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Txr COUNCIL h'oft ToBAcco I3Escaacii-U.S.A., INC. ME'i+IORANDUM TO: Dr. S. C. Sommers and Staff FROM: D. H. Ford HK31166001 December 29, 1981 SUBTCT: Site visit with,Dr. Paul Lewis, Hammersmith Hospital, London, December 16, 1981. Grant No. 1187 M R1; Title: "Effects of Nicotine on Cell Pro- liferation in the Developing Brain." This investigation, which utilized a radiographic technique mea- suring 3H-thymidine incorporation into dividing neurons of the cerebellum and forebrain of rat pups exposed to nicotine in utero, demonstrated that, unless unusually high doses of nicotine were given subcutaneously (above 1 mg/kg acutely), there were essentially no significant effects of nicotine on dividing neurons of the cerebellum and forebrain (daily dose in drinking water = 1.6 mg/kg/ day). There were no differences in eye opening or eruption of the incisors. There were no significant consistent changes in mitotic, pynknotic or labelling indices, brain weight, etc. A slight, but not maintained reduction in the labelling index occurred in the external granule cell layer of the cerebellum on day 6 and there was a slight reduction in the mitotic index in the granule cell layer of the dentate fascia (gyrus) on day 16, neither of vrhich were con- sidered to be of sufficient magnitude or duration to be significant. Biochemical analysis of the forebrain and cerebellum revealed no differences in levels of DNA between control and nicotine-treated rat pups. In an acute study, levels of nicotine exceeding 1 mg/kg led to significar,t decreases in the specific activity of DNA in both the forebrain and cerebellum, but often induced seizure activity in the dams. Doses of up 1600ug of nicotine/day subcutaneously ^ had no effect on total forebrain DNA specific activity, while dose of up to 800ug/kg had no effect on total cerebellar DNA S.A. Higher doses led to a depressed S.A. of the DNA, accompanied by seizure activity in the dams. (Krous reports increased cell death in medulla oblongata neurons in pups from mother rats exposed to 2.6 ag/kg/day of nicotine in their drinking water. How- ever he does not define the percise sites of cell death. Further, cell death is not an extremely rigorous method of determining cell events in neonatal brain tissue, which is very difficult to process, and unless judged by an individual expert in the field of neuronsl growth, differentiation and remodeling through cell loss, difficult to evaluate). Thus, I would conclude that Dr. Lewis' study has demonstrated that nicotine in doses levels equivalent to what a person might get with 20 cigarettes/day (which is in itself difficult to judge, as is what constitutes a rat equivalent dose) has little or no effect on the cell division, DNA sysnthesi:,, etc. In view of Lajtha's observations of depressed protein syn- thesis in neonatal mice (could be a species difference ), the suggestion is that nicotine effects in the developing brain may occur during the growth and differ- entiational phases, effecting ultimate connectivity patterns, rather than during cell division. (This might account for some of the behavioural differences reported

NK®I166002 by the Boulder group following prenatal exposure of pregnant mice to smoke ). In arsy event, Dr. Lewis feels that this project is essentially concluded. Whatever other approaches might be made are beyond his interest and field of competence. Therefore, he has no plans for further studies which we might be izlterested in supporting. A copy of Dr. Lewis' final report will be mailed to us in the near future in the form of a manuscript he plans to submit for publication. A final financial report will be sent on in February after he winds down the staff which he had assigned to the project (Dr. Brian Backhouse started his work on this program during last February and will coaclude this coming February while com- pleting the final verification of statistics, etc.). Comment: This project asked a simple direct question: Does exposure to nicotine at varying levels during pregnancy have any measureable effect on the divisipn of cells which are still undergoing mitosis after birth and which form a reliable index of effect of pharrhacologic agents on CNS development in relation to cell populations? The answer, with doses equivalent to the level of nicotine a smoker might get from 20 cigarettes/day is that there appears to be no overall significant effect. Doses which produce seizures in the Wistar rats used did depress labelling of DNA. The implication is, therefore, that the depression of protein synthesis observed by Lajtha does not relate to any de- crcase in tieuronal number, but to some expression of neuronal growth and maturation. !~he methods used by Dr. Lewis in this study for evaluating an effect on cell division, phases of the cell %:ycle, etc. are quite rigorous and considered by experts in the field to be among the best methods for determing the effect of any pharmacologic agent on neuronal cell populations. They are tedious and time cohsuming, but provide reliable data. Thus, I must agree with Dr. Lewis, that while nicotine may have many pharmacological effects on neurons, it does not appear to irifluence their proliferation. D. H. Ford Associate Research Director DHF:ps