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Vital effects of chronic nicotine absorption and chronic hypoxic stress during pregnancy and the nursing period
Vital effects of chronic nicotine absorption and chronic hypoxic stress during pregnancy and the nursing period R.
FREDERICK
JOAN Pontiac,
C.
BECKER, MARTIN,
Michigan,
and
PH.D.
PH.D. Durham,
North
Carolina
Four experiments were performed in which 3.0 mg. of nicotine per kilogram of body weight twice daily was administered to the grauid rat during the entire gestational period. Other pregnant rats were subjected to hypoxic stress once daily during gestation. Increasing the amount of nicotine administered as weight increased over the gestational period had no greater effect than continuing the dose begun on Day 0. Continuing injections until Day 21 resulted in no additional effects over injecting through Day 20. The offspring of rats given nicotine were significantly more underweight at birth than saline-injected controls; fewer live young were born; and mothers were delivered of their infants later and gained less weight during gestation. Offspring whpse mothers received nicotine throughout the nursing period in addition to the above had a poorer survival chance after birth. Hypoxic young tended to be lighter in weight at birth. The sex ratio was shifted to some extent for both nicotine-injected and hypoxic groups in favor of males.
Method Colony management. The
PRIOR TO THE LAST two years, our studies on nicotine absorption during pregnancy were carried out with Osborne-Mendel rats. When the Department of Veterinary Medicine discontinued breeding this strain in favor of Sprague-Dawley rats, we had to establish a new colony to continue our project. It seemed reasonable to consider the possibility of strain variation in response to the drug. As a consequence, records have been kept on the housekeeping of this new colony and on the findings in respect to nicotine tolerance which might be of interest LO other laboratories. We present such information here in addition to a report on the results of our experiments.
Sprague-Dawley-derived rats were obtained from Holtzmann. The colony was maintained on a la/IO hour light/dark cycle with temperatures maintained between 74’ to 80’ F. Humidity was 50 per cent. The females in this study were virgin, and they were mated as close to 120 days of age as possible. Thus, we are dealing throughout with first litters. At the proper age, females were placed, 6 to 8 in a cage, with a male overnight. The presence of sperm in thr vaginal lavage on the following morning was taken as an indication of pregnancy. Those animals presumed pregnant on this basis were assigned at once to one of 4 treatment cat?gories. Treatment was begun that morning. Th e presumed-pregnant rat was either: (!) given nicotine twice daily throughout pregnancy (N group = nicotine j , (2 J given nicotine twice daily throughout pregnancy and nursing (N-N group = nicotine through
From Michigan State University, College of Osteopathic Medicine, and Duke University, Center for Aging and Human Development. Supported Research American National No. HD
in part by the Committee on Tobacco and Health, Medical Association, and Institutes of Health Grant 00668.
for
by
522
Chronic
nlcotlne
nursing), (3) given saline injections twice daily (S group = control), or (4) subjected to hypoxir stress once daily in a special environmental chamber (H group = hypoxic ) Since handling of animals has been thought to affect their subsequent behavior,ls ’ a salin+injected ,group was considered to be a ntorc realistic control group than one made II~ of stork animals that would be mainly ltxft quiet in the stock cages. The females undergoing any one of these treatments were housed in individual cages. On the twelfth day, they were carefully palllatrd to reconfirm pre happened. Animals still pregnant on thcb twrnty-first day were moved to nesting cages which had solid bottoms covered with pine shavings. Infants were delivered spontanrously; there were no cesarean deliveries. Offspring were counted and weighed on the day of birth. Forty-ei,yht hours later, they were c,ategorized by sex and generally culled hy random techniques to 8 per litter. This was to equalize maternal drain during nursimg ;md to give young mice equitable opportunity tar feeding. If young mice were to be used \ubsequently for behavioral testing, culling was not as random, with an effort being made to preserve at least 4 males per litter. Females were not used in behavioral tests because of the variable effect of the ovarian cycle upon locomotor and emotional behavior. Drug parameters. An empirical, yet practical. dose of nicotine of 3.0 mg. per kilogram of hody weight was chosen for administration to the pregnant females mainly on the basis of prior studies.” Injections twice daily maintained a steadier titer over 24 hours than .I single daily injection. The rat, less sensitive than the human being to the drug, requires a fairly high dosa,ge. Human smokers absorb about I .O to 2.0 mg. of nicotine per kilogram from a pack of cigarettes per day, This dosage is sufficient to produce underweight human offsprimg and to cause a high incitlcncc, of prematurity or abortion in the cast’ of women who smoke heavily during prc’gnaricv. ‘, z
aosorptlon
and
hypoxic
stress
during
pregnancy
523
Yet, to achieve underweight, fetal-like newborn rats or to reduce litter size in the rat, the 3.0 mg. of nicotine per kilograul closr, Riven twice daily seems to be the minimal effective dose, We are not stating that thr total situation in rat and human heing is similar. Obviously, both the administration and absorption routes differ in the 2 cases. The human being receives, as well. by-products of tobacco smoke other than pure nicotine: among them is CO? which may havrk embarrassin,q effects upon the fetus by itscalf. We used Eastman Kodak nicotine. No. 1242. It was redistilled before use. h(*causc it changed color on long standing in ib; nr.ic,rinal containers. The product whc~l treshl) distilled is colorless, but it assumes \.;rrious hues from straw color to deep browri 2s :I function of temperature and shelf a?#‘. Wi* werr assured time and again that thew changes ought not to affect the chltmic;ll composition or chemical reaction of thrs drug in terms of its effectiveness to incite, convulsive behavior at proper dosage lev(xl\. Ytxt. we found lessened behavioral responsivvrtess on the part of our animals as thk. drllp darkened in its aging. We feel strongly, therefort.. that it should be used only in its clear. colorless state. After redistilling the colored product. it could be kept in ;I clear st;ktc by resealing it in small vacuum tllbes which werca kept at -78” C. lmtil rreeclc~tl 'l‘iw temperature for storage may not ht% this critical at all. This just happened to hc the telnpcrature of our particular l:\horator)freezer. Experimental groups. There was 110 h),poxic group (H ! in Experiment 1. In Experiments 2 to 4. animals from all th(b croups mentioned above (N. N-N, H, and S \VWY represented. Since each experimrntal design differed slightly from the others. all ;‘rf‘ dt>scribed separately and briefly below I;:~jwrirnr~~t 1. Rats of the N groulj rrc&cd their daily injections of 3.0 tny. of nicotine. per kilogram of body weight tllrough Day 2 1 The nicotine was +\.cn in i (:In. per milliliter of 0.9 per cent stc.rilt. s;rlincs. and injections were started on thcs :~cornint~ of the (la\; spfsrmatozoa \vcr(’ Uo111ld iI1 tilt.
524
Becker
and
Martin
vaginal lavage, considered to be Day 0. The N-N animals received the same treatment except that treatment was extended beyond pregnancy through the days of weaning to the twenty-first postnatal day. Previous reports” had indicated that nicotine could be transmitted through the milk to the nursing offspring. The total volume of drug obviously increased daily as the weight of the gravid mother increased. It dropped to lower levels, naturally, after parturition in the N-N group where maternal postnatal weight increments in no way matched the daily increments of pregnancy. Controls (S group) were weighed daily and were injected as the others at 9 A.M. and 3 P.M. but with 0.9 per cent saline on a milligrams per kilogram basis. Experiment 2. In this study, the last drug dose was administered to the N group on Day 20 instead of Day 21 in an attempt to avoid the high postnatal mortality rates cncountered among the offspring of Experiment 1. Injection on the day of delivery seemed to leave the mother in a debilitated state so that she failed to take care of the newborn rat. Injections were not resumed in the N-N group until 48 hours after birth. By this time, several pilot studies had been completed in which procedures for inducing chronic hypoxia in pregnant animals had been established. An H group could now be included for contrast of behavior since hypoxic viability parameters for mother and offspring were now known. Evidence7, 6 has been presented that mothers who smoked were prone to deliver offspring in respiratory distress. In pregnant guinea pigs, we had noted that nicotine injection caused blanching and constriction of uterine vasculature. It might, therefore, interfere with fetal oxygenation. Moreover, convulsive episodes of the mother often led to periods of transient apnea which also could disturb fetal oxygenation and possibly induce intrauterine respiratory distress-like behavior in the late fetus in utero. It was for these reasons that it was felt significant to include an H group. Such a group was present in all subsequent studies. The pregnant rats of this group were
placed, S at a time, in an airtight Lucite chamber which had an attached oxygen an:1 nitrogen source. A Beckman 0, analyzer monitored the gas mixture within the chamber throughout the treatment. The oxygen level was reduced from 21 to -l per cent over a 35 to 40 minute period. The pregnant rats were held at this low level for 5 minutes and were then removed from the chamber. Each pregnant rat received one treatment per day for 21 days. Once per day was all that time and apparatus limitations permitted. We recognize that this group was subjected to only one episode per day, whereas the other 3 groups were traumatized twice a day. The 5 per cent level was an empirical one detelrmined to be the lowest compatible state fat viability of both mother and offspring in the apparatus used. Even so, an average mortality rate of 25 to 30 per cent could be cxpetted of mothers in the late stages of pregnancy. Experiment 3. At this time, it was felt that a constant amount of drug based on the weight of the rat at Day 0 more nearly approximated the nicotine absorption parameters of the human smoker. After all, thL% pregnant female smoker does not consume more cigarettes as her weight increases during pregnancy. Thus, in this experiment, a standard dose of 3.0 mg. per kilogram was given, based up the animal’s weight on the day in which sperm was found in the vaginal lavage. While the injected amount of drug could not be exactly equated with the amount likely to be absorbed from cigarette smoke, this change in procedure seemed to be a step in the right direction. Otherwise, conditions were similar to those in Experiment 2. Experiment 4. Animals were injected on the same schedule used in Experiment I, except that a constant dosage level, not an increasing dosage level, was used. Thus, Experiments 1 and 4 with equal time periods could be compared with dosage level as the only variable. Animals in Experiments 2 and 3 formed similar comparison groups. Also, if the amount of drug given was to be more traumatizing than the time interval over which it was spread, the offspring of mothers
Chronic
nicotine
absorption
and
hypoxic
stress
during
525
pregnancy
in Experiment 1 should be more affected than those in Experiments 2, 4, and 3, and effects ought to be less severe in that order. N-Nursing Nicotine Hypoxia Saline Stock
Results Continuation of injections for one additional day, increasing the amount of the drug proportional to weight gain during pregnancy, and 20 rather than 21 days of injections did not result in any added trauma above the minimum level obtained through a constant dosage level based on weight at Day 0. All conditions were equally traumatic. An important effect of nicotine administration was the prolongation of gestation time. A total of 77 per cent of our stock animals were spontaneously delivered of their offspring on the twenty-second day following mating. The remainder were delivered by Day 23. This, then, is the record for normal Sprague-Dawley rats raised in the environment of our animal quarters. All animals of our Experimental Group H were delivered of offspring on the twentysecond day. So were most animals of the S group though some delayed until Day 23. In contrast, the 2 nicotine groups (N and N-N) were delivered of about 40 per cent of their offspring on Day 23, another 40 per cent on Day 24, and the remainder on Day 25 (Fig. 1). The 2 N groups were more variable in terms of delivery date than the other 3 groups (stock animals, hypoxic, and saline-injected) . For analytical purposes, the data on delivery for the N and N-N groups could be combined since experimental treatment of these groups did not differ until after birth. Analyses of variance were performed on each of the 4 experiments: (1) Experiment I-(N + N-N) vs. S yielded F = 33.50, df = 1 and 33, and p = < 0.01; (2) Experiment 2--(N I. N-N) vs. S and H yielded F := 19.90, df = 2 and 42, and p = < 0.01; (3) Experiment 3-(N + N-N) vs. S, H, and stock animals yielded F = 3 1.77, df = 3 and 66, and p =- < 0.01; (4) Experiment & (N + N-N) vs. S, H, and stock yielded F = 60.98, df = 3 and 65, and p = < 0.01. In respect to the matter of accumulated
23 Gestation
Fig. 1. Per cent of deliveries tion
day
across
all
24
25
Day as a function
c*f Resta-
experiments.
weight gain during pregnancy, it was clear that N and N-N treatment groups gained significantly less than S or H treatment groups (Fig, 2). Weight gains in the hypoxic group fell between those of the saline-injected and nicotine-treated animals hut did not differ significantly from levels of the saline-injected group. Pregnant animals in stock are not weighed daily as a rulel but we did get daily weight measures on i0 such animals for comparative purposes. The average weight gained by the 8 animal!: out of 10 which proved to be pregnant was I 14 grams. This fell within thr range of the figures for the saline group. Again, the data for N and N-N groups could be combined for analyses of v:~ri:mce: ( 1) Experiment 1---(N + N-N ) vs. S yielded I: = 30.48, df = 2 and 32, and p = <: 0.01: (2) Experiment 2-(N + N-N I vs. S and H yielded F == 38.30, df = 2 and 42; and p = < 0.01; (3’1 Experiment 3--(N -+ N-N) vs.
526
Becker
and
140
120c
..‘:: n‘il
Martin
-N-Nursing =Nicotine
m w
Hypoxia Soline Stock
3 Experiment
Fig. 2. Cumulative weight gain over the entire pregnancy as a function of treatment. S and H yielded F = 33.99, df = 2 and 57: and p = < 0.01; (4) Experiment 4- (N -IN-N) vs. S, H, and stock yielded F = 29.20, df = 2 and 51, and p = < 0.01. Stillbirths and neonatal and postnatal deaths are recorded in Table I. When the data from all the experiments were combined, we found that 97.8 per cent of our stock offspring were live-born. This figure can be compared with 96.4 per cent for the saline group and 98.1 per cent for the hypoxic group, but with only 67.5 and 64.6 per cent for the nicotine and nicotine-through-nursing groups, respectively. There should be no real difference between the latter two since treatment was the same up to the point of delivery. Some cannibalism was noted in the nicotine groups where mothers were prone to deliver moribund offspring. Chi square analyses on the number of offspring born alive vs. the number stillborn for each experimental group were 164.45, df = 3; 101.75, df = 4; 81.84, df = 4; and 111.07, df = -1 for Experiments 1 through 4. respectively. All cni square values were si,ynificant at p = < 0.005. (Recall that an H group was lacking for Experiment 1.) There seemed to be no question that the nicotine groups formed a population distinct
from At to 8 viable trasted
the rest in respect to fetal kvastagc. 4 days of age when litters were cullctl each, 2.6 per cent of stock offspring. at birth, had died. This fi,qure conwith 3.4 per cent for the hypoxia 11.4 per cent for the saline-injectcad group, group, 29.2 per cent for the nicotine-injected group, and 33.2 per cent for the nicotinethrough-nursing group. More extreme differences became apparent when the number of survivors after culling was compared at the termination of weaning on Day 21. Survival from culling to weaning was 83.7 per cent among stock offspring. This compared with 84.9 per cent for the salintinjected group, 71.9 per cent for the hypoxic group, 85.5 per cent for the nicotine-injectrtl group, an d only 47.1 per cent for the nicctine-through-nursing group. The mothers of this latter group, of course, were being injected with the drug daily. The death toll among the hypoxic group seemed to be due chiefly to the conditions of Experiment 2 which existed at a time when the entire colony was temporarily exposed to a viral infection. Prophylactic measures eliminated this cause before subsequent experiments were undertaken. At this particular time, the hypoxic group suffered more than any other with the exception of the N-N group wherr low rates of survival werr expected under any circumstances. On the whole, Group II offspring tended to survive better than those exposed to nicotine but not as well as young of saline-treated mothers (Table I). Chi square analyses were performed on the number of young which had died between culling and weaning (21 daysi to obtain an estimate of the severity of the postnatal mortality rate. Chi syuare values of 26.22, df = 3; 36.91, df = 4; 30.96, df =4; and 160.53, df = 4 were obtained from the data of the 4 experiments. All chi square levels were significant with p = < 0.005. There have been a number of studies in which rats, mice, and human beings have been subjected to stress and in which a change in sex ratio of resulting offspring has been attributed to the stressful treatment. Such findings usually have been incidental
Chronic
Table I. Mortality
nicotine
absorption
_____ Born aliue 1 Experiment
stress
during
N-N N s Stock
1
N-N N s
I &rre
at cujling (:h)
35.7”
(4 da*ss,) j I
C.‘u”ed at 21anddaJ.5a”t’p (5%‘)
62.7 100.0 95.3
51.4 84.8 K<.S 98.5
76.i 92.5 65.1 !e. I
2
76.4 68.0 91.3 100.0 98.8
59.8 SO.0 86.1 93.4 100.0
,:i.:+t ti4.1t v.9t 4i.91. 65.6:
N-N N s H Stock
3
69.5 66.0 95.3 95.0 99.0
79.3 79.6 98.0 98.2 98.0
T(1.H 92.1 9x.7 95.1 91 :i
N-N N s H Stock
4
66.0 73.8 99.2 99.0 98.6
61.7 54.8 80.6 9H.O 90.H
24.2 78.4 97.7 1t6.1) 96.9
Stock
*Thme females nwe injected twice trclrn offsprinq than in thr nicotine-injected +Cormspondrd
trmpcrally
527
pregr,ancy
~~~~-- -.I ~~ ~~~. ..-...-..~ /
(%)
H
_- ---
hypoxic
data on offspring
. Group
and
with
a virus
on
the day of delivery group which received
in the colony
x+hhicb killed
to the main purpose of these studies and, nlore often than not, have warranted only c,asual mention or a footnote. Table II, in which E and C stand for experimental and control groups, summarizes these findings of others. There is an inkling of a change in sex ratio in some of our data. In Experiment 4. the sex ratio for offspring of our nonhandled stock animals was 0.447. For the saline offspring whose mothers underwent some slight daily trauma, it was 0.476. For the pooled X and N-N groups, the sex ratio was 0.537, and that for the hypoxic group was 0.563. Briefly, then, it appears that trauma to the pregnant framalc rat results in a greater prcponderance of male offspring. A chi-square analysis reached only the 0.15 level of sizIlificance. which is suggestive but not con\.incing. In respect to size of litter, a comparison of Ireated and nontreated groups revealed no diffc,rence in litter size between stock, salineinjected, and hypoxic groups of mothers. Thr ‘1 nicotine ,yroups had fewer live-born oif-
which probably accounts for the last injection the mominq many
of the
the higher of Day 21.
pttrpovtion
rot still-
your,q.
spring per litter. The number of live offspring numbered 9.7 for stock litters, 10.0 for salineinjected animals, and 10.2 for hypoxic mothcrs, but only 5.7 and 5.1 for the nicotinrnursing and nicotine litters, respectively. The number of live offspring per litter is a conservative estimate in the case of these 2 groups since only the litters which produced live offspring were included. The litters in which mothers consumed or killed all of the* young were not used in the analysis. Nom, of the stock, saline-injected, or hypoxia mothers ate their young, but several nicotinetreated moth(brs did so. When both 1il.e ;mcl dead young are included in thr birth count. some differences still exist. The stock and saline-injected litter sizes both averaged 10.3 : the hypoxic group averaged 10.8. T~v figures for the N and N-N groups were 8.1 and 8.8 young per litter, respectively. Thus, nic.otint*injected groups appeared not only IO givca birth to fewer young per litter hut to iracrs more stillborn young than the othrr groups. ‘The F values from the analyses of v:lrinncc on litter sizr including both li\,p and dead
528
Becker
and
Martin
Table II. Effects of environmental _____ No. of births
Experimenter
stress to parent
on XX of offspring Ratio
Species
Stress
2,052
Man
Smoking
Gravid
female
Fewer
males
196 1
2,736
Man
Smoking
0.472
0.528
Gravid
female
Fewer
males
Herriot,
1962
2,745
Man
Smoking
0.492
0.517
Gravid
female
Fewer
males
Man
Smoking
0.457
?
Gravid
female
Fewer
males
Man
Smoking
0.492
0.5 17
Gravid
female
Fewer
males
0.447
0.546
Gravid
female
Fewer
males
female
Terris,
1969
Fraumeni, Neel,
Russell,
1964
6,544
1963
Ounstead, Schuster,
394
1965 1969
1954
E = experimcn:al
C 0.528
Results
Frazier,
1966
/
Sex stressed
E 0.499
Ravenholt,
Man
Irradiation
Man
Irradiation
0.480
0.533
Gravid
Fewer
males
Man
Irradiation
0.523
0.466
Male prior ception
to con-
Fewer
females
Man
Irradiation
0533
0.509
Male prior ception
to con-
Fewer
females
Man
Irradiation
0.539
0.497
Male prior ception
to con-
Fewer
females
Man
Irradiation
0.561
0.515
Male prior ception
to con-
Fewer
females
315
Man
Smoking
0.550
0.377
Gravid
Fewer
females
49
Man
Anxiety about grades
Low
Higher
Male student prior to conception
Fewer
males
Man
Anxiety about grades
High
Lower
Female prior conception
to
Fewer
females
to
Fewer
females
female
Not
given
Rat
Immobilization
0.632
0.472
Female prior conception
Not
given
Rat
Immobiliration
0.286
0.472
Male prior ception
to con-
Fewer
males
Mouse
Irradiation
0.504
0.510
Ma!e prior ception
to con-
Fewer
males
72,472 .qoup:
C = control
group.
offspring were as follows: (1) Experiment 1-F = 3.66, df = 2 and 53, and p < 0.05; (2) Experiment 2-F = 8.34, df = 3 and 65, and p < 0.01; (3) Experiment 3-F x 1.17, df = 3 and 66, and p = not significant; (4) Experiment 4-F = 2.02, df = 3 and 65, and p = not significant. There was considerable variance in litter size among the nicotine groups in Experiments 3 and 4. The reason for this is not known. When an analysis of variance on litter size was performed for all experiments, it appeared that it was not so much litter size but the number of live-born young that was most affected by the treatment.
Our Sprague-Dawley rats injected with saline produced no more stillbirths than stock animals that were unhandled (3.6 vs. 2.2 per cent, a nonsignificant difference). This differed from the old Osborne-Mendel strain where saline injection was a traumatizing factor.3 The most common finding in pregnant women who smoke heavily during pregnancy is that their offspring are significantly lighter in weight at birth than those of nonsmoking women. To date we have found this to be true in rats treated with nicotine.” Table III gives the mean litter weights and the individual weights of offspring at
Chronic
Table III.
Weights
of litters
nicotine
and individual
absorption
offspring
and
hypoxic
stress
during
pregnancy
at birth Average Litter
Experiment
Group
No.
of offspring
weight (Cm.)
weight
of offtfspriny (Gm.)
1
N and s
N-N
110 66
675 418
6.1 6.3
2
N and s
N-N
142 86 76
837 54.5 474
.?.9 ti.:i 6.2
N-N
240 102
1,441 664
ti.0 6.5
1,100 803 421 825
5.9 6.4 6.0 ti.8
H 3
N and s
H 4
529
Weights not taken
N and N-N
185
s I-f St Irk
125 70 122
birth for the Sprague-Dawley rats of th’s study. Stock litters are not weighed habitually, but a stock group was included in Experiment 4 for comparison. Litter weight itself is not very informative, especially if the number of young per litter varies as was the case with the nicotine-injected groups. When individual weights of young were considered, birth weights in the 2 nicotine-injected groups always tended to be lighter than those of the other experimental and control groups. Young of the saline-injected and hypoxic groups were somewhat lighter in weight at birth than stock young, leading one to assume that daily handling and incidental trauma to the mother are likely to affect birth weight. An analysis of variance was performed on the individual birth weights with the nicotine-injected groups combined for analytical purposes : (1) Experiment 1-F = 0.93, df =r 1 and 23, and p = not significant; (2 1 Experiment 2-F = 5.03, df = 2 and 32, and p < 0.05; (3) Experiment 3-F z 12.96, df = 1 and 46, and p < 0.01; (4) Experiment 4-F = 5.49, df = 2 and 48, and p < 0.01. Only Experiment 1 resulted in acceptance of the null hypothesis. Comment
The 4 experiments differed slightly in the ll:ngth of time the drug was administered and in whether the total amount of fluid injected was based on Day 0 of pregnancy or
adjusted in accordance with daily weight gain. No differences in terms of any of our measures of trauma were found among the -1 experiments. It may be that all -! combinations of length of time of injection plus amount were of such a level of severit? that differences which might have been seen with a shorter injection time and;‘or a lessrr, amount of drug were obscured. Certainly, differences between both N groups and controls were found on all measures in all csperiments. Length of gestation. Previous studies in this laboratory with Osborne-Mendel rats clemons:rated that pregnant rats injrctpcl wi:h 3.0 to 5.0 mg. of nicotine pear kilogram twice daily were delivered of their young on the average one or 2 days later tllan s:llineinjected controls and, in some instances. -+ to 5 days later. Numerous studies, both prospective and retrospective, in the human being. emphasize an opposite prematurity risk rather than a delay in delivery of infan:s by women who arc heavy smokers. Usually. pr+ maturity hns been correlated posti\rel>, with the increase in number of cigarettes smokrd daily.‘. *” In studies on human subjwts in which thcb effort is mndc to define, premsturity in terms of the duration of gestation in days rather than in terms of birth weight. the findings have varied. Some studirs indicate no difference in the duration of qtlstation time between pregnant smoktbrs and
530
Becker
and
Martin
nonsmokers.“, lz OthersL” found that more women smokers were delivered before the gestation but that there thirty-sixth week of was no difference in delivery time between smoking and nonsmoking women if gestation progressed beyond 36 weeks. The data of Underwood and associates” would tend to support the first of these observations of Russell and colleagues,‘:’ but Underwood and associates failed to make comparisons at delivery dates later than 36 weeks. There is only one study we have encountered to date which mentions delayed gestation times for women smokers.‘.’ This last study in human beings is the only one to date which bears out our findings in the rat where delayed gestation appears to be the rule. If pregnant rats are given an ineffective treatment dose of nicotine, i.e., 0.5 to 2.0 mg. per kilogram, the dosage range per 24 hours in the heavy human smoker. we find no delay in gestation time at all and no loss in birth weight of the offspring.” Maternal weight loss. Previously it was demonstrated” that pregnant Osborne-Mende1 rats lost significantly more weight during pregnancy than did controls when these rats received 3.0 to 5.0 mg. of nicotine per kilogram twice daily. Our current study with Sprague-Dawley rats indicates that weight gains in the 2 nicotine-injected groups arc significantly less than those in stock or salineinjected controls or in pregnant rats subjected to daily bouts of hypoxia. We reported that saline-injected controls lost a significanr amount of weight during pregnancy. We did not replicate the latter finding in the SpragueDawley animals, and this may be a significant strain difference. The Sprague-Dawley rats were much more docile under daily handling and nowhere near as excitable as the Osborne-Mendel rats when injected. Human studies which have mentioned maternal weight gain have found no significant differences between smoking and nonsmoking women even though the offspring of smokers weighed significantly less at birth.‘. I.* One .human study’” reports that prematurity is correlated with smoking and with low maternal weight, although, unfor-
tunately, smoking and low maternal wciglll were not directly correlated with each othr:l. Pregnant female rats wrrf’ placed irl :I chamber and exposed to cigarette, smoke which contained 15 mg. of nicotine, lettuccxleaf smoke, or lettuce-leaf smoke with 15 mg. of nicotine added. The only gronp which failed to gain weight was the one exposed to cigarette smoke.” One can anticipate the author’s conclusions, namely, that failure to gain weight must bc due to a product 01 products other than nicotine in smoke. We rannot say that nicotine does not trigger some effect, directly or secondarily, which inhibits weight gains during pregnancy. Indeed, all evidence from our studies points to significant weight loss after nicotine absorption in rats. The question remains: Do rats absorb enough nicotine from a smoke-filled chamber to become affected by it? Fetal, neonatal, and postnatal mortalit) rates. Retrospective and prospective stud& on human smokers during pregnancy yielded divided findings on an increased mortality risk for offspring. Some investigatorsI’. Is reported a high mortality rate for offspring of mothers who smoke, but others”, I”. I8 found no difference in the number of stillbirths born to smoking or nonsmoking mothers. Although Yerushalmyll found no differences in neonatal mortality rates between the 2
Chronic
nicotine
abortion (not a factor in the rat) and tho high incidence of stillbirths among women who smoke helped to eliminate many of the high-risk infants. leaving only the healthirr ones to come to term. The infants of nonsmoking mothers would then tend to range from borderline to healthy. This might esresults in which TOMplain Yerushalmy’s birth-weight babies of smokers appeared to have a better chance for survival. These irlfants may have been the cream of the crop. Evidence that increased risk of miscarriage correlates with smoking has been put forth bv certain invcstigators.lO. I9 Previously we found that nicotine-treated Osborne-Mcndel rats were delivered of fewel live young than were controls. Fewer of their live yormg lived 48 hours than those in the control group. This was essentially true in the case of our Sprague-Dawley rats treated with nicotine. \Yhile there were no significallr tlifferctncrs between our nonhandled stock animals and our saline-injected controls in tile number of young born alive, more youn,~ of the saline-injected control group failed (0 su~vi\-e to the fourth postnatal day. This ma)- mean that ivhile Sprague-Hawley rats and injection well enough tolerate handling tmotionally so ;~s not to traumatize the fetus irtjertions near the day of delivery are irksortie enough so that the mother is not willing to take proper care of her newborn young. Si&icantly IJ~W N-N young died than die? N oKspring. In Experiment 1, 92 per cent of N young survived compared with 76 per cc,rlt of N-N. In Experiment 2, which corresponded with a viral infection in the rolor+‘, 64 per cent of N offspring were alive at 21 days compared with only 23 per cent of thr N-N group. Only 63 per cent of saline-injected offspring survived the infection here also. In Experiment 3, 99 per cent ol’ the saline-injected and 92 per cent of the nicotine-injected qoup, but only 71 per cent of N-N rats were survi\~ors at weaning. TOP f&ures for Experiment 4 were 78 per cent of N and 24 per cent of N-N offspring, but 98 PC.J- c’c.nt of saline-injected young survived. On the whole. then, the figures for control and N youn? \\.ere fairly close, but the N-N
absorption
ond
hypoxic
stress
during
pregnarv
531
offspring had a significantly poorer prognosis for survival to weaning. On the other hand, if the N animals survived to culling at -1 dayi. they had a fairly good chance of being ali\-\? at weaning. This made it necessary to h-red significantly more nicotine-through-nursing mothers if WC expected sufficient offspring from this group to reach maturity \\herc, the) could eventually he used in behaGorw1 studies. Sex ratios. Parametric studies on moth~s who smoke make the point that fewer IlaIrs and more females are born to women \vho smoke.“‘, I’ These differences were not usu;~ll~~ significant and were sometimes not ~WIItioned except in a table or a footnote. Ounstead’” reported the opposite findin< in mothers who habitually were c!cli\:crtd r)! “growth-retarded” young. This group WIItained a lar,ge percentage of hra~.v smokt&rs, and they were delivered of OX5 nral~~s ;I% compared with 0.38 mairs for the c(intJr)i group (a ,qroup failing to rlcfivc~r growtliretarded young I Since the Itlale con11.o1 ratio is suspiciously lo\\, compared witlf the usunl findings in the human poplllaricjn. it would seem profitable to analvzc- ftrtur r> fmnparison groups of this kind. In ;Inothvr 3t‘ea,“’ fewer rii~ilc~ ofi’sl>rii,q were produced by WOI~~JI who wtw u ithin a proscribed number of meters fl,orri the hypocenter when the bomb was dropped on Hiroshima and Nagasaki. Neef reported sil IIilar findings in :I review of other st .Idies invol\,ing inadvertent irradiation ot‘ prtsyn;lnt human beings. Paternal irradiation pGc)r tcr conception resulted in an increased inciciellce of male births. Although cliffcrrncc*s \WI’C not always significant in these studies. the\ were in thr reported direction. lf a11 ;:~~~rlt had a mutagenic, effect. a decreilsc’ iti the number of female offspring wo~~ld h pwdictcd following paternal expostlrc.. .Irl(l ,111 increase in the number of female> offsl)riq following female exposure prior to c~oriception would be found.‘)’ Hence. such fc~lales so exposed would produce fewer males. and males so exposed would produce more 91~nh. The findings on smoking mothers tend to support this hypothesis. 21s do thmc o! Ywl
532
Becker
and
Martin
inadvertent irradiation of the parent prior to conception. Retrospective examination of 5 studies of smokers which included widely variant sociologic group?’ found support for this prediction in fathers who smoked but found no differences in sex ratio of offspring between smoking and nonsmoking mothers. Neel also noted that Russell found OPPOsite effects in animals when male mice were irradiated. Fewer male offspring were born. If changed sex ratios are a real phenomenon after stresses of this kind, perhaps their direction is species-specific. A study of the effects of sex ratio in both rats and manz3 found that the partner undergoing the least stress near the time of conception tends to reproduce its own sex in the offspring. These findings cannot be applied directly to either of the human smoking studies or to our own reported here. In our case, the stress was applied after conception. In the human smoker it must be presumed that she smoked prior to, as well as during, pregnancy (hopefully, not at the time of conception). The work of the Schuster+‘” in human beings appears to run counter to most of the human surveys since stress to the mother resulted in a preponderance of males. Our finding of sex ratios of 0.537 and 0,563 in our nicotine-injected and hypoxic respectively, lends support to the groups, data of the Schusters on the rat and Russell and associates on mice, but they are at variance with the studies on human smokers except for the one study by Ounstead. The studies on smoking mothers revealed a lessened proportion of male offspring (Table II). We find that the stresses used in our study tended toward a greater preponderance of male offspring, but, as in most of these studies, while the tendency was in this direcon
Amer.
June J. Ohtet.
15. l!Jil Gynec.
tion, it did not prove statistically significant. Our attention was directed toward sex ratios only as an afterthought. More careful records need to be kept on such data by experimenters in order that the validity or invalidity of this phenomenon may be detrrmined. In the light of the severity of the exposure, our hypoxic offspring did not measure up to our expectations of severe trauma. However, as noted previously, this group was subjected to only one stress episode a day and, therefore, cannot be truly compared to either the nicotine-injected or saline-injected groups. Except for birth weight and sex ratio, they reacted much like the stock and saline-injected controls in all parameters studied. Hypoxic females were delivered of the largest percentage of male offspring of any of the groups ; however, this factor should be reexamined since records of sex ratios werr kept only for Experiment 4. In terms of birth weight, hypoxic young fell between the low weights of the nicotine-injected group and the more normal birth weights of the salincinjected control group. Litter size. The size of the litter, live and dead, at birth is not as indicative of the effects of maternal nicotine injections as is the number of young which were stillborn. Nicotine significantly and adversely affects the offspring’s chances of being born alive. We wish to thank Dr. Kallianos of the Liggett and Myers Research Laboratories, Durham, North Carolina, for redistilling the nicotine used in these studies. We also wish to thank Dr. D. C. Martin and the Biometrics Laboratory of the Department of Statistics, North Carolina State University, Raleigh, North Carolina, for performing the statistical and computer analyses for these studies.
REFERENCES
1. Denenberg, V. H., and Karas, G. G.: Science 130: 629, 1959. 2. Retzlaff, E., Fontaine, J., and Furata, W.: Geriatrics 21: 171, 1966. 3. Becker, R. F., Little, C. R. D., and King,
J.
4. 5.
E.:
AMER.
1968. Buncher, 103: 942, Underwood,
J.
OBSTET.
GYNEC.
C. R.: AMER. J. OBSTET. 1969. P. B., Kesler, K. F.,
100:
957,
GYNEC.
O’Lane,
Chronic
10. 11. 1”. 13. 11. 15
nicotine
D. A.: Obstet. Gynec. J. M., and Callaghan, 29: 1, 1967. Perlman, H. H., and Dannenberg, A. M.: J. A. M. A. 120: 1003, 1942. Heron, H. J.: New Zeal. Med. J. 61: 5 15, 1962. Howyen, H. H.: Virginia Med. Monthly 92: ‘73, 1965. Becker, R. F., King, J. E., and Little, C. R. Il.: AMER. J. OBSTET. GYNEC. 101: 1109. 1968. Hrrriot, A., Billewicz, W. F., and Hytten. F. E.: Lancet 771: 1, 1962. Yrrushalmy. J.: AMER. J. OBSTET. GYNEC. 88: 505. 1964. OLtnstead, M.: Develop. Med. Child. Neural. 7: 479, 1965. Russell, C. S., Taylor, R., and Law, C. F.: Brit. J, Prev. Sot. Med. 22: 119, 1968. Jsrvinen, P. .4., and Osterlund, K.: Ann. Paediat. Fenn. 9-10: 18. 1963-1964. hl., and Gold, E. M.: AMER. J. OBT rrrix, STET. Gvsec. 103: 358, 1969.
absorption
16.
17. 18. 19. 20.
‘1. 2,‘. 23. 24. 25.
and
hypoxic
stress
during
pregnancy
533
Yuunoszai, M. K., Peloso, J.. and t laworth. J. C.: AMF.R. J. OBSTET. Gv~ec. 104: 13(?7. 1969. Cornstock, G. W.. and Lundin. F. E : .\~IER. J. OBSTET. GYNEC. 98: 708. 1967. Mulcahy. R.. and Knaggs, J. I; : .\SII:K. J. OBSTET. GYXEC. 101: 844. 1968. MacMahon. B., Alpert, M., and Salt3s.r. E. J.: Amer. J. Epidem. 82: 247. 1965. Nrel, J, V.: Changing Persprrtivc\ on :he Genetic Effects of Radiation. Chap. 2. Sprin,qfield, Illinois. 1963, Charles c: ThcBlrias, Pui,lisher. Scholte, P. J. I,., and Sobels. F II : Am?;. J. Hum. Genet. 16: 26, 1964. Damon, is.. Nuttall, R. L., Salber, E. .I., t.t al.: Amer. J. Epidem. 83: 5301 1966. Schuster, D. H., and Schuster. I,.: Proc. 77th Ann. Conv.. .4PA, pp. 223. and 3?5, 1969. Martin, J. C.. and Becker, R. F.: i’uvchon. Sci. 19: 59. 1970. Martin, J. C.. and Becker, R. F.: i)r\,elol). Psychohirll. Accepted for publicatioi
FREDERICK
JOAN Pontiac,
C.
BECKER, MARTIN,
Michigan,
and
PH.D.
PH.D. Durham,
North
Carolina
Four experiments were performed in which 3.0 mg. of nicotine per kilogram of body weight twice daily was administered to the grauid rat during the entire gestational period. Other pregnant rats were subjected to hypoxic stress once daily during gestation. Increasing the amount of nicotine administered as weight increased over the gestational period had no greater effect than continuing the dose begun on Day 0. Continuing injections until Day 21 resulted in no additional effects over injecting through Day 20. The offspring of rats given nicotine were significantly more underweight at birth than saline-injected controls; fewer live young were born; and mothers were delivered of their infants later and gained less weight during gestation. Offspring whpse mothers received nicotine throughout the nursing period in addition to the above had a poorer survival chance after birth. Hypoxic young tended to be lighter in weight at birth. The sex ratio was shifted to some extent for both nicotine-injected and hypoxic groups in favor of males.
Method Colony management. The
PRIOR TO THE LAST two years, our studies on nicotine absorption during pregnancy were carried out with Osborne-Mendel rats. When the Department of Veterinary Medicine discontinued breeding this strain in favor of Sprague-Dawley rats, we had to establish a new colony to continue our project. It seemed reasonable to consider the possibility of strain variation in response to the drug. As a consequence, records have been kept on the housekeeping of this new colony and on the findings in respect to nicotine tolerance which might be of interest LO other laboratories. We present such information here in addition to a report on the results of our experiments.
Sprague-Dawley-derived rats were obtained from Holtzmann. The colony was maintained on a la/IO hour light/dark cycle with temperatures maintained between 74’ to 80’ F. Humidity was 50 per cent. The females in this study were virgin, and they were mated as close to 120 days of age as possible. Thus, we are dealing throughout with first litters. At the proper age, females were placed, 6 to 8 in a cage, with a male overnight. The presence of sperm in thr vaginal lavage on the following morning was taken as an indication of pregnancy. Those animals presumed pregnant on this basis were assigned at once to one of 4 treatment cat?gories. Treatment was begun that morning. Th e presumed-pregnant rat was either: (!) given nicotine twice daily throughout pregnancy (N group = nicotine j , (2 J given nicotine twice daily throughout pregnancy and nursing (N-N group = nicotine through
From Michigan State University, College of Osteopathic Medicine, and Duke University, Center for Aging and Human Development. Supported Research American National No. HD
in part by the Committee on Tobacco and Health, Medical Association, and Institutes of Health Grant 00668.
for
by
522
Chronic
nlcotlne
nursing), (3) given saline injections twice daily (S group = control), or (4) subjected to hypoxir stress once daily in a special environmental chamber (H group = hypoxic ) Since handling of animals has been thought to affect their subsequent behavior,ls ’ a salin+injected ,group was considered to be a ntorc realistic control group than one made II~ of stork animals that would be mainly ltxft quiet in the stock cages. The females undergoing any one of these treatments were housed in individual cages. On the twelfth day, they were carefully palllatrd to reconfirm pre
aosorptlon
and
hypoxic
stress
during
pregnancy
523
Yet, to achieve underweight, fetal-like newborn rats or to reduce litter size in the rat, the 3.0 mg. of nicotine per kilograul closr, Riven twice daily seems to be the minimal effective dose, We are not stating that thr total situation in rat and human heing is similar. Obviously, both the administration and absorption routes differ in the 2 cases. The human being receives, as well. by-products of tobacco smoke other than pure nicotine: among them is CO? which may havrk embarrassin,q effects upon the fetus by itscalf. We used Eastman Kodak nicotine. No. 1242. It was redistilled before use. h(*causc it changed color on long standing in ib; nr.ic,rinal containers. The product whc~l treshl) distilled is colorless, but it assumes \.;rrious hues from straw color to deep browri 2s :I function of temperature and shelf a?#‘. Wi* werr assured time and again that thew changes ought not to affect the chltmic;ll composition or chemical reaction of thrs drug in terms of its effectiveness to incite, convulsive behavior at proper dosage lev(xl\. Ytxt. we found lessened behavioral responsivvrtess on the part of our animals as thk. drllp darkened in its aging. We feel strongly, therefort.. that it should be used only in its clear. colorless state. After redistilling the colored product. it could be kept in ;I clear st;ktc by resealing it in small vacuum tllbes which werca kept at -78” C. lmtil rreeclc~tl 'l‘iw temperature for storage may not ht% this critical at all. This just happened to hc the telnpcrature of our particular l:\horator)freezer. Experimental groups. There was 110 h),poxic group (H ! in Experiment 1. In Experiments 2 to 4. animals from all th(b croups mentioned above (N. N-N, H, and S \VWY represented. Since each experimrntal design differed slightly from the others. all ;‘rf‘ dt>scribed separately and briefly below I;:~jwrirnr~~t 1. Rats of the N groulj rrc&cd their daily injections of 3.0 tny. of nicotine. per kilogram of body weight tllrough Day 2 1 The nicotine was +\.cn in i (:In. per milliliter of 0.9 per cent stc.rilt. s;rlincs. and injections were started on thcs :~cornint~ of the (la\; spfsrmatozoa \vcr(’ Uo111ld iI1 tilt.
524
Becker
and
Martin
vaginal lavage, considered to be Day 0. The N-N animals received the same treatment except that treatment was extended beyond pregnancy through the days of weaning to the twenty-first postnatal day. Previous reports” had indicated that nicotine could be transmitted through the milk to the nursing offspring. The total volume of drug obviously increased daily as the weight of the gravid mother increased. It dropped to lower levels, naturally, after parturition in the N-N group where maternal postnatal weight increments in no way matched the daily increments of pregnancy. Controls (S group) were weighed daily and were injected as the others at 9 A.M. and 3 P.M. but with 0.9 per cent saline on a milligrams per kilogram basis. Experiment 2. In this study, the last drug dose was administered to the N group on Day 20 instead of Day 21 in an attempt to avoid the high postnatal mortality rates cncountered among the offspring of Experiment 1. Injection on the day of delivery seemed to leave the mother in a debilitated state so that she failed to take care of the newborn rat. Injections were not resumed in the N-N group until 48 hours after birth. By this time, several pilot studies had been completed in which procedures for inducing chronic hypoxia in pregnant animals had been established. An H group could now be included for contrast of behavior since hypoxic viability parameters for mother and offspring were now known. Evidence7, 6 has been presented that mothers who smoked were prone to deliver offspring in respiratory distress. In pregnant guinea pigs, we had noted that nicotine injection caused blanching and constriction of uterine vasculature. It might, therefore, interfere with fetal oxygenation. Moreover, convulsive episodes of the mother often led to periods of transient apnea which also could disturb fetal oxygenation and possibly induce intrauterine respiratory distress-like behavior in the late fetus in utero. It was for these reasons that it was felt significant to include an H group. Such a group was present in all subsequent studies. The pregnant rats of this group were
placed, S at a time, in an airtight Lucite chamber which had an attached oxygen an:1 nitrogen source. A Beckman 0, analyzer monitored the gas mixture within the chamber throughout the treatment. The oxygen level was reduced from 21 to -l per cent over a 35 to 40 minute period. The pregnant rats were held at this low level for 5 minutes and were then removed from the chamber. Each pregnant rat received one treatment per day for 21 days. Once per day was all that time and apparatus limitations permitted. We recognize that this group was subjected to only one episode per day, whereas the other 3 groups were traumatized twice a day. The 5 per cent level was an empirical one detelrmined to be the lowest compatible state fat viability of both mother and offspring in the apparatus used. Even so, an average mortality rate of 25 to 30 per cent could be cxpetted of mothers in the late stages of pregnancy. Experiment 3. At this time, it was felt that a constant amount of drug based on the weight of the rat at Day 0 more nearly approximated the nicotine absorption parameters of the human smoker. After all, thL% pregnant female smoker does not consume more cigarettes as her weight increases during pregnancy. Thus, in this experiment, a standard dose of 3.0 mg. per kilogram was given, based up the animal’s weight on the day in which sperm was found in the vaginal lavage. While the injected amount of drug could not be exactly equated with the amount likely to be absorbed from cigarette smoke, this change in procedure seemed to be a step in the right direction. Otherwise, conditions were similar to those in Experiment 2. Experiment 4. Animals were injected on the same schedule used in Experiment I, except that a constant dosage level, not an increasing dosage level, was used. Thus, Experiments 1 and 4 with equal time periods could be compared with dosage level as the only variable. Animals in Experiments 2 and 3 formed similar comparison groups. Also, if the amount of drug given was to be more traumatizing than the time interval over which it was spread, the offspring of mothers
Chronic
nicotine
absorption
and
hypoxic
stress
during
525
pregnancy
in Experiment 1 should be more affected than those in Experiments 2, 4, and 3, and effects ought to be less severe in that order. N-Nursing Nicotine Hypoxia Saline Stock
Results Continuation of injections for one additional day, increasing the amount of the drug proportional to weight gain during pregnancy, and 20 rather than 21 days of injections did not result in any added trauma above the minimum level obtained through a constant dosage level based on weight at Day 0. All conditions were equally traumatic. An important effect of nicotine administration was the prolongation of gestation time. A total of 77 per cent of our stock animals were spontaneously delivered of their offspring on the twenty-second day following mating. The remainder were delivered by Day 23. This, then, is the record for normal Sprague-Dawley rats raised in the environment of our animal quarters. All animals of our Experimental Group H were delivered of offspring on the twentysecond day. So were most animals of the S group though some delayed until Day 23. In contrast, the 2 nicotine groups (N and N-N) were delivered of about 40 per cent of their offspring on Day 23, another 40 per cent on Day 24, and the remainder on Day 25 (Fig. 1). The 2 N groups were more variable in terms of delivery date than the other 3 groups (stock animals, hypoxic, and saline-injected) . For analytical purposes, the data on delivery for the N and N-N groups could be combined since experimental treatment of these groups did not differ until after birth. Analyses of variance were performed on each of the 4 experiments: (1) Experiment I-(N + N-N) vs. S yielded F = 33.50, df = 1 and 33, and p = < 0.01; (2) Experiment 2--(N I. N-N) vs. S and H yielded F := 19.90, df = 2 and 42, and p = < 0.01; (3) Experiment 3-(N + N-N) vs. S, H, and stock animals yielded F = 3 1.77, df = 3 and 66, and p =- < 0.01; (4) Experiment & (N + N-N) vs. S, H, and stock yielded F = 60.98, df = 3 and 65, and p = < 0.01. In respect to the matter of accumulated
23 Gestation
Fig. 1. Per cent of deliveries tion
day
across
all
24
25
Day as a function
c*f Resta-
experiments.
weight gain during pregnancy, it was clear that N and N-N treatment groups gained significantly less than S or H treatment groups (Fig, 2). Weight gains in the hypoxic group fell between those of the saline-injected and nicotine-treated animals hut did not differ significantly from levels of the saline-injected group. Pregnant animals in stock are not weighed daily as a rulel but we did get daily weight measures on i0 such animals for comparative purposes. The average weight gained by the 8 animal!: out of 10 which proved to be pregnant was I 14 grams. This fell within thr range of the figures for the saline group. Again, the data for N and N-N groups could be combined for analyses of v:~ri:mce: ( 1) Experiment 1---(N + N-N ) vs. S yielded I: = 30.48, df = 2 and 32, and p = <: 0.01: (2) Experiment 2-(N + N-N I vs. S and H yielded F == 38.30, df = 2 and 42; and p = < 0.01; (3’1 Experiment 3--(N -+ N-N) vs.
526
Becker
and
140
120c
..‘:: n‘il
Martin
-N-Nursing =Nicotine
m w
Hypoxia Soline Stock
3 Experiment
Fig. 2. Cumulative weight gain over the entire pregnancy as a function of treatment. S and H yielded F = 33.99, df = 2 and 57: and p = < 0.01; (4) Experiment 4- (N -IN-N) vs. S, H, and stock yielded F = 29.20, df = 2 and 51, and p = < 0.01. Stillbirths and neonatal and postnatal deaths are recorded in Table I. When the data from all the experiments were combined, we found that 97.8 per cent of our stock offspring were live-born. This figure can be compared with 96.4 per cent for the saline group and 98.1 per cent for the hypoxic group, but with only 67.5 and 64.6 per cent for the nicotine and nicotine-through-nursing groups, respectively. There should be no real difference between the latter two since treatment was the same up to the point of delivery. Some cannibalism was noted in the nicotine groups where mothers were prone to deliver moribund offspring. Chi square analyses on the number of offspring born alive vs. the number stillborn for each experimental group were 164.45, df = 3; 101.75, df = 4; 81.84, df = 4; and 111.07, df = -1 for Experiments 1 through 4. respectively. All cni square values were si,ynificant at p = < 0.005. (Recall that an H group was lacking for Experiment 1.) There seemed to be no question that the nicotine groups formed a population distinct
from At to 8 viable trasted
the rest in respect to fetal kvastagc. 4 days of age when litters were cullctl each, 2.6 per cent of stock offspring. at birth, had died. This fi,qure conwith 3.4 per cent for the hypoxia 11.4 per cent for the saline-injectcad group, group, 29.2 per cent for the nicotine-injected group, and 33.2 per cent for the nicotinethrough-nursing group. More extreme differences became apparent when the number of survivors after culling was compared at the termination of weaning on Day 21. Survival from culling to weaning was 83.7 per cent among stock offspring. This compared with 84.9 per cent for the salintinjected group, 71.9 per cent for the hypoxic group, 85.5 per cent for the nicotine-injectrtl group, an d only 47.1 per cent for the nicctine-through-nursing group. The mothers of this latter group, of course, were being injected with the drug daily. The death toll among the hypoxic group seemed to be due chiefly to the conditions of Experiment 2 which existed at a time when the entire colony was temporarily exposed to a viral infection. Prophylactic measures eliminated this cause before subsequent experiments were undertaken. At this particular time, the hypoxic group suffered more than any other with the exception of the N-N group wherr low rates of survival werr expected under any circumstances. On the whole, Group II offspring tended to survive better than those exposed to nicotine but not as well as young of saline-treated mothers (Table I). Chi square analyses were performed on the number of young which had died between culling and weaning (21 daysi to obtain an estimate of the severity of the postnatal mortality rate. Chi syuare values of 26.22, df = 3; 36.91, df = 4; 30.96, df =4; and 160.53, df = 4 were obtained from the data of the 4 experiments. All chi square levels were significant with p = < 0.005. There have been a number of studies in which rats, mice, and human beings have been subjected to stress and in which a change in sex ratio of resulting offspring has been attributed to the stressful treatment. Such findings usually have been incidental
Chronic
Table I. Mortality
nicotine
absorption
_____ Born aliue 1 Experiment
stress
during
N-N N s Stock
1
N-N N s
I &rre
at cujling (:h)
35.7”
(4 da*ss,) j I
C.‘u”ed at 21anddaJ.5a”t’p (5%‘)
62.7 100.0 95.3
51.4 84.8 K<.S 98.5
76.i 92.5 65.1 !e. I
2
76.4 68.0 91.3 100.0 98.8
59.8 SO.0 86.1 93.4 100.0
,:i.:+t ti4.1t v.9t 4i.91. 65.6:
N-N N s H Stock
3
69.5 66.0 95.3 95.0 99.0
79.3 79.6 98.0 98.2 98.0
T(1.H 92.1 9x.7 95.1 91 :i
N-N N s H Stock
4
66.0 73.8 99.2 99.0 98.6
61.7 54.8 80.6 9H.O 90.H
24.2 78.4 97.7 1t6.1) 96.9
Stock
*Thme females nwe injected twice trclrn offsprinq than in thr nicotine-injected +Cormspondrd
trmpcrally
527
pregr,ancy
~~~~-- -.I ~~ ~~~. ..-...-..~ /
(%)
H
_- ---
hypoxic
data on offspring
. Group
and
with
a virus
on
the day of delivery group which received
in the colony
x+hhicb killed
to the main purpose of these studies and, nlore often than not, have warranted only c,asual mention or a footnote. Table II, in which E and C stand for experimental and control groups, summarizes these findings of others. There is an inkling of a change in sex ratio in some of our data. In Experiment 4. the sex ratio for offspring of our nonhandled stock animals was 0.447. For the saline offspring whose mothers underwent some slight daily trauma, it was 0.476. For the pooled X and N-N groups, the sex ratio was 0.537, and that for the hypoxic group was 0.563. Briefly, then, it appears that trauma to the pregnant framalc rat results in a greater prcponderance of male offspring. A chi-square analysis reached only the 0.15 level of sizIlificance. which is suggestive but not con\.incing. In respect to size of litter, a comparison of Ireated and nontreated groups revealed no diffc,rence in litter size between stock, salineinjected, and hypoxic groups of mothers. Thr ‘1 nicotine ,yroups had fewer live-born oif-
which probably accounts for the last injection the mominq many
of the
the higher of Day 21.
pttrpovtion
rot still-
your,q.
spring per litter. The number of live offspring numbered 9.7 for stock litters, 10.0 for salineinjected animals, and 10.2 for hypoxic mothcrs, but only 5.7 and 5.1 for the nicotinrnursing and nicotine litters, respectively. The number of live offspring per litter is a conservative estimate in the case of these 2 groups since only the litters which produced live offspring were included. The litters in which mothers consumed or killed all of the* young were not used in the analysis. Nom, of the stock, saline-injected, or hypoxia mothers ate their young, but several nicotinetreated moth(brs did so. When both 1il.e ;mcl dead young are included in thr birth count. some differences still exist. The stock and saline-injected litter sizes both averaged 10.3 : the hypoxic group averaged 10.8. T~v figures for the N and N-N groups were 8.1 and 8.8 young per litter, respectively. Thus, nic.otint*injected groups appeared not only IO givca birth to fewer young per litter hut to iracrs more stillborn young than the othrr groups. ‘The F values from the analyses of v:lrinncc on litter sizr including both li\,p and dead
528
Becker
and
Martin
Table II. Effects of environmental _____ No. of births
Experimenter
stress to parent
on XX of offspring Ratio
Species
Stress
2,052
Man
Smoking
Gravid
female
Fewer
males
196 1
2,736
Man
Smoking
0.472
0.528
Gravid
female
Fewer
males
Herriot,
1962
2,745
Man
Smoking
0.492
0.517
Gravid
female
Fewer
males
Man
Smoking
0.457
?
Gravid
female
Fewer
males
Man
Smoking
0.492
0.5 17
Gravid
female
Fewer
males
0.447
0.546
Gravid
female
Fewer
males
female
Terris,
1969
Fraumeni, Neel,
Russell,
1964
6,544
1963
Ounstead, Schuster,
394
1965 1969
1954
E = experimcn:al
C 0.528
Results
Frazier,
1966
/
Sex stressed
E 0.499
Ravenholt,
Man
Irradiation
Man
Irradiation
0.480
0.533
Gravid
Fewer
males
Man
Irradiation
0.523
0.466
Male prior ception
to con-
Fewer
females
Man
Irradiation
0533
0.509
Male prior ception
to con-
Fewer
females
Man
Irradiation
0.539
0.497
Male prior ception
to con-
Fewer
females
Man
Irradiation
0.561
0.515
Male prior ception
to con-
Fewer
females
315
Man
Smoking
0.550
0.377
Gravid
Fewer
females
49
Man
Anxiety about grades
Low
Higher
Male student prior to conception
Fewer
males
Man
Anxiety about grades
High
Lower
Female prior conception
to
Fewer
females
to
Fewer
females
female
Not
given
Rat
Immobilization
0.632
0.472
Female prior conception
Not
given
Rat
Immobiliration
0.286
0.472
Male prior ception
to con-
Fewer
males
Mouse
Irradiation
0.504
0.510
Ma!e prior ception
to con-
Fewer
males
72,472 .qoup:
C = control
group.
offspring were as follows: (1) Experiment 1-F = 3.66, df = 2 and 53, and p < 0.05; (2) Experiment 2-F = 8.34, df = 3 and 65, and p < 0.01; (3) Experiment 3-F x 1.17, df = 3 and 66, and p = not significant; (4) Experiment 4-F = 2.02, df = 3 and 65, and p = not significant. There was considerable variance in litter size among the nicotine groups in Experiments 3 and 4. The reason for this is not known. When an analysis of variance on litter size was performed for all experiments, it appeared that it was not so much litter size but the number of live-born young that was most affected by the treatment.
Our Sprague-Dawley rats injected with saline produced no more stillbirths than stock animals that were unhandled (3.6 vs. 2.2 per cent, a nonsignificant difference). This differed from the old Osborne-Mendel strain where saline injection was a traumatizing factor.3 The most common finding in pregnant women who smoke heavily during pregnancy is that their offspring are significantly lighter in weight at birth than those of nonsmoking women. To date we have found this to be true in rats treated with nicotine.” Table III gives the mean litter weights and the individual weights of offspring at
Chronic
Table III.
Weights
of litters
nicotine
and individual
absorption
offspring
and
hypoxic
stress
during
pregnancy
at birth Average Litter
Experiment
Group
No.
of offspring
weight (Cm.)
weight
of offtfspriny (Gm.)
1
N and s
N-N
110 66
675 418
6.1 6.3
2
N and s
N-N
142 86 76
837 54.5 474
.?.9 ti.:i 6.2
N-N
240 102
1,441 664
ti.0 6.5
1,100 803 421 825
5.9 6.4 6.0 ti.8
H 3
N and s
H 4
529
Weights not taken
N and N-N
185
s I-f St Irk
125 70 122
birth for the Sprague-Dawley rats of th’s study. Stock litters are not weighed habitually, but a stock group was included in Experiment 4 for comparison. Litter weight itself is not very informative, especially if the number of young per litter varies as was the case with the nicotine-injected groups. When individual weights of young were considered, birth weights in the 2 nicotine-injected groups always tended to be lighter than those of the other experimental and control groups. Young of the saline-injected and hypoxic groups were somewhat lighter in weight at birth than stock young, leading one to assume that daily handling and incidental trauma to the mother are likely to affect birth weight. An analysis of variance was performed on the individual birth weights with the nicotine-injected groups combined for analytical purposes : (1) Experiment 1-F = 0.93, df =r 1 and 23, and p = not significant; (2 1 Experiment 2-F = 5.03, df = 2 and 32, and p < 0.05; (3) Experiment 3-F z 12.96, df = 1 and 46, and p < 0.01; (4) Experiment 4-F = 5.49, df = 2 and 48, and p < 0.01. Only Experiment 1 resulted in acceptance of the null hypothesis. Comment
The 4 experiments differed slightly in the ll:ngth of time the drug was administered and in whether the total amount of fluid injected was based on Day 0 of pregnancy or
adjusted in accordance with daily weight gain. No differences in terms of any of our measures of trauma were found among the -1 experiments. It may be that all -! combinations of length of time of injection plus amount were of such a level of severit? that differences which might have been seen with a shorter injection time and;‘or a lessrr, amount of drug were obscured. Certainly, differences between both N groups and controls were found on all measures in all csperiments. Length of gestation. Previous studies in this laboratory with Osborne-Mendel rats clemons:rated that pregnant rats injrctpcl wi:h 3.0 to 5.0 mg. of nicotine pear kilogram twice daily were delivered of their young on the average one or 2 days later tllan s:llineinjected controls and, in some instances. -+ to 5 days later. Numerous studies, both prospective and retrospective, in the human being. emphasize an opposite prematurity risk rather than a delay in delivery of infan:s by women who arc heavy smokers. Usually. pr+ maturity hns been correlated posti\rel>, with the increase in number of cigarettes smokrd daily.‘. *” In studies on human subjwts in which thcb effort is mndc to define, premsturity in terms of the duration of gestation in days rather than in terms of birth weight. the findings have varied. Some studirs indicate no difference in the duration of qtlstation time between pregnant smoktbrs and
530
Becker
and
Martin
nonsmokers.“, lz OthersL” found that more women smokers were delivered before the gestation but that there thirty-sixth week of was no difference in delivery time between smoking and nonsmoking women if gestation progressed beyond 36 weeks. The data of Underwood and associates” would tend to support the first of these observations of Russell and colleagues,‘:’ but Underwood and associates failed to make comparisons at delivery dates later than 36 weeks. There is only one study we have encountered to date which mentions delayed gestation times for women smokers.‘.’ This last study in human beings is the only one to date which bears out our findings in the rat where delayed gestation appears to be the rule. If pregnant rats are given an ineffective treatment dose of nicotine, i.e., 0.5 to 2.0 mg. per kilogram, the dosage range per 24 hours in the heavy human smoker. we find no delay in gestation time at all and no loss in birth weight of the offspring.” Maternal weight loss. Previously it was demonstrated” that pregnant Osborne-Mende1 rats lost significantly more weight during pregnancy than did controls when these rats received 3.0 to 5.0 mg. of nicotine per kilogram twice daily. Our current study with Sprague-Dawley rats indicates that weight gains in the 2 nicotine-injected groups arc significantly less than those in stock or salineinjected controls or in pregnant rats subjected to daily bouts of hypoxia. We reported that saline-injected controls lost a significanr amount of weight during pregnancy. We did not replicate the latter finding in the SpragueDawley animals, and this may be a significant strain difference. The Sprague-Dawley rats were much more docile under daily handling and nowhere near as excitable as the Osborne-Mendel rats when injected. Human studies which have mentioned maternal weight gain have found no significant differences between smoking and nonsmoking women even though the offspring of smokers weighed significantly less at birth.‘. I.* One .human study’” reports that prematurity is correlated with smoking and with low maternal weight, although, unfor-
tunately, smoking and low maternal wciglll were not directly correlated with each othr:l. Pregnant female rats wrrf’ placed irl :I chamber and exposed to cigarette, smoke which contained 15 mg. of nicotine, lettuccxleaf smoke, or lettuce-leaf smoke with 15 mg. of nicotine added. The only gronp which failed to gain weight was the one exposed to cigarette smoke.” One can anticipate the author’s conclusions, namely, that failure to gain weight must bc due to a product 01 products other than nicotine in smoke. We rannot say that nicotine does not trigger some effect, directly or secondarily, which inhibits weight gains during pregnancy. Indeed, all evidence from our studies points to significant weight loss after nicotine absorption in rats. The question remains: Do rats absorb enough nicotine from a smoke-filled chamber to become affected by it? Fetal, neonatal, and postnatal mortalit) rates. Retrospective and prospective stud& on human smokers during pregnancy yielded divided findings on an increased mortality risk for offspring. Some investigatorsI’. Is reported a high mortality rate for offspring of mothers who smoke, but others”, I”. I8 found no difference in the number of stillbirths born to smoking or nonsmoking mothers. Although Yerushalmyll found no differences in neonatal mortality rates between the 2
Chronic
nicotine
abortion (not a factor in the rat) and tho high incidence of stillbirths among women who smoke helped to eliminate many of the high-risk infants. leaving only the healthirr ones to come to term. The infants of nonsmoking mothers would then tend to range from borderline to healthy. This might esresults in which TOMplain Yerushalmy’s birth-weight babies of smokers appeared to have a better chance for survival. These irlfants may have been the cream of the crop. Evidence that increased risk of miscarriage correlates with smoking has been put forth bv certain invcstigators.lO. I9 Previously we found that nicotine-treated Osborne-Mcndel rats were delivered of fewel live young than were controls. Fewer of their live yormg lived 48 hours than those in the control group. This was essentially true in the case of our Sprague-Dawley rats treated with nicotine. \Yhile there were no significallr tlifferctncrs between our nonhandled stock animals and our saline-injected controls in tile number of young born alive, more youn,~ of the saline-injected control group failed (0 su~vi\-e to the fourth postnatal day. This ma)- mean that ivhile Sprague-Hawley rats and injection well enough tolerate handling tmotionally so ;~s not to traumatize the fetus irtjertions near the day of delivery are irksortie enough so that the mother is not willing to take proper care of her newborn young. Si&icantly IJ~W N-N young died than die? N oKspring. In Experiment 1, 92 per cent of N young survived compared with 76 per cc,rlt of N-N. In Experiment 2, which corresponded with a viral infection in the rolor+‘, 64 per cent of N offspring were alive at 21 days compared with only 23 per cent of thr N-N group. Only 63 per cent of saline-injected offspring survived the infection here also. In Experiment 3, 99 per cent ol’ the saline-injected and 92 per cent of the nicotine-injected qoup, but only 71 per cent of N-N rats were survi\~ors at weaning. TOP f&ures for Experiment 4 were 78 per cent of N and 24 per cent of N-N offspring, but 98 PC.J- c’c.nt of saline-injected young survived. On the whole. then, the figures for control and N youn? \\.ere fairly close, but the N-N
absorption
ond
hypoxic
stress
during
pregnarv
531
offspring had a significantly poorer prognosis for survival to weaning. On the other hand, if the N animals survived to culling at -1 dayi. they had a fairly good chance of being ali\-\? at weaning. This made it necessary to h-red significantly more nicotine-through-nursing mothers if WC expected sufficient offspring from this group to reach maturity \\herc, the) could eventually he used in behaGorw1 studies. Sex ratios. Parametric studies on moth~s who smoke make the point that fewer IlaIrs and more females are born to women \vho smoke.“‘, I’ These differences were not usu;~ll~~ significant and were sometimes not ~WIItioned except in a table or a footnote. Ounstead’” reported the opposite findin< in mothers who habitually were c!cli\:crtd r)! “growth-retarded” young. This group WIItained a lar,ge percentage of hra~.v smokt&rs, and they were delivered of OX5 nral~~s ;I% compared with 0.38 mairs for the c(intJr)i group (a ,qroup failing to rlcfivc~r growtliretarded young I Since the Itlale con11.o1 ratio is suspiciously lo\\, compared witlf the usunl findings in the human poplllaricjn. it would seem profitable to analvzc- ftrtur r> fmnparison groups of this kind. In ;Inothvr 3t‘ea,“’ fewer rii~ilc~ ofi’sl>rii,q were produced by WOI~~JI who wtw u ithin a proscribed number of meters fl,orri the hypocenter when the bomb was dropped on Hiroshima and Nagasaki. Neef reported sil IIilar findings in :I review of other st .Idies invol\,ing inadvertent irradiation ot‘ prtsyn;lnt human beings. Paternal irradiation pGc)r tcr conception resulted in an increased inciciellce of male births. Although cliffcrrncc*s \WI’C not always significant in these studies. the\ were in thr reported direction. lf a11 ;:~~~rlt had a mutagenic, effect. a decreilsc’ iti the number of female offspring wo~~ld h pwdictcd following paternal expostlrc.. .Irl(l ,111 increase in the number of female> offsl)riq following female exposure prior to c~oriception would be found.‘)’ Hence. such fc~lales so exposed would produce fewer males. and males so exposed would produce more 91~nh. The findings on smoking mothers tend to support this hypothesis. 21s do thmc o! Ywl
532
Becker
and
Martin
inadvertent irradiation of the parent prior to conception. Retrospective examination of 5 studies of smokers which included widely variant sociologic group?’ found support for this prediction in fathers who smoked but found no differences in sex ratio of offspring between smoking and nonsmoking mothers. Neel also noted that Russell found OPPOsite effects in animals when male mice were irradiated. Fewer male offspring were born. If changed sex ratios are a real phenomenon after stresses of this kind, perhaps their direction is species-specific. A study of the effects of sex ratio in both rats and manz3 found that the partner undergoing the least stress near the time of conception tends to reproduce its own sex in the offspring. These findings cannot be applied directly to either of the human smoking studies or to our own reported here. In our case, the stress was applied after conception. In the human smoker it must be presumed that she smoked prior to, as well as during, pregnancy (hopefully, not at the time of conception). The work of the Schuster+‘” in human beings appears to run counter to most of the human surveys since stress to the mother resulted in a preponderance of males. Our finding of sex ratios of 0.537 and 0,563 in our nicotine-injected and hypoxic respectively, lends support to the groups, data of the Schusters on the rat and Russell and associates on mice, but they are at variance with the studies on human smokers except for the one study by Ounstead. The studies on smoking mothers revealed a lessened proportion of male offspring (Table II). We find that the stresses used in our study tended toward a greater preponderance of male offspring, but, as in most of these studies, while the tendency was in this direcon
Amer.
June J. Ohtet.
15. l!Jil Gynec.
tion, it did not prove statistically significant. Our attention was directed toward sex ratios only as an afterthought. More careful records need to be kept on such data by experimenters in order that the validity or invalidity of this phenomenon may be detrrmined. In the light of the severity of the exposure, our hypoxic offspring did not measure up to our expectations of severe trauma. However, as noted previously, this group was subjected to only one stress episode a day and, therefore, cannot be truly compared to either the nicotine-injected or saline-injected groups. Except for birth weight and sex ratio, they reacted much like the stock and saline-injected controls in all parameters studied. Hypoxic females were delivered of the largest percentage of male offspring of any of the groups ; however, this factor should be reexamined since records of sex ratios werr kept only for Experiment 4. In terms of birth weight, hypoxic young fell between the low weights of the nicotine-injected group and the more normal birth weights of the salincinjected control group. Litter size. The size of the litter, live and dead, at birth is not as indicative of the effects of maternal nicotine injections as is the number of young which were stillborn. Nicotine significantly and adversely affects the offspring’s chances of being born alive. We wish to thank Dr. Kallianos of the Liggett and Myers Research Laboratories, Durham, North Carolina, for redistilling the nicotine used in these studies. We also wish to thank Dr. D. C. Martin and the Biometrics Laboratory of the Department of Statistics, North Carolina State University, Raleigh, North Carolina, for performing the statistical and computer analyses for these studies.
REFERENCES
1. Denenberg, V. H., and Karas, G. G.: Science 130: 629, 1959. 2. Retzlaff, E., Fontaine, J., and Furata, W.: Geriatrics 21: 171, 1966. 3. Becker, R. F., Little, C. R. D., and King,
J.
4. 5.
E.:
AMER.
1968. Buncher, 103: 942, Underwood,
J.
OBSTET.
GYNEC.
C. R.: AMER. J. OBSTET. 1969. P. B., Kesler, K. F.,
100:
957,
GYNEC.
O’Lane,
Chronic
10. 11. 1”. 13. 11. 15
nicotine
D. A.: Obstet. Gynec. J. M., and Callaghan, 29: 1, 1967. Perlman, H. H., and Dannenberg, A. M.: J. A. M. A. 120: 1003, 1942. Heron, H. J.: New Zeal. Med. J. 61: 5 15, 1962. Howyen, H. H.: Virginia Med. Monthly 92: ‘73, 1965. Becker, R. F., King, J. E., and Little, C. R. Il.: AMER. J. OBSTET. GYNEC. 101: 1109. 1968. Hrrriot, A., Billewicz, W. F., and Hytten. F. E.: Lancet 771: 1, 1962. Yrrushalmy. J.: AMER. J. OBSTET. GYNEC. 88: 505. 1964. OLtnstead, M.: Develop. Med. Child. Neural. 7: 479, 1965. Russell, C. S., Taylor, R., and Law, C. F.: Brit. J, Prev. Sot. Med. 22: 119, 1968. Jsrvinen, P. .4., and Osterlund, K.: Ann. Paediat. Fenn. 9-10: 18. 1963-1964. hl., and Gold, E. M.: AMER. J. OBT rrrix, STET. Gvsec. 103: 358, 1969.
absorption
16.
17. 18. 19. 20.
‘1. 2,‘. 23. 24. 25.
and
hypoxic
stress
during
pregnancy
533
Yuunoszai, M. K., Peloso, J.. and t laworth. J. C.: AMF.R. J. OBSTET. Gv~ec. 104: 13(?7. 1969. Cornstock, G. W.. and Lundin. F. E : .\~IER. J. OBSTET. GYNEC. 98: 708. 1967. Mulcahy. R.. and Knaggs, J. I; : .\SII:K. J. OBSTET. GYXEC. 101: 844. 1968. MacMahon. B., Alpert, M., and Salt3s.r. E. J.: Amer. J. Epidem. 82: 247. 1965. Nrel, J, V.: Changing Persprrtivc\ on :he Genetic Effects of Radiation. Chap. 2. Sprin,qfield, Illinois. 1963, Charles c: ThcBlrias, Pui,lisher. Scholte, P. J. I,., and Sobels. F II : Am?;. J. Hum. Genet. 16: 26, 1964. Damon, is.. Nuttall, R. L., Salber, E. .I., t.t al.: Amer. J. Epidem. 83: 5301 1966. Schuster, D. H., and Schuster. I,.: Proc. 77th Ann. Conv.. .4PA, pp. 223. and 3?5, 1969. Martin, J. C.. and Becker, R. F.: i’uvchon. Sci. 19: 59. 1970. Martin, J. C.. and Becker, R. F.: i)r\,elol). Psychohirll. Accepted for publicatioi