella assay with the fly S9. However, these com-pounds were also negative in the assay.
In conclusion, the house fly S9 was shown to be capable of metabolizing several indirect acting mutagens to their active mutagenic intermediates. However, the polychlorinated hydrocarbons and other compounds tested (i.e., other false negatives) were not acti-vated by the house fly S9.
b) The Mutagenicity of Edible Seaweeds.
During the last three years it has been shown that mutagens are present in many of the foods we eat. These include naturally occurring compounds such as quercetin and quercetin-glycosides that are present in many fruits and vegetables, and the very potent mutagens found in broiled meat and fish that are formed during the broiling process. Intensive work is currently being carried out at a number of different laboratories to identify those mutagens in foods that might be significant factors in causing human cancers. A useful approach to the study of dietary causes of cancer is to look at the mutagenic activity of foods that are unique to different population groups.
Seaweeds, an important constituent in the Japanese diet, meet this requirement. They also contain a number of brominated and chlorinated hydrocarbons, a class of compounds that often have high mutagenic and carcinogenic activity. While in Japan, I tested for mutagenicity five different edible seaweeds that are popular in Japanese cooking. Methanol and chloroform: methanol extracts of each seaweed were prepared and then tested in the Salmonella mutagenicity test. All five seaweeds were weakly mutagenic in the assay. Four of the five seaweeds were most active on the Salmonella tester strain TA98, in the presence of S9. While there was not time to identify the active principle in the seaweeds, these results would be consistent with the hypothesis that the mutagenicity was due to the presence of quercetin in the seaweeds. One seaweed was most active on strain TA100 in the absence of S9. Preliminary experiments attempting to concentrate the mutagenic compound(s) resulted in the complete loss of activity.
Based on these preliminary results, studies on the mutagenicity of edible seaweeds may be continued in Dr. Sugimura's laboratory in Japan, or by Dr. Ames in collaboration with Dr. Lowell Hager at the University of Illinois.
1 Compounds tested were dieldrin, aldrin, p,p’DDD, o,p’ and p,p’-DDT, p,p’-DDE, CC14, CHC13 and!!
!and!!
!BHC.4) ARTHUR WEISSBACH, PH.D.,
Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110
Host Institution:
Dr. Katsuro Koike, Cancer Institute (Japanese Foundation for Cancer Research) Sponsored the meeting entitled “Molecular Mechanism of DNA Synthesis in Cancer Cells” which was held at the International House of Japan, Tokyo
Dr. Kiyoshi Kurahashi, Osaka University Dr. Osamu Hayaishi, Kyoto University
Dr. Taijo Takahashi, Aichi Cancer Center
Dates of Visit: February 27, 1980 through March 10, 1980
Summary of Activities:
In conjunction with my participation in the meeting on molecular mechanism of DNA synthesis in cancer cells in Tokyo, I spent additional time in Japan (from February 27-March 10, 1980) visiting and consulting at the Osaka University, Kyoto University and the Aichi Cancer Center. I also presented seminars on my work on manunalian DNA polymerase at these institutions.
The exchange of information with Japanese scientists was valuable in planning future research. It provided information which will enable me to avoid non-productive work and suggested new possibilities to us. The meeting “Molecular Mechanism of DNA Synthesis in Cancer Cells,” held in Tokyo, provided a valuable forum for the exchange of knowledge between the United States and Japanese scientists. Most of the information exchanged had not been previously published and was, therefore, new. Several possible collaborative research projects between my laboratory and some Japanese laboratories were discussed and will, hopefully, be initiated in the near future.
5) DAVID KORN, M.D.,
Department of Pathology, Stanford University School of Medicine, Stanford, California
Host Institution:
Dr. Katsuro Koike Cancer Institute, Tokyo
Dates of Visit: March 6-8, 1980
Summary of Activities:
I wished to let you know that the US-Japan Conference was extremely successful in all respects and I gathered from my subsequent conversations with Drs. Sugano and Sugimura that there is some considerable interest on the Japanese side in trying to continue this series of meetings, on an alternating basis, perhaps every two years. I personally believe such a schedule of meetings would be profitable and justifiable, but obviously, I do not know precisely how such decisions are made under the aegis of the US-Japan Cooperative Program.
In addition to the meeting itself, I very much enjoyed my visits to Kyoto University, Aichi Cancer Center, the Cancer Institute, the National Cancer Center and Saitama Cancer Center, and in the latter four institutions, I delivered lectures that ran between 90 and 120 minutes to a well-informed and gratifyingly interactive audience. I also enjoyed my consultations with workers in each institution who are engaged in relevant research activities pertaining to the regulation of DNA replication and repair in a variety of mammalian cell systems.
The physical facilities in the several cancer institutes were quite impressive, as was the general quality of the science being performed, particularly by the younger generation of investigators.
6) TAKESHI HIRAYAMA, M.D.,
Chief, Epidemiology Division, National Cancer Center Research Institute
I visited the U.S. from August 5-August 25, 1979 under the Scientist Exchange Program of the U.S.-Japan Cooperative Cancer Research Program.
Most of the work was done in the Clinical Epidemiology Branch National Cancer Institute. Precious assistance and cooperation was given by Dr. Robert Miller, the Chief of the Branch.
The main subject studied was the Comparative Epidemiology of Chemical carcinogenesis and Radiation carcinogenesis.
REC (rem-equivalent-chemical) is a recently developed term describing the concentration of a chemical mutagen that produces an amount of genetic damage equal to that produced by I rem of chronic irradiation. This notion was developed from in vivo and/or in vivo experiment. A need was felt to develop a similar concept in humans. For that purpose, a dose-response relationship between life time dose of cigarette smoking and cancer mortality was compared with similar dose-response relationships between the exposure dose of radiation and cancer mortality. The materials used were: (1) Ongoing prospective study for 265,115 adults aged 40 and above in Japan (Hirayama, 1966-78) and (2) Ongoing prospective study for 109,000 A-bomb survivors and controls in Hiroshima and Nagasaki, 1950-74 detailed comparison revealed that the smoking of one cigarette is A equivalent to 1-4 millirad in terms of cancer causing effect. A similar comparison was made for cancers of specific sites (e.g., lung cancer).
On August 13, these findings were reported and discussed in a seminar jointly held by the Environmental Epidemiology Branch and the Clinical Epidemiology Branch, NCI.
On August 15 and 16, a workshop was held on Mutagen Load, with Dr. Hollaender as the Chairman. Both Dr. Sugimura and I attended from Japan. In this workshop again, the above results were discussed. Points raised were as follows:
1. In the case of A-bomb survivors, exposure was only once. In the case of cigarette smoking, it is a continued/repeated exposure. Such differences must be kept in mind.
2. A more suitable mathematical model should be sought in addition to a linear model.
3. Similar studies should be attempted with other chemical carcinogens.
In relation to these points, the lung cancer mortality for smokers and non-smokers in radium miners was studied based on official reports and available literature. For lung cancer, one cigarette was noted to be equivalent to 7.5-15 millirad.
On August 20-24, the Gordon Conference on Cancer Etiology in Humans was attended by both Dr. Sugimura and myself, and several new clues for future epidemiological research on cancer were obtained.
7) KAZUO UMEZAWA,
Institute of Medical Science, University of Tokyo
I visited the United States between August 18, 1979 and October 19, 1979 to study the mechanism of tumor promotion cooperatively with Dr. I. B. Weinstein, College of Physicians and Surgeons of Columbia University
Prior to joining Dr. Weinstein’s research group, I attended the Gordon Cancer Conference in New London, New Hampshire, together with Dr. T. Sugimura, where the mechanism of carcinogenesis at the cellular level was mainly discussed. I went to New York on August 25 and began to work on the cellular action of tumor promoters and membrane active compounds. During my relatively short stay at Columbia University, I completed two research projects as follow:
a) Inhibition of Cellular Binding of Epidermal Growth Factor (EGF) by Staphylococcal Delta-Hemolysin
Recent studies indicate that the potent tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) at nanomolar concentrations specifically inhibits the binding of epidermal growth factor (EGF) to Its cell surface receptor. Staphylococcal delta-hemolysin, a poly-peptide having the molecular weight of about 3500 also inhibited cellular EGF binding. The addition of 2.5 µg/ml of delta-hemolysin resulted in a rapid and marked inhibition of EGF binding in rat embryo cells. The dose required to produce 50% inhibition was about 2 µg/ml and complete inhibition was obtained with about 5 µg/ml. Parallel studies indicated that no cytolytic effect was observed at concentrations as high as 10 µg/ml of delta-hemolysin, after incubation for 2 hours at 37°C, although higher concentrations were toxic.
Delta-hemolysin also inhibited EGF binding in HeLa cells. With these cells 50% inhibition was observed at about 10 µg/ml and again no cytolytic effect was observed at this concentration of delta-hemolysin. HeLa cells displayed slight swelling at 50 µg/ml of delta-hemolysin after I hour incubation. Inhibition of EGF binding by delta-hemolysin was not affected by 50 µg/ml of hydrocortisone or dexamethasone of 25 µg/ml of mepacrine, agents reported to inhibit membrane phospholipases. Studies on the release of arachidonic acid were done with the 10T1/2 mouse embryo cells to compare the effects with that previously reported for TPA. At 1µg/ml delta-hemolysin proved to be a potent inducer of the release or arachidonic acid metabolites from the 3H-arachidonic acid prelabelled cells. Chromatographic analysis of the media showed that free arachidonic acid and significant amounts of prostaglandins E2 and F2!!
!were released in cells exposed to I µg/ ml of delta-hemolysin. These data do not, of course, indicate that the primary site of binding of these compounds is the same. Both TPA and delta-hemolysin stimulate lymphocyte mitosis and both induce epidermal inflamation. It will be of interest to determine to what extent delta-hemolysin shares other effects with tumor promoters.
b) Inhibition of Cellular Binding of Epidermal Growth Factor (EGF) by Phorbol Esters in Various Types of Cell Cultures Inhibition of EGF binding by phorbol esters was studied with hamster, rat, and mouse embryo cells. TPA and phorbol-12,13-didecanoate inhibited EGF binding in 3 cell cultures at 0.05-0.1 µg/ml. Phorbol-12, 13-diacetateand phorbol did not inhibit EGF binding even at 10 µg/ml. 4-0-Methyl-TPA, a weak tumor promotor, inhibited EGF binding at 0.2 µg/ml in 3 primary cell lines. Therefore, the receptors for phorbol esters appear to be similar in the 3 rodent cell cultures; they may differ, however, from the receptors in HeLa cells where 4-0-methyl-TPA did not inhibit EGF binding. Collaborative studies between I. B. Weinstein, T. Sugimura and myself on membrane effects of the compound teleocidin were also begun during this visit and are yielding interesting similarities with the effects of TPA.
8) NORIO MATSUKURA,
National Cancer Center Research
Institute Host Institution:
(1) National Cancer Institute, Bethesda, Md.
(2) Kuakini Medical Center, Honolulu, Hawaii
Dates of Visit: from September 1, 1979 to September 24, 1979
Summary of Activities:
I took specimens of experimental gastric cancer and intestinal metaplasia in animals which were produced at the National Cancer Center Research Institute, Tokyo, to the United States to be examined jointly by Dr. Harold L. Stewart, Scientist Emeritus of the National Cancer Institute (NCI) and myself through the U.S.-Japan Cooperative Cancer Research Program. The following points have been agreed between Dr. Stewart and me.
!C-stem. I also discussed the problem of sequencing of tRNA, using a post-labeling technique, with Dr. Grunberger. My visit to the U.S. turned out to be very productive since the work led to the conclusion that alteration of tRNAPhe in tumor cells is due to the aberrant modification of the Y base, but not due to altered different transcription of tRNAPhe.