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4 Virus, Previous Infections ..--~- #327R~ Committee cf. #59 Kotin, Chm. Activated: 6/15/55 0 Little cf. #200 Lynch Activated: 1/1/59 Wilson Renewed: i/1/60 1/1/61 Activated: 1/1/62 TOBACCO INDUSTRY RESEARCH COMMITTEE 150 East Forty Second Street New York 17, N.Y. Application Renewal for Research Grant Date: November 2, 1962 1. Name of Investigator: a. Philip Cooper, M.D. 2. do4rincipa7, Investigator: Co principa7, znvestigator: Title: b. c. Irene P. Goldring, Ph.D. Herbert Volk, M.D. a. Professor of Clinical Surgery, Albert Einstein College of Medicine Director of Laboratory of Cellular Physio].ogy, Department of Surgery, Albert Einstein College of Medicine, Bronx, New York Chief of Surgical Services, Veterans Administration Hospital, I3ronx, New York b. Research Assistant Professor in Surgery, Albert Einstein College of Medicine, Bronx, New York c. Associate Professor of Surgery, Albert Einstein College of Medicine, Bronx, New York 3. Institution Albert Einstein College of Medicine & Address: Eastchester Road and Morris Park Avenue Bronx 61, New York 4. Project or Subject: Study of the Combined Effect of Injected Viral Agents and Environmental Factors (including smoke and air pollutants) on the Tracheo bronchial Tree and Pulmonary Parenchyma of Experim.ental Animals, and on Tissues in Organ Culture. 5. Detailed Plan of Procedure: During this past year we have been studying the effects of various gases and "human" viruses on the development of lung pathology in experimental animals. The concept that we are investigating in regard to the development of lung cancer in humans is based on the hypothesis that lung cancers develop in individuals who have had some prior damage to their lungs, which predisposes that tissue to neoplastic change when exposed to other agents. We are therefore attempting to reproduce these conditions in laboratory animals in the following manner:

- 2 - • At first, we are subjecting animals to agents which are known to produce lung damage such as viruses, and then are exposing the same animals to a second challenge of a gas. At present we are.using S02 but plan to use smoke gases and other gases such as ozone, oxides of nitrogen, and certain particulate mixtures as found in automobile exhaust, which are air pollutants. The Syrian hamster has been the animal of choice. This animal has an extremely low incidence of spontaneous lung tumors, and has other desirable features for this type of study. It has an extremely short gestation period (16 days) and a total life expectancy of 2 years. This latter feature is particularly desirable for studies involving long-term exposures to experimental agents. It is capable of developing tumors of the lungs following intratracheal inoculation with S.E. Polyoma virus (Rabson, et al. 1960). Trentin has described intrathoracic tumors following the intrathoracic percutaneous 'injection of Adenovirus Type 12 (1962). Heubner (unpublished) has recently reported similar findings with the Adenovirus Type 18. Although newborn hamsters develop tumors in over 80% of animals injected (Trentin - 1962), there is a falling off as the animals get older. Weanlings develop tumors at a much slower rate in about 30% of animals so treated. We have divided these animals into two groups: 1) Weanlings: 1. Control animals - exposed to filtered air only. 2. Animals subjected to known concentrations of a specific gas for predetermined periods. 3. Animals inoculated with a virus and subjected to filtered air only. 4. Animals inoculated with a virus and subjected to a specific gas in same concentration and duration as group 2. All animals are observed daily for signs of respiratory disease. Weekly weight records are kept. Animals that have diedduring the course of an experiment are autopsied and detailed gross examination accomplished on all organs. The tracheo bronchial tree, the lungs and the liver have been routinely prepared for histologic examination. Other organs with suggestive pathology have also been prepared for such examination. Animals that do not die in the course of the experiment, are sacrificed at predetermined intervals, and similar studies conducted., 2) Newborn - (within 24 hours after birth) a. Repetition of work reported by Trentin. Intrapulm.onary (percutaneous) injection of adenovirus Type XII and other adenoviruses as Type 18. b. Similar intrapulmonary (percutaneous) injection of various myxoviruses (as influenza - PR8). Control animals are being maintained (not injected). We intend to expose some of the injected animals to inhalation of gaseous agents, following the initial evaluation of the direct effects of the viral agents alone. All animals that die, or are sacrificed at predetermined intervals are autopsied and studied as noted above. The viral agents are being secured from the American Type Culture Collection, and from Dr. P. Marcus, Micro-biologist and Virologist at the Albert Einstein College of Medicine. The viruses are being titrated prior to their use. The "human" viruses are being maintained and passed in tissue culture. The tissue culture fluid cell is harvested at the proper time, centrifuged, and the supernatant fluid is filtered through a Selas filter candle. That fluid is then used for injection purposes.

So far we have been employing only one inoculation. If it appears advisable we intend to repeat the inoculation in the same animals. The gases are being obtained from commercial sources. The flow rates and the concentrations are established by appropriate flow meters, and other measuring devices. The concentration of the.gas in the chambers is further checked by chemical analysis. We have introduced a Dautrebande generator into the input system in our present exposure chaXnber to create an aerosol of sodium chloride. The particle size is such (0.5a,4 to allow for the deeper penetration of the gas into the lungs of the exposedd animals. This project was begun in January 1962. We have had many problems to solve. Because of the difficulties of securing animals of the right age from commercial supply houses, we initiated our own animal colony. The quarters to house normal- uninfected animals and viral infected animals are separated and staffed by separate personnel. After initial diff iculties with cannabalism of the young an adequate number of breeding females are now on hand. Consultation with Dr. P. Marcus~ Virologist in the Department of Microbiology, resulted in an agreement to furnish us with viruses known to cause lung inflammation in other laboratory animals particularly the mouse. We first tried viruses in the myxovirus group which have been shown to cause respiratory disease and lung consolidation in mice, b'ut no information was available to the effects of those agents in hamsters. We injected these viruses intrathoracically (percutaneous) into newborn hamsters, and administered them by inhalation. Weanling hamsters received virus by intratracheal injection. Histological examination of the tracheo bronchial tree and lungs at this time suggests evidence of acute and chronic changes resulting from the use of•~one of the viral agents employed. We are continuing to study the effects of thIs virus to confirm our present findings. A report by J. J. Trentin which appeared in May indicated that a virus of the adenovirus group, Type 12, when injected into newborn hamsters was capable of producing tumors in the chest. On consultation with Dr. Trentin we secured somee of this virus from the American Type Culture Collection (which was Trentin°s source also) and we repeated his procedure and have obtained similar tumors in newborn animals. Histological examination of the tumors reveals probable sarcomas although the exact identity of the tumors has not been established. That virus has also been injected intratracheally into weanling 3 week-old hamsters. None of the weanling hamsters which received the virus by intratracheal znoculation died. We began to sacrifice them at two week intervals. No gross pathology has been revealed to date and the histological examinations are being continued. Dr. Trentin reported that tumor induction did occur in a small percentage of animals so inoculated (about 30%) but its development was quite slow, upwards of three months. Within the last month Dr. Heubner has reported oncogenesis in Syrian hamsters with adenovirus Type 18. The ATCC has been very cautious about releasing any more of either virus, and therefore, Dr. Marcus has kindly offered to passage it for us in HeLa cells and to titer it for our use. Considerable progress has been made in the re-design of the Leuchtenberger smoking apparatus to deliver gases in known concentration. The apparatus consists of a dual chamber. One houses the control animals which breathe filte,red air and the other the experimental animals which breathe a mixture of the added S02 in air. An aerosol generator (Dautrebande) adds to the streamm of air entering the exposure chamber a sodium chloride aerosol whose particle size is 0.5,t4,. At present we can feed in a stream of 15 lpm of air with an S02 concentration that can'be varied f.~rom 1-5 ppm up to 650 ppm. The concentration of S02 is further monitored by bubbling

4 a known quantity of the air-S02 mixture through 12 and titrating it with thiosulfate using the chemical method described in "Chemistry of Industrial Toxicology," J. Wiley and Sons, Elkin, H.B., ed., 1959, PP• 396-397. Amdur has demonstrated the importance of the addition of an aerosol to gas mixtures to be inhaled. The combination of S02 and a sodium chloride aerosol produce airway resistance eff ects many times greater than S02 alone. Tt is of interest to note that adult hamsters seem to tolerate exposures of 600 ppm for 3 hours a day for several days with no outward untoward effects. The histologic examination of the lungs is not complete as yet. An experiment is in progress now to demonstrate the effects, if any, on weanling hamsters subjected to high conc. of 802 600 ppm for three hours a day for five days/week. Organ culture techniques are planned for this study. Fragments of lesions which develop in the tracheo bronchial tree and lungs at di.fferent stages of development will be transferred to organ culture. The growth characteristics of the tissues in organ culture will be noted. evaluation of early.lesions. This method should contribute significantly to an 6. Budget Plan: a. Salaries S.S. Retire. Total Technician $6,000 195 ,~195 I. P. Goldring . 72500 244 375 8,119 14, 314.00 b. Expendable Supplies Animals, Viral Supplies, Chemicals, Gases 2,000.00 c. Permanent Equipment Additional Exposure Chambers 1,500 Animal Cages 500 Monitory & Measuring Device for G as 3,000 5,000.00 d. Other Travel 800 Equipment Maintenance and.Repairs 250 1,050.00 e. Overhead (15% of a,b, d) 2,,604.60 Total $249968.60 7. Anticipated Duration of Work: 3-5 years. . Facilities and Staff Available: The Laboratory of Cellular Physiology of the Department of Surgery occupies four rooms in the original Science Building of the Albert Einstein College of Medicine. This area includes rooms for the viral studies, f or the gas chambers, for the organ culture work, and for time lapse studies., and for the preparation of glassware, drugs and chemicals. Additional facilities are available for the housing of animals., The viral agents are being stored, cultured and prepared by Dr. Marcus and his staff.

5 9. Additional Requirements: Additional gas chambers, and control equipment for the introduction of gases into the chambers will be required. This will increase the efficiency of the available apparatus, and may allow us to do two studies simultaneously. Additional monitoring equipment will also expedite our work, and should be procured. 10. Additional Information: Additional support is being requested from other.agencies in order to complete this work. This additional support will allow for procurement of additional personnel and for additional equipment and supplies. Mrs. Maria L. Duran Reynals is joining our laboratory on January l, 1963. She will do work on the combined effect of vaccinia and other viruses and chemical carcinogens on skin of laboratory animals. This will be a continuation of the investigation which her husband, Francisco Duran-Reynals, initiated at Yale. She has agreed to participate in our project with the ' Syrian hamsters. . Signature Philip Gooperg M.D. Director of Project Marcus D. Kogel, M.D. Dean ~

s BIBLIOGRAPHY 1. Leuchtenberger, C., Leuchtenberger, R., Zebrun, W., and Shaffer: A Correlated Histological, Cytological and Cytochemical Study of the Sequence of Events in the Bronchial Epithelium from Mice Exposed to Cigarette Smoke. Acta Union Internayionale Contre Le Cancer, Vol. 15, p. 632-638, 1959• 2. Leuchtenberger, C., Leuchtenberger, R., and Doolin, P.F,: A Correlated Histological, Cytological and Cytochemical Study of the Tracheobronchial Tree and Lungs of Mice Exposed to Cigarette Smoke. 1. Bronchitis and Atypical Epithelial Changes in Mice Exposed to Cigarette Smoke. Cancer, Vol. 2, p. 490-506, 1958. 3. Cailleau, R.T., Crocker, R. and Wood, D.A.: Attempted Long-term Culture of Human Bronchial Mucosa and Bronchial Neoplasms. Journal of the National Cancer Institute, Vol. 22, p. 1027-1037, 1959• 4. Rabson, A.S., Branigan, W.J. and Legallias, F.Y.: Lung Tumors Produced by Intratracheal Inoculation of Polyorna Virus in Syrian Hamsters. Journal of National Cancer Znstitute, Vol. 25, p• 937-966, 1960. 5. Wiseley, D.V., Kotin, P., Fowler, P.R. and Trivedi, J.: The Combined Effect of Repeated Viral Infection and Exposure to Carcinogenic Aerosols on Pulmonary Tumor Induction in C57 Black Mice, Abstracts ~ Proceedings of American Association for Cancer Research, 1961. 6. Six Years of Research in Air Pollution, U.S. Department of Health, Education and Welfare, Public Health Serv3.ce, July 1, 1955®June 30, 1961. 7. Amdur, M.O.: The Influence of Aerosols Upon the Respiratory Response of Guinea Pigs to Sulfur Dioxide. Industrial Hygiene Quarterly, P. 149-155, June 1957. 8. Trentin, J.J.: The Quest for Human Viruses. Science, 137:835°°841, 1962.