- Email: [email protected]
Studies on nicotine absorption during pregnancy
OBSTETRICS
Studies on nicotine absorption during pregnancy I. LDso for pregnant and nonpregnant rats
.J. EDWARD KING, A.M. R. F. BECKER, PH.D.* Durham, North Carolina
I N P R E v I o u s reports, l-·l we have shown that pregnant and nonpregnant female rats differ significantly in their tolerance and response to certain drugs, e.g., pentobarbital. In beginning a study on the effects of nicotine during pregnancy, we suspected that similar differences might hold. We also noted that nicotine dosages reported in the literature varied according to strain, and that most of the dosages reported pertained to male animals. We felt it best, therefore, to determine the 50 per cent lethal dose for both pregnant and nonpregnant females of our Osborne-Mendel strain to compare this strain with others before estimating doses that could be administered in chronic studies with these animals. Since we were going to be interested in the effects of the drug upon the fetus and neonate, we elected to determine at the same time the LD 50 for newborn young 6 to 24 hours following their normal undrugged delivery. The latter information would highlight any discrepancy m tolerance levels between adult and new-
born. Since nicotine will cross the placenta, the drug, administered to the mother, might be expected to affect the fetus adversely if the tolerance of the immature newborn was substantially below that of the adult. Preliminary probing with small population samples of 20 pregnant and 20 nonpregnant rats over four dosages ranging from 20 to 40 mg. per kilogram did, indeed, show that we might expect the pregnant rat to tolerate less of a 2 per cent solution (in saline) of Eastman Kodak highest purity nicotine* than the nonpregnant rat. This difference, as reflected in mortality, was in the reverse direction of that which we had noted consistently with barbiturates. Such a finding, in itself, justified an LD 30 testing with larger population samples.
Method Five groups ( 20 pregnant animals in each) received, respectively, 22.5, 25.0, 27.5, 30.0, and 32.5 mg. per kilogram nicotine on the twenty-first day of pregnancy, counting as day zero the day sperm were recovered from the vaginal lavage after overnight mating.
From the Department of Anatomy, Laboratory of Perinatal Studies, Duke University Medical Center. *Supported by grants-in-aid from the Council on Tobacco Research-USA and the Duke University Research Council.
*Nicotine No. 1242 BP 115-117°/12 mm., Kodak Company, Rochester, New York.
508
Eastman
Vohunt' 9:) Nttmber 4
Five groups ( 20 nonpregnant rats of thr same age in each) rec.:eived, resjJectively, 25.0, 30.0, 32.5, 35.0, and 37.5 mg. per kilogram nicotine in one subcutaneous injection, just as had the pregnant rats. Animals were assigned to dosage categories by strict random order: ( 1) when rats were determined to be pregnant by palpation; and (2) at once, in the case of nonpregnant rats. Two groups served as controls: 1. There were 20 untreated pregnant rats kept under the same environmental conditions that would be imposed upon the treated mothers, save for nicotine administration. That is, they were brought around into the laboratory on the twenty-first day in nesting boxes where they would deliver their young under observation. They were also deprived of food and water for 8 hours. Nicotine is purported to cut down on caloric intake. While we hardly expected to see much decrease in neonatal weight when nicotine treatment occurred on the twentyfirst day of gestation and delivery on the twenty-second, if there were a difference, we wished to assign it to drug effect, not to temporary dehydration or lack of maternal food intake over these 8 hours. 2. To equate for the trauma of handling and injection, 9 other pregnant controls received 0.50 c.c. saline subcutaneously. Otherwise, they were treated like the rest of the control rats. Some 300 untreated, newborn young were divided into seven dose groups with an average of 38 young per group, and these received single subcutaneous injections of 10, 11.25, 12.50, 13.75, 15.0, 17.50, or 20 gamma per gram nicotine within 6 to 24 hours of normal delivery (Table II). A similar control group of normal young received 0.075 c.c. saline. All nicotine doses for these small animals were given in volumes between 0.05 and 0.10 ml. using a 0.10 ml. microsyringe. Thus, the saline volume was the midpoint between these values. The volumes were measured to the nearest microliter. From the resulting mortality data, doses
Nicotine absorption during pregnancy
509
wPre converted to log concentratiom and per cent mortality was converted to probit values as recommended by Fisher and Yates.·, According to these authors, this procedure is based upon the supposition "that a normal deviate is linearly dependent upon some observable concomitant measurement, and that an observable frequency is that with which this deviate is exceeded in a normal distribution." Realizing that this would mean as little to the average reader as it did to us, we based the procedure on the supposition that mortality incrPases with increased concentration of the drug. The graphed results are presented as Fig. 1. Observations and results Table I demonstrates the effect of these single heavy doses of nicotine given one day before expected delivery date: ( 1) There were no deaths in the control groups. (2) Mortality increased abruptly above 27.5 mg. per kilogram in pregnant rats; above 32.5 mg. per kilogram in nonpregnant rats. Thus, the nonpregnant female continued to show a greater tolerance for this drug than the pregnant female. ( 3) Over the entire range of doses employed, mortality was 49 per cent for pregnant rats; 40 per cent for nonpregnant. (4) Deaths occurred earlier than one hour with about equal frequency in pregnant (14) and nonpregnant (10) rats, and at about the same time after injection (28.1 to 31.8 minutes). These were convulsive deaths. ( 5) However, certain animals survived for longer periods. While this was true of about equal numbers of pregnant ( 35) and nonpregnant ( 30) rats, thf.' nonpregnant in this delayed-death category died closer to 1.5 hours but the pregnant rats staved off death for 3.0 hours on the average; some for as long as 8, 10, and 16 hours. Were we to set an upper limit for early deaths as high as 2 hours, then only 1 nonpregnant nonsurvivor lived longer than this as compared to 18 pregnant nonsurvivors. Fig. 1 graphically illustrates the data on LD 50 determinations. We can say with a fair degree of certainty that the LDoo for pregnant rats of the Osborne-Mendel strain to
510
King and Becker
Am.
June 15, 1966 & Gynec.
J. Obst.
Table I. Single nicotine injection on twenty-first day of gestation: Effect of dosage on maternal - - - - - - - - - - - - - - - - - - - - - - - - - - · · - · - - - - - - - ···--------Nonpregnant
Early death* Dosage mg./Kg.
No. treated
0
20
25.0
20
0
0
2
30.0 32.5 35.0 37.5 Combined data on nicotine
20 20 20 20
1 0 5 4
4.0 0 49.2 42.3
8 2 7 II
No.
I
Survived
Late deatht
Average time
No.
I
Average time
J
No.
%
20
100
91.0
18
90
111.8 133.0 103.0 91.9
11 18 8 5
55.5 90 40
25
*Early, before I hour. tLate, after 1 hour.
10
9
50% LETHAL DOSE NICOTINE FOR NONPREGNANT RATS
50% LETHAL DOSE NICOTINE FOR PREGNANT RATS
50% LETHAL DOSE NICOTINE FOR NEWBORN RATS
(Age 140-150 OQfS)
(PrimQpQrQs On 21st /}{Jf)
(Age 6·24 HQVfS)
!).,f;j\
\~J..
(\~
L·
Oo\
\
\~L.
~
ICO
0
0::
a..
LOG 10 DOSE
I. 1.438 LOG1o DOSE
LOG 10 DOSE
Fig. 1.
subcutaneous injections of nicotine is 27.4 mg. per kilogram. Certainly with doses below 25.2, most pregnant rats will live; above 29.8, most will die. The center graph of Fig. 1 gives these data. We cannot speak with the same degree of confidence from our sample of normal, nonpregnant rats of this strain. Their data are represented in the left graph of Fig. 1 and in the left half of Table I. The erratic deviate behavior of the 32.5 mg. per kilo-
gram group departs so from the regression line as to widen the limits of the 95 per cent confidence bands considerably. There are two likely explanations for this: ( 1) subgroups of 20 animals per dose provide population samples that are still too small for an adequate assay of reliable dosage effects; ( 2) the step intervals between these high doses are so dose that all of the lower doses in the series produce much the same effect. Yet, when death falls within such narrow
511
Nicotine absorption during pregnancy
Volume 9:> r\umbe, 4
death in rats Pregnant Early death*
Survived
Late deatht -~~-~~~~~~-~
Dosage mg./Kg. 0 0 (0.50 mi. saline) 22.5 25.0 27.5 30 32.5
No. treated
No.
I
Average time
Average time I I I
No.
--··----·--· ·-··
No.
'lr 100 100
20 9
20 9 20 20 20 20 20
-~------
I I
1 1 4 5 3
10.0 30.0 25.0 37.8 37.6
limits with nicotine, there is little alternative but to select narrow step-intervals for an assay. Nevertheless, the LD 50 of 33.5 mg. per kilogram for nonpregnant rats differs from that for pregnant rats. This value falls in line with that reported by Behrend and Thienes 6 and Farris and Griffith 7 for mixed populations of nonpregnant rats, i.e., males and virgin females. We can accept with a high degree of confidence the LD5o of 14.55 mg. per kilogram for newborn young of the OsborneMendel strain shown in the right-hand graph of Fig. 1. The raw mortality data is given in Table II. All adult doses were convulsive, the first convulsion following within a minute of injection. There were from 2 to 5 convulsions before death ensued. In the early phase of convulsion, animals assumed a posture of extreme opisthotonos with the tail curved in a rigid arch over the body. This spastic state was interrupted by fitful, jerking movements, and, at times, by rapid alternate running movements as the animal lay on its side. Exophthalmos occurred in all rats. After a fit, respiration was exceedingly rapid and shallow and, at times, temporary apnea ensued. All animals righted after early convulsions and appeared temporarily recovered. But
3 5
3 10 14
80 70 65.5 25 15
16 14 13 5 3
145.6 133.6 274.6 180.7 189.4
Table II. Neonatal mortality with nicotine* Dose (gamma! gram)
I
No. of rats injected
0 10.00 11.25 12.50 13.75 15.00 17.50 20.00 *Single subcutaneous normal birth.
46 39
37
38 42 40 42 31
Died No.
%
0 6 10 13 18 19 27 29
0 15.38 27.03 34.21 42.86 47.50 64.29 93.55
injection 6
to
24
hours
after
with subsequent fits, they showed progressive malaise and became progressively cyanotic. One would have to say that deaths following terminal convulsions appeared asphyxial. Comment
The nature of death in acute nicotine poisoning of the intact organism is not exactly known. The older idea that it is due primarily to cardiac failure is not tenable, for if artificial respiration is maintained, huge doses of nicotine can be given and the heart continues to beat. Hence, most investigators agree that death is the result of respiratory arrest. 8 But, whether this arrest is centrally provoked by medullary depression, or peripherally, as a result of
512 King and Becker
some curare-like poisoning at the myoneural junction, depends upon which animal experiments one is willing to accept as most valid. In accepting either point of view on the basis of these studies, the enigma still persists for the studies are not so much concerned with nicotine poisoning of the intact organism under normal circumstances, but more with the operated organism under special experimental conditions. As Larson, Haag, and Silvctte' point out, even a unicellular organism reacts acutely to high levels of nicotine, so that in higher forms the cause of death may be multiple~the resultant of many individual toxic effects. Larson and associatesn, 10 may have had this concept in mind when they proposed at least two mechanisms to account for early and late deaths in their studies. They were concerned with the early and late deaths in anesthetized and unanesthetized rats. We have seen in this study that the pregnant rat tends to die later than the nonpregnant rat. For this reason, we cannot agree with Chen, Rose, and Robbins 11 who report that when lethal to near-lethal closes of nicotine are administered to rats, they either die promptly or recover without apparent effect. Probably, much hinges upon the definition of the term, "promptly." If death within 1Y2 to 2 hours is a prompt death, then many of our animals died a prompt and early death. But, we would have to class animals dying at 3, 6, 8, 10, and 16 hours as late deaths, and to note that late death is more peculiar to the pregnant rat. It is not for us to argue here whether these deaths resulted primarily from central respiratory depression, or whether they resulted chiefly from peripheral respiratory paralysis as the careful studies of Franke and Thomas 1 ~ strongly suggest. There is not much in our data to support either view. But in observing the series of convulsions to which the intact rat is subjected prior to death, whether it comes early or late, there appears to be a progressive interference with respiratory movements by a fixation of the respiratory muscles during the spastic seizure. Respiration becomes in-
Am.
June 15, 1966 & Gynrc.
J. Obst.
creasingly more rapid and shallow. Regardless of whether peripheral paralysis sets in before the medullary respiratory center fails, certainly some modicum of central depression remains in the picture. These series of spastic crises contribute to increased oxygen consumption and, probably, to the raising of the co" plasma level sufficient, at times, to induce temporary apnea. The final convulsion which carries the animal off differs in no whit from the first which occurs within a minute or 2 of drug injection~except that apnea is now permanent and irreversible. We would be more concerned with an explanation as to why the pregnant animal appears able to stave off death longer than the nonpregnant animal while at the same time it dies more readily of near-lethal doses of nicotine than the nonpregnant rat. Is it possible that the presence of the fetuses and their adnexa has something to do with the prolongation of the time of death? As the drug leaves the subcutaneous tissue site in a pregnant animal, we can conceive of its building up to a certain titer in fetal tissues, placenta, and even amniotic fluid. Such a compartmentalized, accessory tissue pool is not available to the nonpregnant animal. In such a tissue pool drug can be temporarily bound and released for recirculation to the mother; it can even be partially recirculated through the fetal tissues. But it is probably not detoxified there. There is good evidence that the immature liver lacks enzymes for detoxifying barbiturates and other agentsY· 14 We can make the assumption, until adequate methods are devised for measuring small amounts of nicotine in tissues with reasonable accuracy, that a certain titer of unaltered drug exists in these tissues to be recirculated to the mother allowing her to detoxify her drug load over a longer period of time. Indeed, there is precedence for such a condition to exist as we have shown in our studies of the disappearance of barbiturate from fetal and maternal tissues at various intervals after administration of the drug to the mother. 4 • If, Similar time interval studies were run on nonpregnant animals.
Volume 95 Numbrr 4
In the case of barbiturate, fetal tissue levels reached approximately 75 per cent maternal level in brain and liver. This temporary shunting of part of the drug load into an accessory tissue pool resulted in a significantly less intense reaction to drug on the part of the pregnant rat and in significantly increasing her tolerance for barbiturate beyond that of her nonpregnant counterpart. There is no reason to believe that nicotine should be handled exactly the same way as certain other drugs. Indeed, these data indicate that nicotine is not tolerated as well by the pregnant as by the nonpregnant rat. We suspect, therefore, that nicotine will be by no means equally distributed between maternal and fetal compartments. More of it may remain on the maternal side than is true in the case of some other agents where fetal tissue levels more nearly approximate maternal levels. The fetuses should comprise some 25 to 30 per cent of the total maternal weight by the twenty-first day of pregnancy, and it is this total weight upon which our drug doses were calculated. If the assumption is valid that nicotine will fail to reach reasonably equated levels on the fetal side of the placenta, a much higher titer should result in the maternal tissues, in terms of ga;TJma per gram of tissue, than is implied in the calculated dose given. This would, in part, explain our lower LD 30 for pregnant rats. As a consequence, we might expect these animals to die as quickly, if not more so, as their nonpregnant controls. Instead, there was a tendency for them to die much later. This in itself would suggest that a certain amount of unaltered drug is being recirculated from the fetal pool, tending to maintain relatively heightened titers in maternal tissues for a longer period of time than they are maintained in the nonpregnant rat. In those pregnant rats that 5urvive, the recirculating reserve which the mother must detoxify has not pushed her titer to lethal levels; in those that die, it has. Thus, there may be some individual variation in size of the storage pool and the amount and rate of return, dependent upon litter size.
Nicotine absorption during pregnancy
513
The neonatal LD,, 0 was l +55 mg. per kilogram. Yet when twice this dosage was administered to the mother, fetuses survived better than those neonates who received drug after birth. How can this differential be explained? For one thing, it tends to support our suspicion that titers in fetal tissues do not attain maternal tissue levels. Second, the fetus is not concerned with respiratory activity and does not die a respiratory death in the face of nicotine as does the airbreathing neonate. So long as oxygenation of the fetus is maintained via the maternal supply, the fetus suffers no respiratory embarrassment. It is conceivable that the vasoconstrictor effect of nicotine upon uterine and placental vessels could interfere with the adequate oxygenation of the fetus, and so some fetuses could die an asphyxial death in utero if dosage levels to the mother were high enough. We have no evidence at present that such a vasoconstrictor mechanism actually operates in utero in response to nicotine absorption. The differences in tolerance between adult and neonate may depend upon the low enzyme complement in the neonatal liver microsomes. A system for adequate nicotine detoxification may not yet have developed. Hucker, Gillette, and Brodie 1 ~ have shown that nicotine is converted to its major metabolite, cot1n1ne, by microsomal enzymes in the adult liver. The neonatal liver has been shown to be virtually incapable of detoxifying certain other drugs where similar liver enzyme systems are involved. 13 • 14 The prolongation of death in our pregnant group would lead us to believe that under certain conditions drug is not eliminated from the tissues as rapidly as the recent autoradiographic studies of Hansson and Schmitter!ow 1 ; would have us believe. Summary
Pregnant and nonpregnant rats of the Osborne-Mendel strain were injected subcutaneously with heavy doses of a 2 per cent solution of pure nicotine for the purpose of determining the LD 5 o for females of this strain. Pregnant rats received drug on the
514
King and Becker
June 15, 1966
Am. J. Obst. & Gyncc.
twenty-first day, one day before expected deiivery. The LD 50 for neonates of this strain was also determined within 6 to 24 hours of normal birth. The following values were obtained: Osborne-Mendel rats LD,, 0 Pn·gnant adults, 140 to 150 days of age Nonpregnant females, 140 to 150 days of age Neonates, 6 to 24 hours old
(mg.jKg.) 27.4 33.5 14.55
Pregnant rats tend to die significantly later
than nonpregnant rats, but their tolerance for nicotine is considerably less. The significance of these facts is discussed in terms of maternal-fetal relationships in drug detoxification. Pregnant versus nonpregnant differences in respect to tolerance for nicotine were the reverse of those consistently encountered with barbiturates. \Ve wish to express our thanks for the technical assistance of Robert James and Harold Hayes in the conduct of these studies.
REFERENCES
I. King, J. E., and Becker, R. F.: AM. J. OnsT. & GYNEC. 86: 856, 1963. 2. King, J. E., Becker, R. F., and Marsh, R. H.: AM. ]. 0BST. & GYNEC. 86: 865, 1963. 3. King, J. E., Becker, R. F., and James, R. T.: AM. J. 0BST. & GYNEC. 86: 869, 1963. 4. King, J. E.: AM, ]. On sT. & GYNEC. 89: 1019, 1964. 5. Fisher, R. A., and Yates, F.: Statistical Tables for Biological, Agricultural and Medical Research, New York, 1953, Hafner Publishing Company, pp. 9-12. 6. Behrend, A., and Thienes, C. H.: J. Pharmacol. Exper. Therap. 48: 317, 1931. 7. Farris, E. ]., and Griffith, ]. Q., Jr.: The Rat in Laboratory Investigation, ed. 2, Philadelphia, 1949, ]. B. Lippincott Company, p. 359. 8. Larson, P. S., Haag, H. B., and Silvette, H.: Tobacco, Baltimore, 1961, Williams & Wilkins Company, pp. 245-247; 462-463.
9. Larson, P. S., Finnegan, J. K., Bibb, J. C., and Haag, H. B.: Fed. Proc. 8: 313, 1949. 10. Larson, P. S., Finnegan, ]. K., and Haag, H. B.: J. Pharmacol. & Exper. Therap. 95: 506, 1949. 11. Chen, K. K., Rose, C. L., and Robbins, E. B.: Proc. Soc. Exper. Bioi. 38: 241, 1938. 12. Franke, F. E., and Thomas, J. E. A.: J. Pharmacol. & Exper. Therap. 48: 199, I 933. 13. Fouts, ]. R., and Adamson, R. H.: Science 129: 897, 1959. 14. JondorL W. R., Maickel, R. P., and Brodie, B. B.: Pharmacology 1: 352, 1959. 15. King, ]. E., Torgerson, K. E., and Becker, R. F.: Anat. Rec. 151: 372, 1965. 16. Hucker, H. B., Gillette, J. R., and Brodie, B. B.: J. Pharmacol. & Exper. Therap. 129: 94, 1960. 17. Hansson, E., and Schmitterlow, C. G.: ]. Pharmacol. & Exper. Therap. 137: 91, 1962.
Studies on nicotine absorption during pregnancy I. LDso for pregnant and nonpregnant rats
.J. EDWARD KING, A.M. R. F. BECKER, PH.D.* Durham, North Carolina
I N P R E v I o u s reports, l-·l we have shown that pregnant and nonpregnant female rats differ significantly in their tolerance and response to certain drugs, e.g., pentobarbital. In beginning a study on the effects of nicotine during pregnancy, we suspected that similar differences might hold. We also noted that nicotine dosages reported in the literature varied according to strain, and that most of the dosages reported pertained to male animals. We felt it best, therefore, to determine the 50 per cent lethal dose for both pregnant and nonpregnant females of our Osborne-Mendel strain to compare this strain with others before estimating doses that could be administered in chronic studies with these animals. Since we were going to be interested in the effects of the drug upon the fetus and neonate, we elected to determine at the same time the LD 50 for newborn young 6 to 24 hours following their normal undrugged delivery. The latter information would highlight any discrepancy m tolerance levels between adult and new-
born. Since nicotine will cross the placenta, the drug, administered to the mother, might be expected to affect the fetus adversely if the tolerance of the immature newborn was substantially below that of the adult. Preliminary probing with small population samples of 20 pregnant and 20 nonpregnant rats over four dosages ranging from 20 to 40 mg. per kilogram did, indeed, show that we might expect the pregnant rat to tolerate less of a 2 per cent solution (in saline) of Eastman Kodak highest purity nicotine* than the nonpregnant rat. This difference, as reflected in mortality, was in the reverse direction of that which we had noted consistently with barbiturates. Such a finding, in itself, justified an LD 30 testing with larger population samples.
Method Five groups ( 20 pregnant animals in each) received, respectively, 22.5, 25.0, 27.5, 30.0, and 32.5 mg. per kilogram nicotine on the twenty-first day of pregnancy, counting as day zero the day sperm were recovered from the vaginal lavage after overnight mating.
From the Department of Anatomy, Laboratory of Perinatal Studies, Duke University Medical Center. *Supported by grants-in-aid from the Council on Tobacco Research-USA and the Duke University Research Council.
*Nicotine No. 1242 BP 115-117°/12 mm., Kodak Company, Rochester, New York.
508
Eastman
Vohunt' 9:) Nttmber 4
Five groups ( 20 nonpregnant rats of thr same age in each) rec.:eived, resjJectively, 25.0, 30.0, 32.5, 35.0, and 37.5 mg. per kilogram nicotine in one subcutaneous injection, just as had the pregnant rats. Animals were assigned to dosage categories by strict random order: ( 1) when rats were determined to be pregnant by palpation; and (2) at once, in the case of nonpregnant rats. Two groups served as controls: 1. There were 20 untreated pregnant rats kept under the same environmental conditions that would be imposed upon the treated mothers, save for nicotine administration. That is, they were brought around into the laboratory on the twenty-first day in nesting boxes where they would deliver their young under observation. They were also deprived of food and water for 8 hours. Nicotine is purported to cut down on caloric intake. While we hardly expected to see much decrease in neonatal weight when nicotine treatment occurred on the twentyfirst day of gestation and delivery on the twenty-second, if there were a difference, we wished to assign it to drug effect, not to temporary dehydration or lack of maternal food intake over these 8 hours. 2. To equate for the trauma of handling and injection, 9 other pregnant controls received 0.50 c.c. saline subcutaneously. Otherwise, they were treated like the rest of the control rats. Some 300 untreated, newborn young were divided into seven dose groups with an average of 38 young per group, and these received single subcutaneous injections of 10, 11.25, 12.50, 13.75, 15.0, 17.50, or 20 gamma per gram nicotine within 6 to 24 hours of normal delivery (Table II). A similar control group of normal young received 0.075 c.c. saline. All nicotine doses for these small animals were given in volumes between 0.05 and 0.10 ml. using a 0.10 ml. microsyringe. Thus, the saline volume was the midpoint between these values. The volumes were measured to the nearest microliter. From the resulting mortality data, doses
Nicotine absorption during pregnancy
509
wPre converted to log concentratiom and per cent mortality was converted to probit values as recommended by Fisher and Yates.·, According to these authors, this procedure is based upon the supposition "that a normal deviate is linearly dependent upon some observable concomitant measurement, and that an observable frequency is that with which this deviate is exceeded in a normal distribution." Realizing that this would mean as little to the average reader as it did to us, we based the procedure on the supposition that mortality incrPases with increased concentration of the drug. The graphed results are presented as Fig. 1. Observations and results Table I demonstrates the effect of these single heavy doses of nicotine given one day before expected delivery date: ( 1) There were no deaths in the control groups. (2) Mortality increased abruptly above 27.5 mg. per kilogram in pregnant rats; above 32.5 mg. per kilogram in nonpregnant rats. Thus, the nonpregnant female continued to show a greater tolerance for this drug than the pregnant female. ( 3) Over the entire range of doses employed, mortality was 49 per cent for pregnant rats; 40 per cent for nonpregnant. (4) Deaths occurred earlier than one hour with about equal frequency in pregnant (14) and nonpregnant (10) rats, and at about the same time after injection (28.1 to 31.8 minutes). These were convulsive deaths. ( 5) However, certain animals survived for longer periods. While this was true of about equal numbers of pregnant ( 35) and nonpregnant ( 30) rats, thf.' nonpregnant in this delayed-death category died closer to 1.5 hours but the pregnant rats staved off death for 3.0 hours on the average; some for as long as 8, 10, and 16 hours. Were we to set an upper limit for early deaths as high as 2 hours, then only 1 nonpregnant nonsurvivor lived longer than this as compared to 18 pregnant nonsurvivors. Fig. 1 graphically illustrates the data on LD 50 determinations. We can say with a fair degree of certainty that the LDoo for pregnant rats of the Osborne-Mendel strain to
510
King and Becker
Am.
June 15, 1966 & Gynec.
J. Obst.
Table I. Single nicotine injection on twenty-first day of gestation: Effect of dosage on maternal - - - - - - - - - - - - - - - - - - - - - - - - - - · · - · - - - - - - - ···--------Nonpregnant
Early death* Dosage mg./Kg.
No. treated
0
20
25.0
20
0
0
2
30.0 32.5 35.0 37.5 Combined data on nicotine
20 20 20 20
1 0 5 4
4.0 0 49.2 42.3
8 2 7 II
No.
I
Survived
Late deatht
Average time
No.
I
Average time
J
No.
%
20
100
91.0
18
90
111.8 133.0 103.0 91.9
11 18 8 5
55.5 90 40
25
*Early, before I hour. tLate, after 1 hour.
10
9
50% LETHAL DOSE NICOTINE FOR NONPREGNANT RATS
50% LETHAL DOSE NICOTINE FOR PREGNANT RATS
50% LETHAL DOSE NICOTINE FOR NEWBORN RATS
(Age 140-150 OQfS)
(PrimQpQrQs On 21st /}{Jf)
(Age 6·24 HQVfS)
!).,f;j\
\~J..
(\~
L·
Oo\
\
\~L.
~
ICO
0
0::
a..
LOG 10 DOSE
I. 1.438 LOG1o DOSE
LOG 10 DOSE
Fig. 1.
subcutaneous injections of nicotine is 27.4 mg. per kilogram. Certainly with doses below 25.2, most pregnant rats will live; above 29.8, most will die. The center graph of Fig. 1 gives these data. We cannot speak with the same degree of confidence from our sample of normal, nonpregnant rats of this strain. Their data are represented in the left graph of Fig. 1 and in the left half of Table I. The erratic deviate behavior of the 32.5 mg. per kilo-
gram group departs so from the regression line as to widen the limits of the 95 per cent confidence bands considerably. There are two likely explanations for this: ( 1) subgroups of 20 animals per dose provide population samples that are still too small for an adequate assay of reliable dosage effects; ( 2) the step intervals between these high doses are so dose that all of the lower doses in the series produce much the same effect. Yet, when death falls within such narrow
511
Nicotine absorption during pregnancy
Volume 9:> r\umbe, 4
death in rats Pregnant Early death*
Survived
Late deatht -~~-~~~~~~-~
Dosage mg./Kg. 0 0 (0.50 mi. saline) 22.5 25.0 27.5 30 32.5
No. treated
No.
I
Average time
Average time I I I
No.
--··----·--· ·-··
No.
'lr 100 100
20 9
20 9 20 20 20 20 20
-~------
I I
1 1 4 5 3
10.0 30.0 25.0 37.8 37.6
limits with nicotine, there is little alternative but to select narrow step-intervals for an assay. Nevertheless, the LD 50 of 33.5 mg. per kilogram for nonpregnant rats differs from that for pregnant rats. This value falls in line with that reported by Behrend and Thienes 6 and Farris and Griffith 7 for mixed populations of nonpregnant rats, i.e., males and virgin females. We can accept with a high degree of confidence the LD5o of 14.55 mg. per kilogram for newborn young of the OsborneMendel strain shown in the right-hand graph of Fig. 1. The raw mortality data is given in Table II. All adult doses were convulsive, the first convulsion following within a minute of injection. There were from 2 to 5 convulsions before death ensued. In the early phase of convulsion, animals assumed a posture of extreme opisthotonos with the tail curved in a rigid arch over the body. This spastic state was interrupted by fitful, jerking movements, and, at times, by rapid alternate running movements as the animal lay on its side. Exophthalmos occurred in all rats. After a fit, respiration was exceedingly rapid and shallow and, at times, temporary apnea ensued. All animals righted after early convulsions and appeared temporarily recovered. But
3 5
3 10 14
80 70 65.5 25 15
16 14 13 5 3
145.6 133.6 274.6 180.7 189.4
Table II. Neonatal mortality with nicotine* Dose (gamma! gram)
I
No. of rats injected
0 10.00 11.25 12.50 13.75 15.00 17.50 20.00 *Single subcutaneous normal birth.
46 39
37
38 42 40 42 31
Died No.
%
0 6 10 13 18 19 27 29
0 15.38 27.03 34.21 42.86 47.50 64.29 93.55
injection 6
to
24
hours
after
with subsequent fits, they showed progressive malaise and became progressively cyanotic. One would have to say that deaths following terminal convulsions appeared asphyxial. Comment
The nature of death in acute nicotine poisoning of the intact organism is not exactly known. The older idea that it is due primarily to cardiac failure is not tenable, for if artificial respiration is maintained, huge doses of nicotine can be given and the heart continues to beat. Hence, most investigators agree that death is the result of respiratory arrest. 8 But, whether this arrest is centrally provoked by medullary depression, or peripherally, as a result of
512 King and Becker
some curare-like poisoning at the myoneural junction, depends upon which animal experiments one is willing to accept as most valid. In accepting either point of view on the basis of these studies, the enigma still persists for the studies are not so much concerned with nicotine poisoning of the intact organism under normal circumstances, but more with the operated organism under special experimental conditions. As Larson, Haag, and Silvctte' point out, even a unicellular organism reacts acutely to high levels of nicotine, so that in higher forms the cause of death may be multiple~the resultant of many individual toxic effects. Larson and associatesn, 10 may have had this concept in mind when they proposed at least two mechanisms to account for early and late deaths in their studies. They were concerned with the early and late deaths in anesthetized and unanesthetized rats. We have seen in this study that the pregnant rat tends to die later than the nonpregnant rat. For this reason, we cannot agree with Chen, Rose, and Robbins 11 who report that when lethal to near-lethal closes of nicotine are administered to rats, they either die promptly or recover without apparent effect. Probably, much hinges upon the definition of the term, "promptly." If death within 1Y2 to 2 hours is a prompt death, then many of our animals died a prompt and early death. But, we would have to class animals dying at 3, 6, 8, 10, and 16 hours as late deaths, and to note that late death is more peculiar to the pregnant rat. It is not for us to argue here whether these deaths resulted primarily from central respiratory depression, or whether they resulted chiefly from peripheral respiratory paralysis as the careful studies of Franke and Thomas 1 ~ strongly suggest. There is not much in our data to support either view. But in observing the series of convulsions to which the intact rat is subjected prior to death, whether it comes early or late, there appears to be a progressive interference with respiratory movements by a fixation of the respiratory muscles during the spastic seizure. Respiration becomes in-
Am.
June 15, 1966 & Gynrc.
J. Obst.
creasingly more rapid and shallow. Regardless of whether peripheral paralysis sets in before the medullary respiratory center fails, certainly some modicum of central depression remains in the picture. These series of spastic crises contribute to increased oxygen consumption and, probably, to the raising of the co" plasma level sufficient, at times, to induce temporary apnea. The final convulsion which carries the animal off differs in no whit from the first which occurs within a minute or 2 of drug injection~except that apnea is now permanent and irreversible. We would be more concerned with an explanation as to why the pregnant animal appears able to stave off death longer than the nonpregnant animal while at the same time it dies more readily of near-lethal doses of nicotine than the nonpregnant rat. Is it possible that the presence of the fetuses and their adnexa has something to do with the prolongation of the time of death? As the drug leaves the subcutaneous tissue site in a pregnant animal, we can conceive of its building up to a certain titer in fetal tissues, placenta, and even amniotic fluid. Such a compartmentalized, accessory tissue pool is not available to the nonpregnant animal. In such a tissue pool drug can be temporarily bound and released for recirculation to the mother; it can even be partially recirculated through the fetal tissues. But it is probably not detoxified there. There is good evidence that the immature liver lacks enzymes for detoxifying barbiturates and other agentsY· 14 We can make the assumption, until adequate methods are devised for measuring small amounts of nicotine in tissues with reasonable accuracy, that a certain titer of unaltered drug exists in these tissues to be recirculated to the mother allowing her to detoxify her drug load over a longer period of time. Indeed, there is precedence for such a condition to exist as we have shown in our studies of the disappearance of barbiturate from fetal and maternal tissues at various intervals after administration of the drug to the mother. 4 • If, Similar time interval studies were run on nonpregnant animals.
Volume 95 Numbrr 4
In the case of barbiturate, fetal tissue levels reached approximately 75 per cent maternal level in brain and liver. This temporary shunting of part of the drug load into an accessory tissue pool resulted in a significantly less intense reaction to drug on the part of the pregnant rat and in significantly increasing her tolerance for barbiturate beyond that of her nonpregnant counterpart. There is no reason to believe that nicotine should be handled exactly the same way as certain other drugs. Indeed, these data indicate that nicotine is not tolerated as well by the pregnant as by the nonpregnant rat. We suspect, therefore, that nicotine will be by no means equally distributed between maternal and fetal compartments. More of it may remain on the maternal side than is true in the case of some other agents where fetal tissue levels more nearly approximate maternal levels. The fetuses should comprise some 25 to 30 per cent of the total maternal weight by the twenty-first day of pregnancy, and it is this total weight upon which our drug doses were calculated. If the assumption is valid that nicotine will fail to reach reasonably equated levels on the fetal side of the placenta, a much higher titer should result in the maternal tissues, in terms of ga;TJma per gram of tissue, than is implied in the calculated dose given. This would, in part, explain our lower LD 30 for pregnant rats. As a consequence, we might expect these animals to die as quickly, if not more so, as their nonpregnant controls. Instead, there was a tendency for them to die much later. This in itself would suggest that a certain amount of unaltered drug is being recirculated from the fetal pool, tending to maintain relatively heightened titers in maternal tissues for a longer period of time than they are maintained in the nonpregnant rat. In those pregnant rats that 5urvive, the recirculating reserve which the mother must detoxify has not pushed her titer to lethal levels; in those that die, it has. Thus, there may be some individual variation in size of the storage pool and the amount and rate of return, dependent upon litter size.
Nicotine absorption during pregnancy
513
The neonatal LD,, 0 was l +55 mg. per kilogram. Yet when twice this dosage was administered to the mother, fetuses survived better than those neonates who received drug after birth. How can this differential be explained? For one thing, it tends to support our suspicion that titers in fetal tissues do not attain maternal tissue levels. Second, the fetus is not concerned with respiratory activity and does not die a respiratory death in the face of nicotine as does the airbreathing neonate. So long as oxygenation of the fetus is maintained via the maternal supply, the fetus suffers no respiratory embarrassment. It is conceivable that the vasoconstrictor effect of nicotine upon uterine and placental vessels could interfere with the adequate oxygenation of the fetus, and so some fetuses could die an asphyxial death in utero if dosage levels to the mother were high enough. We have no evidence at present that such a vasoconstrictor mechanism actually operates in utero in response to nicotine absorption. The differences in tolerance between adult and neonate may depend upon the low enzyme complement in the neonatal liver microsomes. A system for adequate nicotine detoxification may not yet have developed. Hucker, Gillette, and Brodie 1 ~ have shown that nicotine is converted to its major metabolite, cot1n1ne, by microsomal enzymes in the adult liver. The neonatal liver has been shown to be virtually incapable of detoxifying certain other drugs where similar liver enzyme systems are involved. 13 • 14 The prolongation of death in our pregnant group would lead us to believe that under certain conditions drug is not eliminated from the tissues as rapidly as the recent autoradiographic studies of Hansson and Schmitter!ow 1 ; would have us believe. Summary
Pregnant and nonpregnant rats of the Osborne-Mendel strain were injected subcutaneously with heavy doses of a 2 per cent solution of pure nicotine for the purpose of determining the LD 5 o for females of this strain. Pregnant rats received drug on the
514
King and Becker
June 15, 1966
Am. J. Obst. & Gyncc.
twenty-first day, one day before expected deiivery. The LD 50 for neonates of this strain was also determined within 6 to 24 hours of normal birth. The following values were obtained: Osborne-Mendel rats LD,, 0 Pn·gnant adults, 140 to 150 days of age Nonpregnant females, 140 to 150 days of age Neonates, 6 to 24 hours old
(mg.jKg.) 27.4 33.5 14.55
Pregnant rats tend to die significantly later
than nonpregnant rats, but their tolerance for nicotine is considerably less. The significance of these facts is discussed in terms of maternal-fetal relationships in drug detoxification. Pregnant versus nonpregnant differences in respect to tolerance for nicotine were the reverse of those consistently encountered with barbiturates. \Ve wish to express our thanks for the technical assistance of Robert James and Harold Hayes in the conduct of these studies.
REFERENCES
I. King, J. E., and Becker, R. F.: AM. J. OnsT. & GYNEC. 86: 856, 1963. 2. King, J. E., Becker, R. F., and Marsh, R. H.: AM. ]. 0BST. & GYNEC. 86: 865, 1963. 3. King, J. E., Becker, R. F., and James, R. T.: AM. J. 0BST. & GYNEC. 86: 869, 1963. 4. King, J. E.: AM, ]. On sT. & GYNEC. 89: 1019, 1964. 5. Fisher, R. A., and Yates, F.: Statistical Tables for Biological, Agricultural and Medical Research, New York, 1953, Hafner Publishing Company, pp. 9-12. 6. Behrend, A., and Thienes, C. H.: J. Pharmacol. Exper. Therap. 48: 317, 1931. 7. Farris, E. ]., and Griffith, ]. Q., Jr.: The Rat in Laboratory Investigation, ed. 2, Philadelphia, 1949, ]. B. Lippincott Company, p. 359. 8. Larson, P. S., Haag, H. B., and Silvette, H.: Tobacco, Baltimore, 1961, Williams & Wilkins Company, pp. 245-247; 462-463.
9. Larson, P. S., Finnegan, J. K., Bibb, J. C., and Haag, H. B.: Fed. Proc. 8: 313, 1949. 10. Larson, P. S., Finnegan, ]. K., and Haag, H. B.: J. Pharmacol. & Exper. Therap. 95: 506, 1949. 11. Chen, K. K., Rose, C. L., and Robbins, E. B.: Proc. Soc. Exper. Bioi. 38: 241, 1938. 12. Franke, F. E., and Thomas, J. E. A.: J. Pharmacol. & Exper. Therap. 48: 199, I 933. 13. Fouts, ]. R., and Adamson, R. H.: Science 129: 897, 1959. 14. JondorL W. R., Maickel, R. P., and Brodie, B. B.: Pharmacology 1: 352, 1959. 15. King, ]. E., Torgerson, K. E., and Becker, R. F.: Anat. Rec. 151: 372, 1965. 16. Hucker, H. B., Gillette, J. R., and Brodie, B. B.: J. Pharmacol. & Exper. Therap. 129: 94, 1960. 17. Hansson, E., and Schmitterlow, C. G.: ]. Pharmacol. & Exper. Therap. 137: 91, 1962.