Biochemical changes, early brain growth suppression and impaired detour learning in nicotine-treated chicks

DEVELOPMENTAL BRAIN RESEARCH

ELSEVIER

Developmental Brain Research 83 (1994) 181-189

Research report

Biochemical changes, early brain growth suppression and impaired detour learning in nicotine-treated chicks Sam N. Pennington a,b,*, Lorraine P. Sandstrom a, Ivan A. Shibley Jr. a, Sheree D. Long ~, Kelly R. Beeker a, Carlton P. Smith Jr. a, Kim Lee a, Tabitha A. Jones c, Kevin M. Cummings c, Larry W. Means c Department of Biochemistry, School of Medicine, Greent:ille, NC 27858, USA b Department of Pediatrics, School of Medicine, Greent:ille, NC 27858, USA c Department of Psychology, East Carolina Unirersily, Greenrille, NC 278.58, USA Accepted 19 July 1994

Abstract

Fetal growth suppression associated with chronic maternal intake of cigarette smoke is frequently observed in humans and studies using animal models suggest that in utero nicotine exposure is an important component of this growth suppression. The developing fetal central nervous system (CNS) is sensitive to the growth inhibitory effect of nicotine and morphological as well as functional CNS deficits may result from fetal nicotine exposure. The studies presented here show that nicotine exposure during early embryonic development ultimately inhibits the ability of 7-11 day old chicks to learn a detour task. The brain growth suppression caused by nicotine is paralleled by a failure of the early embryo brain to express the normal developmental increase in ornithine decarboxylase (ODC) activity. This biochemical change may be germane to the mechanism of nicotine-induced growth inhibition and/or nicotine-induced behavioral changes because the appropriate expression of ODC activity is essential to normal growth and differentiation in the fetal CNS. In the chick embryo, nicotine exposure alters several important signaling pathways that regulate ODC expression. For example, nicotine exposure lowers embryonic brain glucose levels and causes significant decreases in whole brain cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels and in cyclic AMP binding proteins (protein kinase-A regulatory activity). Also, in cultured chick cells, nicotine inhibits the ability of a potent mitogen (insulin) to induce ODC activity, but, paradoxically, in ovo nicotine exposure increased insulin binding and stimulated insulin rcceptor autophosphorylation in brain membranes.

Keywords: Nicotine; Detour learning; Growth; cAMP; cAMP-dependent protein kinase; Ornithine decarboxylase; Insulin: Insulin binding

1. Introduction

An extensive literature suggests that smoking has adverse effects on female fertility, conception, implantation and on fetal development. Maternal smoking causes a significant increase in fetal and perinatal morbidity [2,3,14,15,26,29,33,52] and while not all of the active agents have been identified, nicotine is thought to be a major contributor to the adverse fetal effects of cigarette smoke. H u m a n studies have shown

* Corresponding author. Department of Biochemistry, ECU, School of Medicine, Greenville, NC 27858, USA. Fax: (1) (919) 816-3388. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 3 8 0 6 ( 9 4 ) 0 0 1 3 5 - 9

that in utero exposure to the components of cigarette smoke is growth inhibitory to the developing central nervous system (CNS) and is associated with behavioral deficits in the offspring of mothers who smoke during pregnancy [14,15,52]. Although the exact quantitation of smoking's effect on fetal growth is difficult in humans because of the compounding factors of concurrent alcohol, marijuana and caffeine consumption [3,14,15], studies that factor out such interactions have shown that cigarette smoking and specifically nicotine, makes a singular contribution to growth reduction. Thus, neonates whose mothers smoked even moderately during pregnancy are hypertonic and have increased CNS excitability at 9 and 30 days of life [15]. In

182

S.N. Pennington et a L / Dez'clopmental Brain Research 83 (1094) 181-18q

addition, children exposed to nicotine during early development have been shown to have lower mental scores at 12 and 24 months of age [14] and to have lower auditory and visually evoked responses [52]. Therefore, the suppression of CNS growth associated with nicotine exposure appears to result in functional deficits within the CNS that are characteristic of a reduced rate of maturation. These data are consistent with the view that nicotine is a behavioral teratogen but it remains to be determined whether individual mothers a n d / o r fetuses differ in their biochemical response to nicotine and whether abstinence is the only effective preventive measure. Unfortunately, for the nicotine-dependent individual, abstinence is often difficult to achieve. Furthermore, repeated contact with smokers may result in significant passive exposure to nicotine [10]. For these reasons it is important to identify the biochemical pathway(s) by which nicotine alters CNS development. Because of the difficulty in quantitating drug exposure in human populations, animal models have been used to examine the effects of nicotine on fetal development. Using primarily rats as a model, the laboratories of Becker [5,6], Slotkin [34,48,49], Abel [1], Persaud [17,56] and several others [22,44] have shown that fetal nicotine exposure in animals has effects similar to those in humans. Maternal nicotine exposure (up to 5.0 m g / k g ) results in reduced embryo growth, delayed implantation and retarded parturition in rats. These effects are dependent on the strain of rats used and at the higher nicotine doses ( > 3.0 m g / k g ) a significant decrease in maternal food intake occurs. Furthermore, many of these studies used combinations of drugs, e.g. nicotine and ethanol, but carefully controlled experiments, including pair feeding of the pregnant animals, have provided strong support for the hypothesis that fetal nicotine exposure results in significant fetal growth deficiency. Similar growth suppression has been observed in developing chick embryos [16,17,25], a model that circumvents such factors as litter size and the nutritional and behavioral consequences of maternal nicotine intake while allowing characterization of the direct effects of nicotine and its interaction with nutrient availability. In an attempt to understand the molecular mechanism(s) by which nicotine inhibits fetal growth, a variety of factors have been examined. From these studies, it is known that nicotine readily crosses the placenta and accumulates in fetal lung, adrenals, kidney and brain. In fetal brain, the appearance of nicotine receptors follows a specific developmental pattern [26] and the n u m b e r of fetal nicotine receptors increases as a result of chronic nicotine exposure [34]. The work of Slotkin's group [34,48,49] and the article by Lichensteiger et al. [26] have defined the role of the nicotine receptor. These studies have shown that nicotine-in-

duced changes in receptor levels are correlated with changes in brain ornithine decarboxylase (ODC) activity and D N A levels, parameters that may bc more sensitive indicators of the effects of nicotine than growth suppression. Using a chick model, our laboratories are examining the biochemical changes and behavioral consequences resulting from fetal stress. These studies include the effects of exposure to drugs of abuse that cause growth suppression [7,32,36,37,40,41]. The hypothesis for these studies is that inappropriate cell death or retarded neuronal/glial growth in the developing brain could cause anatomical abnormalities a n d / o r mental dysfunction were the hypoplasia to occur during critical periods of cell division, migration a n d / o r differentiation. Previous studies using a chick model have shown that nicotine inhibits growth, causes several types of malformations [16,17], and may result in embryonic death [17,25]. Using the same model, we have reported [45] that a single dose of ethanol inhibits early (72-168 hours) embryo growth but, if allowed to hatch, the ethanol-treated chicks were comparable in weight to the vehicle-treated controls. However, these chicks were significantly impaired in their ability to perform a detour learning task [30-32]. Further, we have reported [8,46] that several drugs which inhibit embryonic growth (including nicotine and ethanol), decreased the expression of ornithine decarboxylase (ODC) enzymatic activity during the early stages of chick development (96-144 hours). Because O D C activity plays a central role in the development of the CNS, we now report the effect of nicotine on early embryo growth, on neonatal behaviors and on biochemical components of several pathways thought to regulate the expression of O D C activity, i.e. tissue glucose levels, tissue cyclic A M P and cyclic A M P binding proteins, tissue insulin binding levels and insulin receptor autophosphorylation. We have also examined the effect of nicotine on the ability of insulin to induce O D C activity in cultured embryonic cells.

2. Materials and methods 2.1. Embryo incubation and nicotine dosing

Unincubated fertile eggs (Arbor Acre or Hubbard strains, Webber's Hatchery, Goldsboro, NC) were stored at 10°C for no more than 7 days prior to incubation. Eggs (n = 10 to 24 per group as indicated in the text) were incubated at 37.5°C in 90% relative humidity in a forced air incubator and turned every 4 h. A single dose of nicotine free base (dose levels given in Fig, 1) was injected into the air space of the eggs at the start of incubation (day 0) using sterile chick Ringer's solution as the vehicle. Vehicle-treated embryos received chick Ringer's solution only and a total volume of 200 izl was used for both the vehicle and nicotine injections. Untreated (sedentary) eggs were incubated to control for handling and seasonal

S.N. Pennington et al. / Det,elopmental Brain Research 83 (1994) 181-189

~ , ~ c ~,

1400 Fq

g:

E 3/ F--

120o

.)

1000 •

Z I,I

~1 ,:: p

so i ~c9

8o0

[3_

NICO;IN£ DOSE (mg/eg(~}

600

-< o

3> Z E) ED :::U :> --

z

(3 2

EID :ZJ

400

':::

Frl

N O

32 200

CD

~--I

5" 0 ,/? ,i,

~

~.,: ,~] N ~

TREATMENT GROUP

Fig. 1. The effect of a single dose of nicotine on the brain weight (A) and on the total weight (B) of developing chick embryos. Eggs were injected at the start of incubation (day 0) with the indicated dose of nicotine dissolved in sterile chick Ringer's solutions as described under 'Materials and methods'. The eggs were opened on day 7 of development and the embryos removed and weighed. Each data point represents the mean of 18 to 21 embryos for each dose with "plus' the standard error indicated at each point as an error bar. The brain weights were significantly (inversely) related to the nicotine dose. If growth was measured as either decreased crown to rump length (nicotine = 24.8 +_ 1.3 mm versus vehicle = 29.8 _+0.1 mm, n = 12, f = 13.4, P = 0.0043); as decreased brain weight (nicotine = 324.2 +9.9 mg; vehicle = 408.2_+9.4 rag, f = 37.4, P < 0.0001); or as decreased whole embryo weight (nicotine = 990.5_+30 mg; vehicle = 1256+ 49 mg, f = 20.9, P = 0.0010) nicotine had a profound impact on early embryo growth. The inverse correlation between total embryo weight and nicotine dose was significant and the 25 # g / e m bryo dose resulted in a statistically significant decrease in embryo weight relative to the vehicle-dosed embryos.

variations in fertility and viability (average > 95% and 75%, respectively) based on previous experiments.

2.2. Embryo and brain wet weight At various times of development, individual eggs were opened from the blunt end and the embryo removed and freed of the associated membranes. The embryos were blotted and weighed to the nearest milligram. The cranial tissue was isolated, dissected and also weighed to the nearest milligram. For the cyclic AMP assay, embryos were dropped directly into liquid nitrogen prior to isolation of the brain.

183

five consecutive days, 12 h food-deprived chicks were given trials lasting a maximum of 240 s. Latency to enter a lighted, warm, social chamber through a detour tunnel from a dark, cool, isolated chamber was recorded for each trial. Finally, as a test of visual discrimination, food-deprived 12 day old chicks were given one session to peck a maximum of 60 times at starter grain spread on a board that had small pebbles, the same size as the starter grain, glued onto the board. The number of pecks correctly aimed at starter grain kernels during the first, second and third blocks of 20 pecks was recorded.

2.4. Cyclie A M P assay Cyclic AMP was assayed in 1:10 (weight to volume) 10C,; trichloroacetic acid (TCA) extracts of frozen brain tissue using a [3]RIA kit purchased from Diagnostic Products Corp. (Los Angeles, CA). The isolation of brain tissue by rapid freezing in liquid nitrogen prior to the TCA extraction was an important precaution to prevent post mortem generation of cyclic AMP. Tissue manipulation at higher temperatures, e.g. at 0-4°C, resulted in rapid increases in cyclic AMP levels [39].

2.5. Cyclic A M P binding (protein kinase-A regulatory) acticity Total cyclic AMP binding was measured by the method of Gill and Watson [18] as previously used in one of our labs [40].

2.6. Glucose assay Tissue glucose levels were estimated by the use of the Sigma Chemical glucose oxidase-coupled spectrophotometric assay (Bulletin #510).

2. Z Membrane preparation For membrane isolation, fresh embryonic brain tissue was isolated as described above and homogenized (1 : 111) in a sucrose (0.25 M ) / T R I S (20 mM) buffer, pH 7.4, containing 1 mM EDTA, I mM PMSF, 2 /xM leupeptin, 2 /zM pepstatin, and 1 m g / m l bacitracin using a glass/glass homogenizer. The homogenate was centrifuged at 600× g for 30 rain and the supernatant further spun at 15,000× g for 60 min in a Beckman ultracentrifuge (50 Ti rotor). The resulting pellet was washed and used as the crude membrane fraction for the study of insulin binding and receptor autophosphorylation after the determination of protein content.

2.8. Tyrosine kinase acticity assay Receptor-specific tyrosine kinase autophosphorylation activity was assayed as described by Grunberger et al. [19] and modified by Sinha et al. [47].

2.3. Behat:ioral testing 2.9. Insulin receptor assay by chemical cross linking A separate group of sedentary, vehicle and nicotine-treated chicks (total n = 88) were allowed to hatch and were tested on three behavioral paradigms between days 2 and 12 post hatching. Firstly, as a test of passive avoidance, 2-3 day old chicks were given a baseline trial to peck at a 3-mm diameter bead immediately followed by a learning trial when they were allowed to peck at the bead after the bead had been dipped in methyl anthranilate whose taste elicits a disgust response. Twenty-four hours later the chicks were give a test trial. Latency to first peck and the number of pecks at the bead were recorded for all three trials. Secondly, 7-11 day old chicks were tested for their ability to perform a detour learning task [30,32]. On

Nicotine-induced differences in insulin receptor levels were assayed in crude CNS membranes by a chemical-cross linking procedure using disuccinimidyl suberate as the cross linking reagent. After an overnight incubation of the tissue (20 /zg) with ~251-radiolabeled insulin (1.0/zCi) with and without cold insulin (10 4 M), the samples were diluted with sample buffer containing dithiothreitol (DTT) as the reducing agent and boiled for 5 min. Samples from vehicle and nicotine-treated embryos were separated in side by side lanes using SDS-PAGE (8% cross linked). Receptor location and the relative amount of bound insulin were determined by autoradiography.

18-I

S.N. Pennington et al./ Det,elopmental Brain Research 83 (1994) 181-189

2.10. Embryo tissue isolation for ODC assay At 96, 120, and 168 h of incubation, individual eggs (n = 10 for each treatment group at each time point) were opened at the blunt end and the embryos removed and freed of the associated membranes. The embryos were blotted and weighed to the nearest milligram. For embryos older than 72 h, the cranial tissue was also isolated and weighed to the nearest milligram. This tissue was collected on wet ice and assayed immediately for ODC activity.

2.11. Tissue culture media The basal media for tissue culture consisted of a 50/50 (v/v) mixture of Dulbecco's Modified Eagle Medium (DME) and Ham's F-12 media and was used for all experiments to maintain a fixed nutrient level in view of ODC's sensitivity to the depletion of basic nutrients, e.g. amino acids or glucose. For most experiments, the basal DME/F-12 media ( D / F ) was supplemented with ITS (insulin, transferrin, and selenium). A 'mitogen-rich' media was prepared for certain cultures by adding fetal calf serum (FCS, 5%) to the basal D / F media.

2.12. Culture of chick embryo tissue Untreated embryos (120 h of development) were isolated under sterile conditions. A sufficient number of embryos were pooled to yield 6 x 107 cells and the tissue minced and incubated in sterile trypsin solution (0.25%, at 37°C for 10 min), followed by washing in basal D / F media containing 5% FCS as a trypsin inhibitor. The tissue was removed from the trypsin inhibitor, washed in basal D / F media to remove the FCS and dissociated by repeatedly flushing through a fire polished Pasteur pipette. The cells were counted and viability estimated using Trypan blue exclusion. The cells were then diluted in basal D / F media plus ITS ([insulin]= 1.6× 10.-6 M) and the cells were plated at 1 x 106 cells per well in 24 well plates.

2.13. Thymidine uptake As a measure of DNA synthesis, the amount of [3H]thymidine incorporated into cultured chick cells was measured. Embryonic cells (1 x 106 cells per well) were cultured in basal D / F media plus ITS and grown in 24 well plates as described above. The cells were synchronized by being placed in D / F media without ITS for 24 h and subsequently assayed for DNA synthesis by adding 3H-labeled thymidine (35 Ci/mM) to the cells at the same time they were refed a mitogen-containing media ( D / F plus insulin). The cells were harvested at various times after mitogen addition as indicated in the figure. The cells were washed ( x 2) in ice-cold PBS and collected on filter paper using a Skatron cell harvester. The radioactivity associated with the filter paper was quantitated by liquid scintillation counting.

placed above the incubation mixture in a plastic well. At the end of 1he 15 min reaction period, 200 ~1 of 50% TCA were injected into each tube and the tubes incubated an additional 15 min at room temperature. The NaOH-saturated filter paper was rem~wed and the associated radioactivity determined by liquid scintillation counting. For tissue in culture, the cells were rinsed in PBS (×2) and a substrate solution added (0.2 ml/well) that contained 50 mM Tris (pH 7.4), 5 mM NaF, 2 mM DTT, 0.1 mM pyridoxal-5-phosphate, 0.08 mM EDTA plus 1-14C ornithine (0.1 /zCi, 58 mCi/mM). The culture plates were incubated for 60 min at 37°C after being covered by a single sheet of filter paper saturated with 2 N NaOH. After 60 rain, 50 /~1 of 50% TCA were added to each well and the plate incubated at room temperature for an additional 30 min. The filter paper was removed and the positions of the various wells lightly marked with a lead pencil. The radioactivity associated with the filter paper was quantitated by the use of a Raytest model 68000 TLC scanner. For some assays, the ODC specific inhibitor-difluoromethylornithine (DFMO, 1.0 mM) was added to the assay mixture.

2.15. Assay of mitogenic induction of ODC Using embryonic tissue cultured as described above, the effect of nicotine on insulin's ability to induce ODC activity in cultured embryonic cells was examined. After an initial 24 h period in the basal D / F media plus ITS, cultures (n = 3 for each treatment at each time point) were washed ( x 2) in PBS and synchronized by placing them in D / F media only for the next 24 h. The cultures were then refed D / F plus ITS and the ODC activity determined at 0, 2, 3, and 4 h after the initiation of the refeeding. Nicotine (100 tzM) was present during the synchronization period, during the refeeding period or both.

2.16. Statistical analyses of data Group means and standard errors as well as post hoc testing of significant differences between means for the various treatments was calculated using the general linear model procedure of the SAS/PC statistical program. Statistically significant differences between group means for vehicle and drug-treated preparations were determined by the appropriate ANOVA with P < 0.05 accepted as significant. Significant differences between individual groups were determined by the use of the Least Squares Means post-hoc test. Correlation coefficients were calculated using a non-weighted least squares procedure of the same SAS/PC program.

3. Results and discussion 3.1. Physical a n d behavioral effects A s s h o w n in Fig. 1 A a s i n g l e i n o v o d o s e o f n i c o t i n e

2.14. Ornithine decarboxylase assay ODC was assayed by one of two radiochemical methods. Embryonic brain tissue was rinsed in phosphate-buffered saline (PBS) and homogenized (using a polytron) in a buffer containing 50 mM Tris (pH 7.4), 5 mM NaF, 2 mM DTT, 0.1 mM pyridoxal-5-phosphate, and 0.08 mM EDTA. The protein content of the homogenate was determined by the BCA procedure using a kit purchased from Pierce Chemical, Rockford, IL. A 50 /zl aliquot of the homogenate was used to assay ODC activity. ODC activity was measured at 37°C as the release of 14CO2 from added 1-14C ornithine (0.1 /.LCi plus 0.3 mM cold ornithine). Using an air-tight reaction container, the released 14CO2 was trapped on a 2 N NaOH-saturated filter paper

g i v e n at t h e s t a r t o f i n c u b a t i o n (0 h ) r e s u l t e d i n s i g n i f i c a n t g r o w t h s u p p r e s s i o n a t 168 h o f d e v e l o p m e n t . I f t h e g r o w t h s u p p r e s s i o n w a s m e a s u r e d as d e c r e a s e d c r o w n t o r u m p l e n g t h , a s d e c r e a s e d b r a i n w e i g h t o r as decreased whole embryo weight, nicotine had a prof o u n d i m p a c t o n e a r l y e m b r y o g r o w t h . A s s h o w n i n Fig. 1B, t h e g r o w t h i n h i b i t i o n w a s p r o p o r t i o n a l t o t h e n i c o t i n e d o s e . A s p r e v i o u s l y r e p o r t e d [8], c h i c k s g i v e n a s i n g l e in o v o d o s e o f n i c o t i n e a n d s u b s e q u e n t l y a l l o w e d t o h a t c h , w e r e c o m p a r a b l e in w e i g h t t o v e h i c l e - t r e a t e d c h i c k s ( n i c o t i n e w e i g h t , 44.0 __ 0.7 g v e r s u s v e h i c l e

S.N. Pennington et al. / Det,elopmental Brain Research 83 (1994) 181-189

weight, 44.3_+ 0.6 g). This finding is similar to that reported by Slotkin et al. for rat fetuses exposed to nicotine during gestational days 4-13 [50]. Furthermore, nicotine appears to accumulate in fetal lung, adrenals, kidney and brain of placental animals but no data are currently available as to the distribution of nicotine in the chick embryo. It has been shown that prior to 190 h of incubation, the chick embryo has little functional liver mass and that even a volatile drug like ethanol is cleared only slowly before 200 h of incubation [40] in this model. The behavior of the nicotine-treated chicks was not different from the vehicle animals in a passive avoidance test or a visual discrimination test (Fig. 2B). However, the nicotine-treated chicks were impaired in their ability to perform a detour learning task (Fig. 2A). The nicotine-treated chicks exhibited a flatter learning curve and were significantly slower to reach the lighted, warm, feeding compartment on trials 4 and 5 compared to the sedentary or vehicle control chicks. The nicotine-exposed chicks were deficient in only the most complex of the three tasks on which they were tested, suggesting that the exposure produced subtle

d 4c!

B

~

SEOEN TARY

~

NrCOTiNE

A

lo o

,If) > ~

48

0

'1

-AVOIDANCED~SCRIMINATION

25o

(D Z m I-._J

4 ~2

d ~4 i;

3OO

03

>_

v

R7

200

Z w

15o

100

-3

DETOUR

4

3o •

-

'

-

•

vehicle 7

nicotine

25

E v

2O

Q_ 0

15

0

I 1

0

c~ o

5

i

100

120

140

160

E M B R Y O AGE ( h o u r s ) Fig. 3. Comparison of brain ODC activity in vehicle and nicotinetreated embryos as a function of age. For this particular experiment, there was an overall difference in ODC activity (df - 5,12; f = 10.8; P < 0.001) but the effect was due entirely to time of incubations ( f = 25.1, P < 0.0001 for 'hours'; f = 1.86, P = 0.19 for the drug effect and f = 1.12, P = 0.35 for the d r u g x h o u r ) but studies using additional animals did show significant differences as a function of drug treatment. Note: this figure is redrawn from ref. [8] by permission of the publisher.

4o

350 w O~ i ~ 24F,

.E E

185

5

behavioral effects. The detour task was the most complex, involving both cognitive demands (learning to use an indirect route) and the actual motor response required (locomotion as opposed to just pecking). The detour task also required that the chicks inhibit their unlearned tendency to go directly toward a visible goal, something that the nicotine-treated chicks appeared unable to do. Due to the complexity of the detour task, it is the most sensitive of the three task to retarded maturation. That the chicks were impaired on the most complex task suggests that nicotine exposure had a limited effect on the maturation of neural processes mediating behavioral plasticity or that only processes unique to the detour problem (cognitive mapping, inhibition of unlearned responses) were affected. We have previously shown that detour learning in the chick is sensitive to in ovo ethanol exposure [30,32].

LEARNING TRIALS

Fig. 2. The effect of nicotine dosing on learning behaviors in the chick. Embryos were dosed with nicotine in ovo as described in the text and the behavior of the resulting chicks tested on a detour learning task (A) and on passive avoidance and visual discrimination (B). There were no nicotine-dependent differences in the chick weights at hatching ( s e d = 44.9 g; veh = 44.3 g; etoh = 44.0 g; df = 2,54; f = 1.63; P = 0.217) or in visual discrimination or in passive avoidance but the nicotine-treated animals were markedly slower to learn a detour task. Their overall learning curve was flatter and the number of trials to criterion was significantly higher for the nicotine-treated animals on days 4 and 5 (dr = 12,348; f = 2.4; P = 0.0058) Note: the error bar were deleted from part A of the figure to aid the comparison of individual data points.

3.2. Molecular effects O D C catalyzes the conversion of ornithine to putrescine, the rate-limiting step in the synthesis of the polyamines (putrescine, spermine and spermidine) [24,51] and the polyamines are essential to fetal growth and development. Fig. 3 shows that nicotine-treatment inhibited the developmental increase in ODC activity normally seen during early embryo development. This nicotine-induced loss of ODC activity could be part of the early embryonic growth suppression, a point sup-

1~6

S,N. Pennington et al. / Decelopmental Brain Research 83 (1994) 181-189

ported by the recent finding that exogenous polyamine supplementation can prevent CNS growth suppression produced by drugs of abuse [45,46]. In like manner, the inhibition of ODC activity by a low dose of difluoromethylornithine (DFMO), a potent and specific inhibitor of ODC, causes a dose-dependent decrease in both whole embryo and brain weights in early chick embryos (see refs. [46,48]). As previously reported [8], embryonic ODC activity recovers during incubation (data not shown); again, a response similar to that observed in fetal rats exposed to nicotine during early development [50] and one that correlates with the recovery of normal size by hatching. ODC enzyme levels are responsive to a variety of mitogenic factors [9,13,21,42] and studies have shown that exposure to drugs of abuse [23] alters embryonic trophic factor (mitogen) levels and also lowers tissue responsiveness to mitogenic factors. Thus, the molecular mechanism by which nicotine prevents the normal developmental increase in ODC activity could involve changes in the embryonic response to mitogenic modulation of ODC. To test this point, we examined components of three pathways thought to regulate the level of ODC activity in developing tissue. These studies examined the effect of nicotine dosing on tissue glucose levels, the effect of nicotine on tissue cyclic AMP content and cyclic AMP binding proteins, and the effect of nicotine on insulin's mitogenic activity. These latter experiments included the determination of nicotine's effect on the ability of insulin to induce ODC activity in cultured embryonic tissue as well as the determination of nicotine's effect on tissue insulin binding and insulin receptor autophosphorylation in whole brain membrane preparations. As shown in Fig. 4A, the early chick embryo brain contains high levels of glucose. In ovo nicotine treatment significantly lowered embryo tissue glucose content, with the decrease being proportional to the nicotine dose (Fig. 4B). Such changes in tissue glucose concentration have been reported to regulate ODC expression [9,27]. Unfortunately, the present data do not differentiate between cause and effect in the nicotine/glucose relationship. However, preliminary studies do suggest that in ovo nicotine exposure inhibits embryonic tissue glucose uptake (data not shown). Changes in the concentration of cyclic AMP a n d / o r in the activity of cyclic AMP-dependent protein kinase control the expression of ODC in certain cell types [20,35,54], e.g. in cultured chick cells, the stimulation of cyclic AMP synthesis by forskolin leads to dose dependent induction of ODC activity (Fig. 5). The synthesis of cyclic AMP in response to beta-adrenergic agonists is known to be altered as the result of exposure to growth inhibitory drugs, e.g. ethanol [53], and Rabin [43] has shown that chronic drug exposure lowers tissue cyclic AMP levels in fetal brain cells grown in

80

1

a

c @ o

70

6o

ven\cle

T

8 ~

50

~ 0q

4o

,f £

t

o (D _J ©

.58

2c.

Z f~ m

\

\ \

10

\

0 ' 50

1O0

150

200

250

300

350

/~00

45(}

EMBRYO AGE ( h o u r s ) Fig. 4. The effect of nicotine on embryo brain glucose levels. Embryos were dosed as described in the text and tissue glucose assayed at the indicated times. There was an overall difference in tissue glucose (df = 7,13; f = 10.62; P < 0.0002) as well as hour- and drugdependent difference ( f = 19.1; P < 0.0001 and f = 9.12; P < 0.0098, respectively). There was no hour x drug interaction ( P = 0.098). The differences in glucose levels for the 120 and 192 h nicotine-treated embryos compared to the appropriate vehicle-treated group was significant (P = 0.0064 and P = 0.0001, respectively)

culture. We have reported decreased tissue levels of cyclic AMP and the loss of cyclic AMP-dependent regulatory and catalytic activity in embryonic brains

-j 0

~3 ~D bJ 0 E o._ o

4

>p-

N

(.9 ~

o

2,3

i vT!,lCI

, E

0 i1:

FORSKOLIN

,

,

(" i

DOSE

':

(uM)

Fig. 5. Induction of ODC activity by forskolin. O D C activity was assayed in cultured embryonic cells that were treated with forskolin and assayed as described in the text. The treatment also increased tissue cyclic AMP levels as measured by R I A (dat a not shown). These result support the postulate that increased adenylate cyclase activity (cyclic AMP levels) would be mitogenic in these cells:

S.N. Pennington et al. / Det,elopmental Brain Research 83 (1994) 181-189

"• E

F.

2000

350

I ~oo

:tD

0 G3

200

:

::

EL

<

Z

(~

0

.

.

.

.

.

.

.

.

.

.

.

.

150

!

%.1 5 o o

t~

250

~!~

Z

<

o r~ Z

.

i

VEHICLE

"---- NICOTINE

E

.

187

,

r-

rY=

f3 > ~,

0 ~ IX. o n," t.o

i [

-o

S

]:

{3 o

~IO00

___-.. hi E G. z~

;"

500

~,

: /

,

(D 100 (.D >~ 0

o

T

i

v

5O cAMP BOUND

BRAIN cAMP

Fig. 6. The effect of nicotine on cyclic AMP levels and on cyclic AMP-dependent protein kinase regulatory subunit (cyclic A M P binding) activity in 192 h embryonic brain. Tissue cyclic A M P content was measured by RIA and cyclic AMP binding was measured by a competitive binding assay as described in the text. Bar heights indicate group mean values with 'plus' the standard error indicated as an error bar (n = 6 for each group). The difference between vehicle and nicotine-treated embryos for the cyclic A M P levels and cyclic AMP binding proteins are significant ( P < 0.05 in both cases by A N O V A ) . This figure has been redrawn and corrected from data that appeared in ref. [39].

exposed to growth-inhibitory drugs, including nicotine [7,38,39]. Thus, the cyclic AMP cascade continues to be a focus of research in growth regulation involving ODC (for a review see ref. [55]). In the present study, in ovo nicotine exposure significantly lowered brain cytoplasmic fraction cyclic AMP levels (Fig. 6, right bars) and cyclic AMP-dependent protein kinase regulatory (binding) activity (Fig. 6, left bars). Since the binding of cyclic AMP was reduced by greater than 40% as the result of nicotine exposure (304 versus 185 cpm/mg protein) the data indicate a major loss of brain protein kinase regulatory subunit activity as the result of nicotine. Decreases in tissue cyclic AMP levels and protein kinase regulatory subunit activity suggest that nicotine exposure results in the disruption of a crucial second messenger pathway known to be important for normal fetal growth and development. Other studies from our lab [38] have shown that drug-induced growth inhibition is associated with decreased in vitro phosphorylation of endogenous brain proteins in growth inhibited embryonic chick brain thus suggesting that the decreases in tissue cyclic AMP and kinase regulatory activity are translated into decreases in cyclic AMP-dependent protein kinase catalytic activity. Cellular response to tyrosine kinase-linked mitogens are also altered by drug exposure. For example, insulin is a potent mitogen in chick embryo cells [4,11,12] (Fig. 7) and insulin is an effective inducer of ODC activity in embryonic cells [28,42]. Fig. 8 shows that nicotine blocks the ability of insulin to induce the expression of ODC in these cells. For embryonic chick tissue grown in serum-free media, in vitro nicotine dosing for 24 h

.

.

.

.

.

.

.

0.0

1.0

:

"

•

-

2.0

3.0

4.0

5.0

HOURS AFTER INSULIN ADDITION Fig. 7. Stimulation of thymidine uptake in cultured 120 h embryonic cells by insulin. The cells, growing in culture, were mitogen starved for 24 h and then exposed to insulin for the indicated times. The amount of [3H]thymidine incorporated by the cells was then assayed as described in the text. From these data, It is apparent that insulin is a effective mitogen for embryonic chick cells.

significantly inhibited the ability of insulin to induce ODC activity in synchronized cultures. Compared to control cultures, the presence of nicotine during the synchronization period caused a marked inhibition of the subsequent insulin-induced expression of ODC enzymatic activity. If nicotine was present during both the synchronization and the 4 h mitogen refeeding periods, little further decline in ODC was seen (data not shown). The molecular mechanism by which nicotine exposure resulted in the loss of insulin responsiveness does not appear to involve a decrease in insulin's ability to

O JZ

-:X

•

O,)

-

•

-

"' NICOTINE

CONTROL

CELLS TREATED CELLS

EO O 4 ¸ :i

E C3L

/

/i

L)

g

/ /

2 7:?

< g 0

1 L:'::'

i •

'

4

TIME AFTER I N S U L I N ADDITION ( h r s ) Fig. 8. The effect of nicotine on the induction of O D C activity by insulin. Embryonic cells were isolated at 120 h of development and cultured as described in the text. Exposure to nicotine (100 /xM) during the mitogen starvation period clearly suppressed the ability of insulin to induce O D C activity in these cells. Exposure to nicotine during the mitogen refeeding period or during both the starvation and refeeding periods gave similar results (data no shown).

t88

S.N. Pennington et al. / Developmental Brain Research 83 (1994) 181-189

c r e a s e d insulin b i n d i n g a n d s t i m u l a t e d i n s u l i n - d e p e n d ent r e c e p t o r a u t o p h o s p h o r y l a t i o n (a finding r e p o r t e d for o t h e r g r o w t h - i n h i b i t o r y drugs). T a k e n t o g e t h e r , the d a t a suggest that e i t h e r nicotine e x p o s u r e causes multip l e signaling defects, any one or all of which could block fetal growth or, t h a t n i c o t i n e blocks fetal growth at an, as yet, u n r e c o g n i z e d step that in some way i n t e r a c t s with m u l t i p l e o t h e r signaling pathways.

/

~/~ 3250

g

C3

3OC 2v~0

0,S

Z m

Z --2 O3 Z

05

1.o

~

NICOTINE BOSE (mg/egg) 2500

References

2250

I.d > 200C

5k J n,-

VEHICLE

NICOTINE

TREATMENT CROUP

Fig. 9. Nicotine-induced changes in insulin receptor function. The membrane fractions from 168 h embryo brains were isolated as described in the text and assayed for insulin binding (A) and basal and insulin-dependent receptor autophosphorylation (B). The increase in insulin binding in the nicotine-treated membranes was calculated as the net difference between binding in the presence and absence of excess cold insulin and is significant (df = 1,10; f = 6.48; P = 0.029). The correlation of decreased basal receptor autophosphorylation with the nicotine dose is significant (r =-0.952, P = 0.047) as is the increase in insulin-dependent labeling (r = 0.951, P = 0.048). i n t e r a c t with t h e cells. Fig. 9 A shows t h a t m e m b r a n e s f r o m e m b r y o s e x p o s e d to n i c o t i n e in ovo h a d i n c r e a s e d r e c e p t o r - m e d i a t e d b i n d i n g o f insulin. F u r t h e r , n i c o t i n e e x p o s u r e i n c r e a s e d i n s u l i n - m e d i a t e d r e c e p t o r aut o p h o s p h o r y l a t i o n (Fig. 9B). T h e s e d a t a suggest t h a t t h e n i c o t i n e - i n d u c e d b l o c k in O D C i n d u c t i o n is a p o s t r e c e p t o r event t h a t occurs d o w n s t r e a m from t h e insulin r e c e p t o r b u t p r i o r to t h e g e n e r a t i o n o f t h e f e e d b a c k signal(s) t h a t r e g u l a t e insulin r e c e p t o r levels. I n s u m m a r y , n i c o t i n e e x p o s u r e at t h e start o f incub a t i o n results in significant, e a r l y g r o w t h s u p p r e s s i o n in t h e d e v e l o p i n g chick e m b r y o . I f t h e chicks a r e given no a d d i t i o n a l nicotine, g r o w t h r e t u r n s to n o r m a l and, at hatching, t h e chicks a r e n o t g r o w t h s u p p r e s s e d c o m p a r e d with v e h i c l e - d o s e d controls. H o w e v e r , a l t h o u g h n o r m a l in size, t h e n i c o t i n e - t r e a t e d chicks a r e significantly slower to achieve c r i t e r i o n for a d e t o u r l e a r n i n g task. A t t h e m o l e c u l a r level, n i c o t i n e e x p o s u r e inhibits the e m b r y o ' s ability to express n o r m a l levels o f O D C e n z y m a t i c activity a n d t h e loss o f O D C is c o r r e l a t e d with t h e g r o w t h s u p p r e s s i o n . S e v e r a l p a r a m e t e r s t h a t have b e e n r e p o r t e d to r e g u l a t e O D C e x p r e s s i o n w e r e a l t e r e d by nicotine. Thus, b r a i n cyclic A M P levels a n d tissue glucose c o n t e n t w e r e b o t h d e c r e a s e d by n i c o t i n e a n d b o t h o f t h e s e p a r a m e t e r s have b e e n r e p o r t e d to r e g u l a t e O D C levels. F u r t h e r m o r e , n i c o t i n e e x p o s u r e b l o c k e d t h e ability o f insulin to i n d u c e O D C activity in c u l t u r e d e m b r y o n i c chick cells b u t t h e m e c h a n i s m o f this effect is u n c l e a r b e c a u s e n i c o t i n e e x p o s u r e in-

[1] Abel, E.L., Dintcheff, B.A. and Day, N.L., Effects of in utero exposure to alcohol, nicotine and alcohol plus nicotine, on growth and development in rats, Neurobehav. Toxicol., 1 (1979) 153-159. [2] Ahlsten, G., Cnattingius, S. and Lindmark, G., Cessation of smoking during pregnancy improves foetal growth and reduces infant morbidity in the neonatal period. Apopulation-based prospective study, Acta Paediatr., 82 (1993) 177-182. [3] Anokute, C.C., Epidemiology of spontaneous abortions: the effects of alcohol consumption and cigarette smoking, J. Natl. Med. Assoc., 78 (1986) 771-775. [4] Bassas, L., Girbau, M., Lesniak, M.A., Roth, J. and De Pablo, F., Development of receptors for insulin and insulin-like growth factor I in head and brain of chick embryos: autoradiographic localization, Endocrinology, 125 (1989) 2320-2327. [5] Becket, R.F., Little, C. and King, J.E., Experimental studies on nicotine absorption in rats during pregnancy, Am. J. Obstet. Gynecol., I00 (1968) 957-968. [6] Becker, R.F. and Martin, J.C., Vital effects of chronic nicotine absorption and chronic hypoxia stress during pregnancy and the nursing period, Am. J. Obstet. Gynecol., 110 (1971) 522-533. [7] Beeker, K., Deans, D., Elton, C. and Pennington, S.N., Ethanol-induced growth inhibition in embryonic chick brain is associated with changes in cyclic AMP-dependent protein kinase regulatory subunit, Alcohol Alcoholism, 23 (1988) 477-482. [8] Beeker, K., Smith Jr., C.P. and Pennington, S.N., Effect of cocaine, ethanol or nicotine on ornithine decarboxylase activity in early chick embryo brain, Dev. Brain Res., 69 (1992) 51-57. [9] Butler-Gralla, E. and Herschman, H.R., Glucose uptake and ODC activity in tetradecanoyt phorbol acetate non-proliferative variants, J. Cellular Physiol., 114 (1983) 317-320. [10] Curvall, M. and Enzell, C.R., Monitoring absorption by means of determination of nicotine and cotinine, Archl Toxicol., 9 (1986) 88-102. [11] De Pablo, F., Girbau, M., Gomez, J.A., Hernandez, E. and Roth, J., Insulin antibodies and insulin accelerates growth and differentiation in early embryos, Diabetes, 34 (1985) 1063-1067. [12] De Pablo, F., Scott, L.A. and Roth, J., Insulin and insulin-like growth factors in early development: peptides, receptors and biological events, Endocr. Rev., 11 (1990) 558-577. [13] Eggo, M.C., Higgins, B.P., Tam, D., Bachrach, L.K. and Burrow, G.N., Induction of ornithine decarboxylase activity by growth and differentiation factors in FRTL-5 cells, Yale J. Biol. Med., 62 (1989) 435-444. [14] Fried, P.A. and Watkinson, B., 12- and 24-month neurobehavioral follow-up of children prenatally exposed to marihuana, cigarettes and alcohol, Neurobehav. Toxicol. Teratol., 10 (1988) 305 -313. [15] Fried, P.A., Watkinson, B., Dillon, R.F. and Dulberg, C.S., Neonatal neurological status in a low-risk population after prenatal exposure to cigarettes, marijuana, and alcohol, Dev. Behay. Peds., 8 (1987) 318-326. [16] Gatling, R.R., Effects of nicotine on chick embryo, Arch. Pathol., 78 (1967) 652-657.

S.N. Pennington et al. / Det:elopmental Brain Research 83 (19941 181-189 [17] Gilani, S.H. and Persaud, T.V.N., Chick embryo development following exposure to caffeine and nicotine, Anat. Anz..lena, 161 (1986) 23-26. [18] Gill, G.N. and Walton, G.M., Assay of cyclic nucleotide-dependent protein kinases, AdL:. Cyclic Nucleotide Res., 10 (1979) 93 - 106. [19] Grunberger, G., Comi, R.J., Taylor, S.I. and Gorden, P., Tyrosine kinase activity of the insulin receptor of patients with type A extreme insulin resistance: studies with circulating mononuclear cells and cultured lymphocytes, J. Clin. Endocrin. Metab., 59 (1984) 1152-1158. [20] Haddox, M.K. and Russell, D.H., Cyclic AMP-dependent protein kinase implicated in the transcriptional induction of ODC. In O.M. Rosen and E.G. Kreds (Eds.), Protein Phosphorylation, Cold Springs Harbor, MA, 1981, pp. 1013-1035. [21] Hama, T., Huang, K. and Guroff, G., Protein kinase C as a component of a nerve growth factor-sensitive phosphorylation system in PC12 cells, Proc. NatL Acad. Sci. USA, 83 (1986) 2353-2357. [22] Hammer, R.E. and Mitchell, J.A., Nicotine reduces embryo growth, delays implantation and retards parturition in rats, Proc. Sot:. Exp. Biol. Med., 162 (1979) 333-336. [23] Heavton, M., Swanson, D., Paiva, M. and Walker, D., Ethanol exposure affects trophic factor activity and responsiveness in chick embryos, Alcohol, 9 (1992) 161-166. [24] Janne, J., Alhonen, L. and Leinonen, P., Polyamines: from molecular biology to clinical applications, Trends Mol. Med., 23 (1991) 241-259. [25] Landauer, W., Nicotine-induced malformations of chicken embryos and their bearing on the phenocopy problem, J. Exp. Zool., 143 (1960) 107-122. [26] Lichtensteiger, W., Ribary, U., Schlumpf, M., Odermatt, B. and Widmer, H., Prenatal adverse effects of nicotine on the developing brain, Prog. Brain Res., 73 (1988) 137-157. [27] Lundgren, D.W. and Porkay, S.L., Glucose elevates ODC expression in Vero Cells, J. Cellular Physiol., 137 (1988) 469-475. [28] Manzella, M.J., Rychlik, W., Rhoads, R.E., Hersery, J.W. and Blackshear, P.J., Insulin induction of ornithine decarboxylase, J. Biol. Chem., 266 (1991) 2383-2389. [29] Martin, J.C., An overview: maternal nicotine and caffeine consumption and offspring outcome, Neurobehac. Toxicol. TeratoL, 4 (1982) 421-427. [30] Means, L.W., Burnette, M. and Pennington, S.N., The effect of embryonic ethanol exposure on detour learning in the chick, Alcohol, 5 (1988) 305-308. [31] Means, L.W., Henson, J.L. and Pennington, S.N., The effects of ethanol on embryonic and hatching behaviors in the chick, IRCS Med. Sci., 14 (1986) 142-143. [32] Means, L.W., McDaniel, K. and Pennington, S.N., Embryonic ethanol exposure impairs detour learning in the chick, Alcohol, 6 (1989) 327-330. [33] Meyer, M. and Tonascia, J., Maternal smoking, pregnancy complications, and perinatal mortality, Am. J. Obstet. GynecoL, 128 (1977) 494-502. [34] Navarro, H.A., Seidler, F.J., Schwartz, R.D., Baker, F,E., Dobbins, S.S. and Slotkin, T.A., Prenatal exposure to nicotine impairs nervous system development at a dose which does not affect viability or growth, Brain Res. Bull., 23 (1989) 187-192. [35] Nishiguchi, S., Otani, S., Matsui-Yuasa, I., Morisawa, S., Monna, T., Kuroki, T. and Kobayashi, K., Inhibition of ODC induction by interferon and its reversal by dibutryl-adenosine 3', 5'-monophosphate, Eur. J. Biochem., 172 (1988) 287-292. [36] Pennington, S.N., Ethanol-induced growth inhibition: the role of cyclic AMP dependent protein kinase, Alcoholism: Clin. Exp. Res., 12 (19881 125-130. [37] Pennington, S.N., Alcohol metabolism and fetal brain hypoplasia, Alcohol, 5 (1988) 91-94.

189

[38] Pennington, S.N., Molecular changes associated with ethanol-induced growth suppression in the chick embryo, Alcoholism: Clin. Exp. Res., 14 (1990) 832-837. [39] Pennington, S.N., Ethanol-induced teratology and second messenger signal transduction. In M.W. Miller (Ed.), De~,elopment of the Central Nert~ous System: Effects of Alcohol and Opiates, Wiley-Liss, New York, NY, 1992, pp. 1890-2007. [40] Pennington, S.N., Boyd, J.W., Kalmus, G. and Wilson, R., The molecular mechanism of fetal alcohol syndrome (FAS). I. Ethanol-induced growth suppression, Neurobehac. Toxicol., 5 (1983) 259-262. [41] Pennington, S.N. and Kalmus, G., Brain growth during ethanolinduced hypoplasia, Drug Alcohol Depend, 20 (1988) 279-286. [42] Potter, V.R., Evanson, T.R., Gayda, D.P. and Gurr, J.A., Cultured hepatoma cells for the study of enzyme regulation: induction of ODC by insulin and asparagine, In Vitro, 2(1 (1984) 723-731. [43] Rabin, R.A., Direct effects of chronic ethanol exposure on beta-adrenergic and adenosine-sensitive adenylate cyclase activities and cyclic AMP content in primary cerebellar cultures, J. Neurochem., 55 (1990) 122-128. [44] Riesenfeld, A. and Oliva, H., The effect of nicotine and alcohol on the fertility and life span of rats, Acta Anat., 128 (1987) 45-50. [45] Sandstrom, L.P. and Pennington, S.N., Embryonic growth inhibition induced by cocaine is associated with the suppression of ornithine decarboxylase activity, Proc. Soc. Exp. BioL Med., 202 (1993) 491-498. [46] Sandstrom, L.P., Sandstrom, P.A. and Pennington, S.N., Ethanol-induced insulin resistance suppresses the expression of embryonic ornithine decarboxylase activity, Alcohol, 10 (1993) 1-8. [47] Sinha, M.K., Pories, W.J., Flickinger, E.G., Meelheim, D. and Caro, J.F., Insulin-receptor kinase activity of adipose tissue from morbidly obese humans with and without NIDDM, Diabetes, 36 (1987) 620-625. [48] Slotkin, T.A. and Bartolome, J.V., Role of ornithine decarboxylase and polyamines in the nervous system development: a review, Brain Res. Bull., 17 (1986) 307-320. [49] Slotkin, T.A., Greet, N., Faust, J., Cho, H. and Seidler, F.J., Effects of maternal nicotine injections on brain development in the rat: ODC activity, nucleic acids and proteins in discrete brain regions, Brain Res. Bull., 17 (1986) 41-50. [50] Slotkin, T.A., Lappi, S.E. and Seider, F.J., Impact of fetal nicotine exposure on development of rat brain regions: critical sensitive periods or effects of withdrawal?, Brain Res. Bull., 31 (1993) 319-328. [51] Tabor, C. and Tabor, H., Polyamines, Annu. ReL'. Biochem., 53 (1984) 749-790. [52] Tansley, B.W., Fried, P.A. and Mount, H.T.J., Visual processing in children exposed prenatally to marihuana and nicotine: a preliminary report, Can. J. Public Health, 77 (1986) 72-78. [53] Valverius, P., Hoffman, P. and Tabakoff, B., Effects of chronic ethanol ingestion on mouse brain beta-adrenergic receptors (BAR) and adenylate cyclase, AdL,. Alcohol Subst. Abuse, 7 (1988) 99-101. [54] Willey, J.C., Laveck, M.A., McClendon, I.A. and Lechner, J.F., Relationship of ODC activity and cAMP metabolism to proliferation of normal human bronchial epithelial cells, J. Cellular Physiol., 124 (1985) 207-212. [55] Wilson, J., Protein phosphorylation: involvement in brain function, Brain. Biochem., S. Kumar (Ed.), Pergamon, New York, NY, 1980, pp. 523-544. [56] Woo, N.D. and Persaud, T.V.N., Rat embryogenesis following exposure to alcohol and nicotine, Acta Anat., 131 (1988) 122126.