M. K. YOUNOSZAI

JEAN PELOSO

J. C. HAWORTH*

Winnipeg, Manitoba, Canada

Groups of Pregnant rats were placed in a smoking chamber for 4 minutes from the third to the twenty-second day of pregnancy five times daily and exposed to the smoke from one of three types at cigarettes: (1) tobacco cigarettes each containing 15 mg. at nicotine; (2) lettuce leaf cigarettes (nonnicotine); (3) lettuce teat cigarettes, to each of which 15 mg. at nicotine had been added. The per cent carbon monoxide saturation of the hemoglobin was used as an index of the amount at smoke inhaled by the rats. This was maintained between 2 and 8 per cent in all rats. Fetuses Of all the smoked rats were growth retarded compared to control animals, those exposed to tobacco cigarette smoke being most severely affected. The amount at food consumed by the rats exposed to cigarette smoke was reduced. The food intake at several other groups at Pregnant rats was restricted to various levels of that consumed by control animals. There was a significant direct relation between fetal body weight and the average amount of food eaten daily during pregnancy. Fetal weight was reduced in proportion to the decrease in maternal food intake in the two groups of rats exposed to the lettuce leaf cigarette smoke (with and without nicotine). In the rats exposed to the smoke of tobacco cigarettes, fetal weight was reduced more than that expected from the decrease in maternal food intake. Since all the rats inhaled similar amounts of carbon monoxide and therefore probably Similar amounts of smoke and of nicotine, it is unlikely that these substances were directly responsible for the fetal growth retardation. It seems possible that tobacco smoke contains factors as yet unrecognized which have fetal growth retarding effects additional to that produced by diminishing the appetite of the mother.

The pathogenesis of the fetal growth retarding effect of maternal cigarette smoking is unknown. There has, however, been speculation that nicotine which is present in cigarette smoke may cause uterine vasoconstriction, reduce maternal appetite, or in some way produce metabolic changes in the mother and/or fetus. Some have also suggested that carbon monoxide inhaled from cigarette smoke, because of its enzyme-inhibiting action and reduction of the oxygen-carrying capacity of hemoglobin, may affect the fetus adversely. Most of the other tobacco smoke constituents are present in very small amounts and it has been generally considered that they are not harmful to the fetus.

From the Research Foundation, The Children's Hospital, Winnipeg, and the Department of Pediatrics, University of Manitoba.

Supported by a grant from the Imperial Tobacco Company of Canada Limited.

*Address: Children's Hospital, 685

Bannatyne Avenue, Winnipeg 3,

Manitoba, Canada.

The present investigation was undertaken to study whether or not fetal growth retardation in pregnant rats exposed to tobacco smoke is primarily due to a reduction in food intake or to the absorption of nicotine and carbon monoxide.

Material and methods

Sperm positive Holtzman albino rats weighing 200 to 250 grams were used, the time of conception being known to within 8 hours. Groups of 2 to 4 rats were housed in cages measuring 12 x 12 x 9 inches and from the third to the twenty-second day of pregnancy were exposed to smoke from the following types of cigarettes: (a) regular tobacco cigarettes, stated by the manufacturers to contain approximately 15 mg. Of nicotine per cigarette; (b) nonnicotine cigarettes made with leaves of a variety of lettuce (Lactuca sativa); and (c) nonnicotine cigarettes as above to each of which 15 mg. of nicotine was added by the injection of half a milliliter of a freshly prepared aqueous solution containing 30 mg. of nicotine per milliliter along the length of the cigarette and then drying overnight at room temperature. Filter tips when present were removed before igniting the cigarette. All cigarettes contained approximately 950 mg. of tobacco or lettuce leaf.

The rats were forced to inhale cigarette smoke by placing their cages in a smoking chamber, a box measuring 18 x 15 x 11 inches. This box was not airtight, having holes 1 inch in diameter in two opposite sides, and was covered by a glass lid through which the rats could be observed. A cigarette was fixed into a hole drilled in a cork and the lit end was introduced into a glass separatory funnel (Fig. 1). Air, blown through the funnel by a one-way rubber bulb attached to it by tubing, passed through the cigarette carrying the smoke into the box.

The per cent carbon monoxide saturation of hemoglobin was measured by the method described by Natelson and taken as air index of the amount of smoke inhaled. It was found that the exposure of rats for 4 minutes to the smoke of 3/4 of a cigarette (either tobacco or lettuce leaf) resulted in 7 to 8 per cent carbon monoxide saturation of the hemoglobin. Within 2 hours this level had detoms in the rats exposed to smoke from the lettuce leaf cigarettes with added nicotine were not as marked and disappeared within 5 minutes of removal from the smoke. The rats exposed to the lettuce leaf cigarette smoke had much milder reactions; there was no wheezing or dyspnea, sweating was minimal, and they behaved normally after removal from the box. In 4 to 5 days the fur of all the rats showed a yellowish discoloration. Apart from the above signs and symptoms, all the rats remained in apparent good health during the investigation. No convulsions or tremors were noted.

In Fig. 2 the body and organ weights of the fetuses in the various experimental groups are expressed as percentage deviations from those of the respective control animals. Fetal body weight was significantly reduced in all experimental groups. The mean weight of the fetuses of the rats exposed to tobacco smoke was significantly less than that of fetuses of rats exposed to the smoke from lettuce leaf cigarettes with and without added nicotine. Fetal weights in the latter two groups were not significantly different. Fetal brain and kidney weights were also significantly reduced in all groups, but significant liver weight reduction was not seen in the fetuses of mothers exposed to lettuce leaf cigarettes.

The relation between fetal weight and maternal food intake was assessed from the data obtained from all the control groups of rats and those fed a restricted diet. Regression lines were calculated using the mean daily food intake per rat per cage and the mean fetal body and organ weights per cage.

Fig. 3 shows that there was a significant direct relation between the average daily food intake per rat, expressed as a percentage of the maximum food intake observed, and the mean fetal body weight per cage, Fig. 4 shows that significant direct relations existed between the mean weights of each of the three fetal organs and body weight.

From the relations thus obtained it became possible to predict fetal body and organ weight for a given average daily amount of food consumed by the mother during pregnancy. When the fetal body and organ weights in the groups of rats exposed to lettuce leaf cigarettes with and without added nicotine were compared to the weights predicted from the relations noted above, it was found that fetal body and organ weights were reduced in proportion to that expected from the amount of food consumed by the mothers (Figs. 3 and 4). In the rats exposed to the smoke of the tobacco cigarettes, fetal weight was reduced more than that expected from the reduction in the maternal food intake.

The results of the present investigation confirm that fetal growth is retarded by restricting the maternal food intake. In addition, a direct relation between fetal weight at term and the average amount of food consumed daily by the mother rats during pregnancy has been demonstrated (Fig. 3).

All the rats in the smoking groups ate less food than the controls and their fetuses at term weighed less than the control fetuses. The fetal growth retardation in the rats exposed to the smoke of the lettuce leaf cigarettes with and without added nicotine was no greater than that predicted from the decreased food intake of their mothers. However, in the rats who inhaled tobacco cigarette smoke, the fetal growth retardation was greater than that resulting from the decrease of maternal food intake alone (Fig. 3). The weight gain of these animals during pregnancy was much less than in the control animals and those eating comparable amounts of food. The cause of this was not determined. They had no diarrhea and did not appear to be ill except for the symptoms noted during and immediately after exposure to the smoke. Although fluid balance was not measured, they did not appear dehydrated. It is possible that these animals had a metabolic derangement which affected the growth of their fetuses in excess of that which could be explained by dietary restriction alone.

The experimental technique of compelling the rats to inhale cigarette smoke was obviously not very comparable to the human cigarette smoker who inhales one puff of smoke at a time and breathes air in between the puffs. The rats were exposed to a heavily smoke-filled environment for 4 minutes at a time. This "fumigation" procedure might have been expected to result in an irritation of air passages and perhaps some degree of hypoxia. Fetal growth retardation explainable on this basis is unlikely because the rats exposed to lettuce leaf smoke, which was just as dense as that of the regular tobacco cigarettes, showed negligible symptoms of distress. The smoke was doubtless responsible for their anorexia, however, and we believe the growth retardation seen in the fetuses of these animals was due to their reduced food intake. What part the carbon monoxide played in the symptomatology of the rats is unknown.

Nicotine was not measured in the tissues of body fluids of the rats exposed to the nicotine-containing cigarette smoke (tobacco and lettuce leaf -with added nicotine) but the fact that the blood levels of carbon monoxide in these two groups were in the same range suggests that they had inhaled similar amounts of smoke and it is reasonable to assume that they had also inhaled similar amounts of nicotine. The injection of nicotine into rats in doses greater than 1mg. per kilogram produces convulsions and death. Smaller doses (0.5 to 1 mg. per kilogram) results in restlessness, dyspnea, "circling," and tremors. The rats exposed to tobacco smoke showed, on the whole, mild symptoms compared to the above and it seems likely that they absorbed less than 0.5 mg. per kilogram of nicotine.

The symptoms in the two groups of rats exposed to the smoke of the nicotine-containing cigarettes could have been partly produced by nicotine, On the other hand the rats which inhaled lettuce leaf cigarette smoke with added nicotine had much milder symptoms than those which inhaled tobacco smoke. This suggests that tobacco smoke contains substances other than nicotine and carbon monoxide which cause immediate symptoms and may be involved in fetal growth retardation.

Comparison between the smoking rat experiments and the situation of the cigarette smoking woman and the effects on the offspring would obviously be unwarranted. Infants of smoking women weigh 6 per cent less at birth on the average than infants of nonsmokers, whereas in these rats, fetal weight was reduced by about 17 per cent. A group of infants born to mothers who smoked more than 20 cigarettes per day showed no biochemical signs of nicotine in their body fluids. They did, however, have a mild metabolic acidosis and slightly higher blood hematocrit levels. The latter could have been the result of mild chronic carbon monoxide poisoning. The results of the rat experiments, however, suggest that nicotine and carbon monoxide inhaled during exposure to cigarette smoke are not the prime agents affecting fetal growth. Whether this is also true in humans remains to be shown.

REFERENCES

Simpson, W. J.: Am. J. OBST. & GYNEC. 73: 808, 1957.

Lowe, C. R.: Brit. M. J. 2: 673, 1959.

Zabiskie, J. R.: Obst. & Gynec. 21: 405, 1963.

Yershalmy, J.: Am. J. OBST. & GYNEC. 88: 505, 1964.

Ravenholt, R. T., Levinski, M. J., Nellist, J., and Takenaga, M.: Am. J. OBST. & GYNEC. 96: 267, 1966.

Underwood, P. B., Kesler, K. F., O'Lane, M., and Callagan, D. A.: Obst. & Gynec. 29: 1, 1967.

Butler, N. R.: J. Obst. & Gynaec. Brit. Comm. 72: 1001, 1965.

Russell, C. S., Taylor, R., and Maddison, R. J. Obst, & Gynaec. Brit. Comm. 73: 742, 1966.

Comstock, G. W., and Lundin, F. E.: Am. J. OBST. & GYNEC. 98: 708, 1967.

Goldstein, H., Goldberg, 1. D., Frazier, T. M., and Davis, G. E.: Pub. Health Rep. 79: 553, 1964.

Haddon, W., Jr., Nesbitt, R. E. L., and Garcia, R.: Obst. & Gynec. 18: 262, 1961.

Essenberg, J. M., Schwind, S. V., and Patras, A.R.: J. Lab. & Clin. Med. 23: 708, 1940.

Schoeneck, F. J.: New York State J. Med. 41: 1945, 1941.

King, J. E., and Becker, R. F.: Am. J. OBST. & GYNNC. 95: 508, 1966.

Becker, R. F., Little, C. R. D., and King, J.E.: Am. J. OBST. & GYNEC. 100: 957, 1968.

Chow, B. F., and Lee, C. J.: J. Nutrition 82: 10, 1964.

Berg, B. N.: J. Nutrition 87: 344, 1965.

Hsuch, A. M., Agustin, C. E., and Chow, B. F.: J. Nutrition 91: 195, 1967.

Natelson,, S.: Microtechniques of Clinical Chemistry, ed. 2, Springfield, Illinois, 1963, Charles C Thomas, Publisher, p. 158.

Younoszai, M. K., Kacic, A., and Haworth, J. C.: Canad. M. A. J. 99: 197, 1968.

685 Bannatyne Avenue

Winnipeg 3, Manitoba, Canada

(In French) Tobacco substitutes IV. Lettuce cigarettes In the U.SA.

Rev. Tabacs, Fr., 1969, 267, p. 18.

It is largely because tobacco enjoys the fairly exceptional botanical property of having proteolytic diastases in its leaves that it can acquire a pleasant aroma during curing, fermentation or aging, the albuminoid materials, which give the smoke of other plants a green, peppery, bitter taste and a smell of burnt horn, are hydrolyzed, NH3 is released and amines are formed ; this reaction does riot occur with substitutes, and only badly in low quality tobacco. In 1963, P.C. Torigian filed a patent (no 1,372,143) for the use of a proteolytic ferment such as papain or trypsin. This method applied to lettuce leaves gave rise to the Bravo cigarette. The Roswell Park Memorial Institute is carrying out trials to find which plant substances give the lowest possible level of carcinogenous tars when burnt. A French patent, no 1,409,919, filed by Berthiot Laboratories in 1964, relates to a cigarette containing Lobelia.

MEAN CHANGES IN PLUS-RATE AND BLOOD-PRESSURE
AND ANALYSES OF VARIANCE

d.f.=degrees of freedom

s.s.=sum of squares

m.s.=mean sum of squares

n.s.=not significant

F=Fisher's F

P=Probability

CIRCULATORY EFFECTS

OF NICOTINE AEROSOL INHALATIONS

AND CIGARETTE SMOKING IN MAN

A. HERXHEIMER

M.B. Lond.

SENIOR LECRURER IN PHARMACOLOGY

R. L. GRIFFITHS B. HAMILTON MARION WAKEFIELD

MEDICAL STUDENTS

THE LONDON HOSPITAL MEDICAL COLLEGE, E.1

Inhalations of smoke from a filter-tip Summary cigarette and of nicotine aerosol in approximately equivalent amounts (with respect to nicotine) from a pressurized container, produced similar increases in pulse-rate and blood-pressure in healthy volunteers. Inhalations from nicotine-free cigarettes and of aerosol propellant alone had no effects on the circulation.

Introduction

The circulatory effects of cigarette smoking are probably due to inhalation of nicotine, the most active alkaloid in the smoke (Clark et al. 1965). If these effects could be matched by inhalation of an aerosol containing nicotine without the other constituents of the smoke, this might help people dependent on cigarettes, as has been suggested by Collier (1965). We have compared the effects of smoking a commercially available filter-tip cigarette (' Piccadilly ') and a filter-tip cigarette made from cured lettuce leaves which contain no nicotine (' Bravo Smokes '), with the effects of inhaling an aerosol containing 53 g. nicotine per metered puff from a specially prepared ' Medihaler ', and a control aerosol containing only the propellant.

Methods

Preliminary experiments with different rates of inhalation from cigarettes indicated that one dose every 30 seconds was effective and convenient. Two puffs of the nicotine aerosol every 30 seconds seemed to compare more closely with inhalations from a cigarette than did single puffs at the same rate. We therefore used a dose of two puffs of the aerosol given in two successive inspirations every 30 seconds.

Five healthy volunteers (four men, one woman), aged 20-21, each received all four preparations. All five had been smoking 5-15 cigarettes daily for 3 years, and were " inhalers ". They did not smoke for 2 hours before each experiment. Experiments with the active treatments were separated by at least 1 day. Occasionally, when two experiments on the same subject were done in succession (i.e., only one with nicotine), they were separated by at least 30 minutes; after this time pulse-rate and blood-pressure had returned to the level of the first control observations.

The observations were made at about the same time of day with the volunteer comfortably seated in a quiet room. The pulse-rate was counted and the blood-pressure measured with a sphygmomanometer in alternate minutes during a control period of 10 minutes or longer until the readings became stable. The volunteer then took one of the preparations for 7, or in some experiments, 8 minutes; pulse-rate and blood-pressure were recorded as before, and were followed until they had returned to the control levels.

For each experiment the mean of the three control measurements immediately preceding the treatment was taken as the basal value. The measurements of pulse-rate and blood pressure made from the second minute to the end of the exposure period were used to calculate mean values for this period. The differences between the basal and treatment means were taken as measures of the response, and subjected to analyses of variance.

Results

Inhalation of cigarette smoke and nicotine aerosol significantly increased pulse-rate and blood-pressure during the period of inhalation, and these two treatments produced similar increases; the control smoke and the control aerosol had no effect (see table). The between volunteer range of basal values for pulse-rate was 55-84 beats per 1 minute, and for blood-pressure 102/56 to 127/76 mm. Hg. There was little difference between the basal values on different occasions in the same volunteer.

The effects became apparent 1 or 2 minutes after the subject started to inhale nicotine, and then remained fairly constant. However, the peak effect on pulse-rate and blood-pressure tended to occur later during cigarette smoking than with nicotine aerosol inhalations.

Discussion

Since inhalation of tobacco smoke and of nicotine aerosol increased pulse-rate and blood-pressure in all volunteers, and inhalations of the control preparations did not, these effects cannot be ascribed to an irritant action of the smoke or the propellant. Nicotine produced a greater percentage increase in pulse-rate than in blood pressure, as it did in earlier studies, for example, by Roth et al. (1944).

The circulatory effects of inhaling nicotine aerosol at the rate of two puffs (106 g.) every 30 seconds did not differ significantly from those of inhaling cigarette smoke. The nicotine content of cigarette smoke is about 100 g. per puff (A. K. Armitage, personal communication, 1967), so that the amounts of nicotine inhaled by the two methods were similar. However, the equivalence can only be approximate because the nicotine content of cigarette smoke increases greatly as the cigarette is smoked. This increasing yield of nicotine presumably explains why the peak effects on the circulation tended to occur later with cigarette smoking than with inhalation of the aerosol.

Since the circulatory effects of inhaling nicotine aerosol so closely match those of cigarette smoking, the use of nicotine aerosol as a substitute deserves investigation. It might be particularly useful in patients with respiratory disease who have difficulty in giving up cigarettes.

We thank the volunteers for their excellent cooperation, and Dr. E. M. Glaser and Mr. J. Sloan of Riker Laboratories Ltd. for supplying the aerosol preparations at our request, and for assaying them.

Requests for reprints should be addressed to A. H.

REFERENCES

Clark, M. S. G., Rand, M. J., Vanov, S. (1965) Archs int. Pharmacodyn. 156, 363.

Collier, H. 0. J. (1965) New Scientist, 27, 826.

Roth, G. M,, McDonald, J. B., Sheard, C. (1954) Y. Am. med. Ass. 125, 761.