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Effect of maternal ethanol ingestion on serum growth hormone in the developing rat
Nuurophorm,,‘~,loR?. Vol 18. pp 821 to 826 Pergamon Pres Ltd 197Y. Prmted m Great EMam
EFFECT OF MATERNAL ETHANOL INGESTION ON SERUM GROWTH HORMONE IN THE DEVELOPING RAT* and S. M. SCHANBERG~
PUSHPA V. THADANI~~ Department
of Pharmacology,
Duke
University
(Acccyred
Medical
I I April
Center.
Durham.
NC 27710. U.S.A.
1979)
Summary-Pregnant rats were kept on a liquid diet containing ethanol (6.8% v/v) from the 13th day of gestation and serum GH levels were determined in the offspring. In neonates exposed to ethanol throughout development, serum growth hormone (GH) secretion was initially increased and then declined to subnormal levels compared to controls. The levels returned to normal after weaning. Withdrawal from ethanol at birth produced similar alterations in serum GH concentrations to those seen in neonatal rats exposed to ethanol continuously. Postnatal exposure to ethanol alone also caused a decrease in GH concentration in rat pups. These data indicate that exposure to ethanol during gestation causes an alteration in the regulation of secretion of GH in the developing pups. A single dose of ethanol given to pups postnatally also caused a marked decline in serum GH levels demonstrating that in the developing pup ethanol can interfere acutely with GH release.
Many studies have shown that the activity of ornithine decarboxylase (ODC), the rate-limiting step in polyamine biosynthesis. Tabor and Tabor (1964); Pegg and Williams-Ashman (1968); Russell and Snyder (1968) is elevated during tissue growth or stimulation and after administration of several drugs and hormones, including growth hormone (GH), Janne, Raina and Siimes (1968); Russell and Snyder (1968); Cohen, O’MalIey and Stastny (1970); Levine, Nicholson. Liddle and Orth (1973); Richman, Dobbins, Voina, Underwood, Mahaffee, Gitelman, Van Wyk and Ney (1973); Byus and Russell (197.5). Administration of a drug or hormones including GH during the early development of brain in rat produced an alteration in the characteristic developmental pattern of ODC activity. Anderson and Schanberg (1972); Roger, Schanberg and Fellows (1974): Anderson and Schanberg (1975); Butler and Schanberg (1975). Kuhn. Butler and Schanberg (1978) have shown that maternal deprivation caused a selective decline in serum immunoreactive CH and this decrease in GH was associated with a decrease in brain ODC activity. A recent clinical study reported that infants born to alcoholic mothers showed a hypersecretion of GH after insulin-induced hypoglycemia and arginine infusion, Tze, Friesen and MacLeod (1976). The present authors have reported previously
that maternal ethanol ingestion produced an alteration in the developmental pattern of brain and heart ODC activity in the developing rat, Thadani, Lau, Slotkin and Schanberg (1977a, b). This study was undertaken io determine whether chronic maternal ethanol ingestion by pregnant rats’ affects the serum GH level in the offspring. The effect of a single dose of ethanol given to developing rats on serum GH and brain and heart ODC activity was also studied. METHODS Chronic
*Supported by NIAAA Grant No. 5 ROI AA0293502. ‘i’Postdoctoral Fellow supported by Research Training Program in Toxicology (NIEHS). Grant No. 5 T32 ESO7002. $ Present address: Mount Sinai Medical School. City University of New York, Institute of Computer Science. New York. U.S.A. aRecipient of an NIMH Research Scientist Development Award, USPHS Grant No. 50K 5 MH 06489.
ethanol
experiments
Timed pregnant Sprague-Dawley rats (Zivic-Miller Laboratories, Allison Park, Pennsylvania) were housed individually in breeding cages without access to water and were fed a nutritionally complete liquid diet (sustacal) from the 1ith day of gestation. On the 13th day of gestation and thereafter, the experimental group received ethanol (6.87; v/v) in sustacal while controls received sustacal, made isocaloric and isonutritional to the ethanol diet by the .addition of sucrose. To ensure that both groups ate the same amount. the control intake was restricted to that consumed by the ethanol group throughout the study; consumption was measured daily and averaged approximately 65 ml/day. In cross-fostering studies, some pups born of ethanol-treated mothers were transferred at birth to control mothers and control pups, to ethanoltreated mothers, giving rise to four groups of pups: (1) control (2) those exposed to ethanol.both prenatally and postnataliy (3) those withdrawn at birth (4) those exposed to ethanol postnatally starting at birth. Maternal care in both control and ethandl groups appeared normal and no rejection of pups by foster
811
822
PUSHPAV. THADANIand S. M. SCHANBERG
mothers was evident. In every case, the litter size was made equivalent in control and experimental groups. After weaning, rats were housed two to three per cage and allowed standard chow and water ad libitum. The pups were killed by decapitation at intervals of several days beginning from the 20th day of gestation to the 37th day of postna~l age. Blood samples were collected in polystyrene tubes, allowed to clot at 4”C, centrifuged and the serum samples stored at -20°C until analyzed for GH. All experiments were carried out between 9:00 and 1O:OOa.m. To minimize the effects of stress, the rats were brought to the laboratory at least 16 hr before the experiment. Acute ethanol experiments
Timed pregnant Sprague-Dawley rats (Zivic-Miller Laboratory) were obtained at least 4 days before delivery and housed individually in breeding cages with access to food and water ad ~~itum. Pups along with their mothers were brought to the laboratory at least 16 hr before the experiment and each litter was divided into three groups. One group was injected subcutaneously with ethanol 2g/kg as 20% saline solution, the second, received the same amount of saline and the third group was used as control and received no treatment. Thirty minutes after the injection, half of the pups from each group were killed by decapitation. The remaining pups were killed 85 min after ethanol injection and the brain and heart was analyzed for ODC activity. Blood was collected in polystyrene tubes, allowed to clot at 4”C, centrifuged and serum was analyzed for GH.
Materials
DL(~-‘~C) ornithine monohydrochloride (sp. activity 43.1 mCi/mmol) was obtained from New England Nuclear Corporation, L-ornithine monohydrochloride and pyridoxal S-phosphate from Sigma Chemical Company and dithiothreitol from Bachem Feinchemikalien AC. The GH kit was supplied by NIAMDD Rat Pituitary Program.
RESULTS Eflect of ethanol, acute ‘and chronic, on serum growth hormone levels
In control pups; the serum GH levels decreased from 522 ng/ml in 20 day old fetus to 230ng/ml a few hours after birth (Fig. 1). A further decline in GH level was observed until postnatal day 3 after which the levels remained Unchanged through 14 days of age: At 20 postnatal day, GH level was si~ificantly less than at 14 days of age. These values were stiil higher than those found. at day 37. In neonates exposed to ethanol from day 13 of gestation, serum GH levels were significantly elevated compared to controls (Fig. 1) in 20 days old fetus and in pups 2 and 3 postnatal days of age. The levels were normal
SERUM GROWTH HORMONE LEVELS
cmmi
Assays
The brain and heart were weighed, homogenized in 20 vol of ice-cold 10mM Tris-HCl (pH 7.2) and the homdgenate centrifuged at 26,OOOg for 20min. An aliquot of the supernatant was assayed for ODC activity by generating 14C0, from DL(1-i4C)-ornithine using a modification of the method of Russell and Snyder (1968). The incubation medium for brain contained final concentrations of 0.5 mM dithiothreipyridoxal S-phosphate, 0.25 &i toi, 0.5 mM DL(l-14C~ornithine and 125 pM unlabelled t-ornithine. In order to increase the sensitivity of the assay for heart, no unlabelled ornithine was added to the incubation medium. The 14C02 was trapped using hyamine hydroxide and counted by liquid scintillation spectrometry. Growth hormone was assayed in each serum sample by radioimmunoassay with reagents supplied by NAIMDD Rat Pituitary Program. Assays were performed according to the recommendations sup plied with NIAMDD Kit. Results are expressed in terms of NIAMDD Rat GH-RP 1. Data are presented as means and standard errors; levels of significance calculated by paired and unpaired student’s t-test.
Birlh
POSTNATAL AGE ( days)
Linq
Fig. 1. Serum growth hormone levels in developing rats. Continuous ethanol ingestion from 13th day of gestation and continued for the duration of the experiment. Pups born or ethanol-treated niothers were transferred to control mothers at birth (withdrawn group) and control pups were reared by ethanol-treated mothers (started from 13th day of gestation) mothers (postnatal group) as indicated. Points and bars represent mean + SE of 5-15 determinations at each age; asterisks denote significant differences vs control (at least P < 0.05 by unpaired r-test).
823
Ethanol and serum growth hormone SERUM
GROWTH
HORMONE
LEVELS
AFTER
ACUTE
ETHANOL
DOSE
0 Control
63 Solinc q 30 minr,ofln
Eloti dose 85 mins. ofler CIoH dose
days
5 days
II days
3 days
30 days
Fig. 2. Etfect of acute ethanol dose (2 g/kg) on serum growth hormone levels in developing rats after 30 or 85 min. Bars represent mean + SE of 12-24 determinations at each age; asterisks denote significant difference vs saline/untreated-control unpaired f-test).
between 5 and 6 days and then declined significantly until 13 postnatal day after which the levels became supranormal again. However, no change was seen in GH levels after weaning. in cross-fostering studies, withdrawal at birth produced similar changes in GH levels to those seen in the pups continuously exposed to ethanol. Exposure of pups to ethanol postnatally also produced a signific~t decrease in serum GH levels (paired t-test P c 0.01 through 13 days). As there was no statistical difference in the serum GH levels in the pups injected with saline 30 or 85 BRAIN
0
(at least P -C 0.05 by
minutes before killing, the data for these two groups were pooled together (Fig. 2). The results for control group were also pooled. A single dose of ethanol given 30min prior to killing produced a significant decrease in GH levels of S-, 7-, ll- or 13-day-old rats compared to their respective control or saline groups (Fig. 2). Similar decreases were also observed in neonates given a single dose of ethanol 85 minutes prior to killing. While the GH levels measured tended to be lower in 3O-day-old rats after ethanol injection, the changes were not significant statistically. ODC
ACTIVITY
Control
hp Soline 30 mins after OoH in&lion q 85 mins oflet EtoHinjection 4.0 .g 5
3.2
9 2
2.4
g c I .6
0.8 -i I
days
I doys
13 doys
Fig. 3. Effect of acute ethanol dose (Zgikg) on brain omithine decarboxylase activity in developing rats after 30 or 85 min. Bars represent mean + SE of 4-12 determinations
at each age; asterisks denote significant difference vs control (untreated)/saIine (at least P c 0.05 N.P.
18/1&-F
by unpaired t-test).
824
PUSHPAV. THADAN~and S. M. SCHANBERG
(1975). fn additions some of these children show a bypers~cr~tio~ of GH after insulin-indeed hypoglycemia and arginine infusion, Tze et al. (1976). In animinsofler Eloti mjeetion mals, chronic maternal ethanol ingestion alters. in the mins after Eto H injtclron offspring, both biochemical and morphological development in the brain and heart, Sandor (1968); Sandor and Amels (1971); Branchey and Friedhoff (1975); Rawat (1975); Thadani et al. (1977a, b. c), including ODC activity and development of synaptic function in central noradrenergic systems. Previously, this laboratory has shown that GH administration induces an increase in ODC activity in brain and heart, Roger et al. (1974); Butler and Sch~~~~ (1975) and its decrease appears to mediate the decline in ODC activity in brain and heart observed after maternal deprivation, Kuhn et al. (1978). As alcohol administration also alters ODC activity, Thadani et al. (1977a b, c) the present study 7 days days 3 days was undertaken to investigate whether acute and/or chronic (from 13th day of gestation) exposure to ethaFig. 4. Effect ‘of acute ethanol dose (2g/kg) on heart ornithine decarboxylase activity in developing rat 30 or nol affects serum GH levels. In control rat pups, 85 min after ethanol injection. Bars represent mean + SE serum GH levels were high at 20th gestation day and of 512 determinations at each age; asterisks denote signi- then a dramatic decline was observed until 20th postficant difference vs saline~u~treated cantrol (at least natal day con~rmi~g previous findings, Blazquez, P < 0.05 by unpaired t-test). Simon, Blazquez and Foa (1974); Rieutort (1974); Walker, Dussault, Alvarado-~rbina and Dupont (1977). However, the age at which serum GH concentrations reach the basal adult level appear to be different in various laboratories probably due to difference As there was no statistical difference in brain ODC in species and nutritional status, Blazquez et al. activity of the pups injected with saline 30 or 85 min (1974); Rieutort (1974); Walker ef al. (1977). The prior to killing, the data for both groups were pooled maternal ethanol ingestion from 13th day af gestation together (Fig. 3). The results for control groups were caused an initial increase and then a decrease in also pooled. In neonates given a single dose of ethaserum GH concentration in the offspring as compared nol 30 min prior to killing brain ODC activity was to control pups indicating that there may be an altermarkedly decreased at 5, 7 or 11 postnatal days compared to respective control or saline groups [Fig. 3). ation in the development of hypothalamic regulation on GH secretion during the early neonatal period. Similar decreases were also observed in pups given These abnormal levels of serum GH seen in neonates ethanol 85 minutes before killing with an exception exposed to ethanol throughout deveIopme~t were not that, itt this time period, a .marked decrease in ODC prevented by ter~natio~ of ethanol at birth (withactivity was seen also in 13-day-old neonatal rats drawal group). whereas no change was observed after 30 min. Heart ODC activity for pooled control and saline To determine whether a significant effect of ethanol could be achieved by solely postnatal exposure, anigroups is shown in Figure 4. Thirty minutes after a single dose of ethanol, heart ODC activity was demals born to control mothers were transferred to creased only in 7-day-old rats compared to control ethanol-treated mothers (from 13th day of gestation) or saline rats (Fig. 4). However, 85 min after ethanol, at birth. A decrease in serum GH concentration was a marked decline in the activity was observed in all observed starting from day 5. Similarly, a single dose ethanol-treated groups (7-, 1l- and 13-day-old}. of ethanol given to the developing rats produced a marked decrease in serum GH level within 3~min (except in 3~ay-olds) indicating that ethanol can interfere .~~utely with GH secretion. However, it is not clear from these data whether this action of ethEthanol crosses the ~la~nta~ barrier, Ho, Fritchie, anol is due to a direct effect on the bypotbalami~Idaupan-He~kkila and Moissac (I972); Waltman and pituitary axis regulation of GH secretion or to the Iniquez (1972) and, therefore, could effect the developrelease of biogenicbamines or whether it is secondary ment of the fetus directly. Studies in humans suggest that infants born to alcoholic mothers experience disto a ‘“nonspecific” stress effect produced by ethanol. Earlier investigations have shown that in adult and turbances in development and function of the central developing rats, GH secretion is regulated by the bionervous system as well as retardation in general genie amines, Muller, Dal Pra and Pecile (1968); Ng, growth, Jones and Smith (1973); Jones, Smith, StreissChase, Colburn and Kopin (1970); Collu, Fraschini, guth and Myrianthopolitus (1974); Jones and Smith
Ethanol and setun 1 growth hormone Visconti and Martini (1972); Smythe and Lazarus (1973); Smythe, Br~dstate~ and Lazarus (1975); Stuart, Lazarus, Smythe, Moore and Sara (1977). In adult rats, acute and chronic ethanol administration has been shown to alter biogenic amines in brain, Corrodi, Fuxe and Hokfelt (1966); Duritz and Truitt (1964); Tyce, Flock, Taylor and Gwen (1970); Hunt and Majchrowicz (1974); Pohorecky (1974); Thadani, Kulig, Brown and Beard (1976); Thadani and Truitt (1977d). However, it is of interest that adult rats differed from neonatal pups and that acute ethanol admi~is~ation to the adult was relatively ineffective in lowering serum GH ~u~ntration as compared to its effect on pups, Tba~~~ and ~hau~rg (unp~blished observations), It has been reported previously (Thadani et al., 1976, 1977a-d) that maternal ethanol exposure causes a perturbation in the developmental pattern of ODC activity in heart and brain of the pups, with the most persistant effect being a decrease in activity. In the present experiments, decreases in brain and heart ODC activity also occurred after acute ethanol administration to the pups. This decrease correlates both in time and effect with the s~ppressioo of GH levels in the serum. It has been reported that GH functions in the regulation of ODC activity in various tissues (Shaw, 1979). Similarly, the present data is consistent with the hypothesis that decreased secretion of GH might mediate the alcohol-induced alterations in organ tissue ODC activity reported previously, Thadani et al. (1976); Thadani et al. (1977a-d). Such a mechanism would appear to be similar to that proposed for the mediation of the decrease in ODC activity occurring in various organs after maternal deprivation, Kuhn et al. (1978). In conclusion, these results indicate that maternal ethanol exposure causes an alteration in serum GH secretion in the offspring which persists until we~ing. This al~ratjo~ is not reversed by terrn~n~t~on of ethanol at birth. Postnatal exposure to ethanol alone also causes a decline in serum GH levels. These data also show that a single dose of ethanol administered to developing rats causes a decline in serum GH levels which correlates with a decrease in brain and heart ODC activity. It is possible that a suppression of GH release by ethanol may contribute to the “failure to thrive” growth pattern evident in the foetal alcohol
syndrome.
Acknowledgements-The
authors wish to thank Mrs Edith Harris for technical assistance. We also wish to thank Dr Parlow ~~~t~i~ry hormone ~bor~tories, ~IA~D~) for supplying us with the Growth Hormone Kit. REFERENCES
Anderson, T. R. and Schanberg, S. M. (1972). Orthinine decarboxylase activity in developing rat brain. J. Neurothem. 19: 1471-1481. Anderson, T. R. and Schanberg, S. M. (1975). Effect of thyroxine and cortisol on brain ornithine decarboxylase activity and swimming behaviour in developing rat. Biothem. Pharmac. 24: 495-501.
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Blazquez. E., Simon, F, A., Bfazquez, M. and Foa, P. P. (1974). Changes in serum ~owth hormone levels from fetal to adult age in the rat. Proc. Sac. exp. Biol. Med. 147: 780-783.
Branchey, L, and Friedhoff, A, J. (1975). The influence of ethanol administered to pregnant rats on tyrosine hydroxylase activity of their offspring. Psychopharmacology 32: 151-156.
Butler, S. R. and Schanberg, S. M. (1975). Effect of maternal morphine administration on neonatal rat brain omithine decarboxylase. Biochem. Pharmac. 24: 1915-1918. Byus, C. V. and Russell, D. H. (1975). Ornithine decarboxylase activity: control by cycfic nucleotides. Science, iVel\r York 187: 65%-652.
Cohen, S., ~Ma11ey, B. W. and Stastny, M. (1970). Estrogenie i~dMctio~ of o~~~thine de~r~xy1a~ in viuu and in vitro. Science,New York 170: 336-338. Coilu, R.. Fraschini, F., Visconti, P. and Martini, L. (1972). Adrcnergic and serotoninergic control of growth hormone secretion in adult male rats. Endocrinology 90: 1231-1237.
Corrodi, H,, Fuxe, K. and Hokfelt, T. (1966). The effect of ethanol on the activity of central catecholamine neurons in rat brain. J. Pharm. Pharmac. 18: 82f823.
Duritz, G. and Truitt, E. B., Jr (1966). Importance of acetaldehyde in the action of ethanol on brain norepinephrine and S-~ydroxytryptamine~ Biochem. ~~arrnac. 15: 71-72. Ho, B. T., Fritchie, G. E., Idaupan-~eikkila, J. E. and M&sac, We M. (1972). ~la~~ta1 transfer and tissue distribution of ethanol-1-“C. Q. JI Stud. Alcohol 33: 485-493. Hunt, W. A. and Majchrowicz, E. {1974). Alterations in the turnover of the brain norepinephrine and dopamine in alcohol-dependent rats. 3. Neurochem. 23, 549-552. Janne. J., Raina, A. and Siimes, M. (1968). Mechanism of stimulation of polyamine synthesis by growth hormine in rat liver. Biochem. biophys. Acta 166: 419426. Jones, K. L. and Smith, D. W. (1973). Recognition of thefetal alcohol syndrome in early infancy. Lancet 2: 999-1001. Jones, K. L. and Smith, D. W. (1975). The fetal alcohol syndrome, ~eratoi~y It: l-10. Jones, K. L., Smith, D. W., Streissgutb, A. P. and Myriant~opo1i1~s~N. C. (1974). outcome in o~sprio~ of chronic alcoholic women. Lancer 1: ~~7~~078. Kuhn, C. M., Butler, S. R. and &ha&erg. S. M. (1978). Selective depression of serum growth hormone during maternal deprivation in rat pups. Science 201: 1034-1036. Levine, J. II., Nicholson, W. E., Liddle, G. W. and Orth, D. N. (1973). Stimulation of adrenal ornithine decarboxylam by adrenocorticotropin and growth hormone. Endocrinalogy 92: 1089-1095. Mutler, E. E,, Dal Pra, P. and Pecile, A. (1968). Influence of brain neurohumours injected into the lateral ventricle of the rat on growth hormone release. ~~dacrj)~~log~ 83: 893496. Ng, K. Y., Chase, T. N., Colburn, 8. W. and Kopin, I. J, (1970). ~~dopa-indu~d release of cerebral monoamines. Science, New York 70: 76-77. Pegg, A. E. and Wil1~am~A~man. H. G. (1968). Biosynthesis of putrescine in prostate gland of the rat. ~i~rh~~~~. 3. 108: 533-539. Pohorecky, L. A. (1974). Effects of ethanol on central and peripheral noradrenergic neurons. J. Pharmac. e.wp.rhrr. 189: 380-391. Rawat, A. K. (1975). Ribosomal protein synthesis in the fetal rat brain as influenced by maternal ethanol consumption. Res. Commun. Chem. Path. Pharmac. 12: 723-732.
PUSHPAV. THADANIand S. M. SCHANBERG
826
Richman, R. A., Dobbins, C., Voina, S. J.. Underwood, L. E., Mahaffee, D.. Gitelman, H. J., Van Wyk, J. J. and Ney, R. (1973). Regulation of adrenal ornithine decarboxylase by adrenocorticotropic hormone and cyclic AMP. J. C/in. invest. 52: 2007-2015. Rieutort, M. (1974). Pituitary content and plasma levels of growth hormone in fetal and weanling rats. J. Endocrinol. 60: 261-268. Roger, J., Schanherg, S. M. and Fellows, R. E. (1974). Growth and lactogenic hormone stimulation of ornithine decarboxylase in neonatal rat brain. E~docri~oiogy 99: 9@4-91 t.
Russell, D. H. and Snyder, S. H. (1968). Amine synthesis in rapidly growing tissues: Ornithine decarboxylase activity in regenerating rat liver. chick embryo, and various tumors. Proc. Natn. Acad. Sci.. U.S.A. 60: 1420-1427. Sandor, S. (1968). The influence of aethyl alcohol on the developing chick embryo II. Rev. Roum. Embryo/. 5: 167-171. Sandor, S. and Amels, D. (1971). The action of aethanol on the praentatal development of albino rats. Rev. Roum. Embryo!. 8: 105-I 18. Shaw, G. G. (1979). The polyamines in the central nervous system. Bioche~. Pharm~. 28: l-6. Smythe, G. A. and Lazarus, L. (1973). Growth hormone regulation by melatonin and serotinin. Nature, Lend. 244 : 230-23 I. Smythe, G. A., Brandstater, J. F, and Lazarus, L. (197.5). Serotoninergic control of rat growth hormone secretion. Neuroendocrinology
17: 245-257.
Stuart, M., Lazarus, L., Smythe, G. A., Moore, S. and Sara, V. (1977). Biogenic amine control of growth hormone secretion in the fetal and neonatal rat. Neuroendocrinology 22: 337-342. Tabor, H. and Tabor, C. W. (1964). Spermidine, spermine and related amines. Pharmac. Rev. 16: 245-300.
Thadani, P. V., Kulig, B. M., Brown, F. C. and Beard. J. D. (1976). Acute and chronic ethanol induced alterations in brain norepinephrine metabolites in the rat. Biochem. Pharmac. 75: 93-94.
Thadani, P. V.. Lau, C., Slotkin, T. A. and Schanberg. S. M. (l977a). Effect of maternal ethanol ingestion on neonatal rat brain and heart ornithine decarboxylase. Biothem. Ph~mac. 26: 523-527. Thadani, P. V., Lau, C., Slotkin, T. A. and Schanberg, S. M. (1977b). Effects of maternal ethanol ingestion on amine uptake into synaptosomes of fetal and neonatal rat brain. J. Pharmac. exp. Ther. 200: 292-297. Thadani, P. V., Slotkin, T. A. and Schanberg, S. M. (1977~). Effects of late prenatal or early postnatal ethanol exposure on ornithine decarboxylase activity in brain and heart of developing rats. Neuropharmacoloyy 16: 289-293. Thadani. P. V. and Truitt, E. B., Jr (l977d). Effect of acute ethanol or acetaldehyde administration on the uptake, release, metabolism and turnover rate of norepinephrine in rat brain. Biochem. Pharmac. 26: I 147-l 150. Tyce, G. M., Flock, E. V., Taylor. W. F. and Owen, C. A., Jr (1970). Effect of ethanol on S-hydroxytryptamine turnover in rat brain. Proc. Sot. exp. Biol. Med. 134: 4@44. Tze, W. J., Friesen, H. G. and MacLeod, P. M. (1976). Growth hormone response in fetal alcohol syndrome. Archs Dis. Child. 51: 703-706.
Waftman, R. and Iniquez, E. S. (1972). Placental transfer of ethanol and its elimination at term. Obstet. Gynecol. 40: 185-189. Walker, P., Dussault, J. H., Alvarado-Urbina, G. and DuPont, A. (1977). The development of the hypothalamopituitary axis in the neonatal rat. Hypothalamic somatostatin and pituitary and serum growth hormone concentrations. Endocrinology 101: 782-787.
EFFECT OF MATERNAL ETHANOL INGESTION ON SERUM GROWTH HORMONE IN THE DEVELOPING RAT* and S. M. SCHANBERG~
PUSHPA V. THADANI~~ Department
of Pharmacology,
Duke
University
(Acccyred
Medical
I I April
Center.
Durham.
NC 27710. U.S.A.
1979)
Summary-Pregnant rats were kept on a liquid diet containing ethanol (6.8% v/v) from the 13th day of gestation and serum GH levels were determined in the offspring. In neonates exposed to ethanol throughout development, serum growth hormone (GH) secretion was initially increased and then declined to subnormal levels compared to controls. The levels returned to normal after weaning. Withdrawal from ethanol at birth produced similar alterations in serum GH concentrations to those seen in neonatal rats exposed to ethanol continuously. Postnatal exposure to ethanol alone also caused a decrease in GH concentration in rat pups. These data indicate that exposure to ethanol during gestation causes an alteration in the regulation of secretion of GH in the developing pups. A single dose of ethanol given to pups postnatally also caused a marked decline in serum GH levels demonstrating that in the developing pup ethanol can interfere acutely with GH release.
Many studies have shown that the activity of ornithine decarboxylase (ODC), the rate-limiting step in polyamine biosynthesis. Tabor and Tabor (1964); Pegg and Williams-Ashman (1968); Russell and Snyder (1968) is elevated during tissue growth or stimulation and after administration of several drugs and hormones, including growth hormone (GH), Janne, Raina and Siimes (1968); Russell and Snyder (1968); Cohen, O’MalIey and Stastny (1970); Levine, Nicholson. Liddle and Orth (1973); Richman, Dobbins, Voina, Underwood, Mahaffee, Gitelman, Van Wyk and Ney (1973); Byus and Russell (197.5). Administration of a drug or hormones including GH during the early development of brain in rat produced an alteration in the characteristic developmental pattern of ODC activity. Anderson and Schanberg (1972); Roger, Schanberg and Fellows (1974): Anderson and Schanberg (1975); Butler and Schanberg (1975). Kuhn. Butler and Schanberg (1978) have shown that maternal deprivation caused a selective decline in serum immunoreactive CH and this decrease in GH was associated with a decrease in brain ODC activity. A recent clinical study reported that infants born to alcoholic mothers showed a hypersecretion of GH after insulin-induced hypoglycemia and arginine infusion, Tze, Friesen and MacLeod (1976). The present authors have reported previously
that maternal ethanol ingestion produced an alteration in the developmental pattern of brain and heart ODC activity in the developing rat, Thadani, Lau, Slotkin and Schanberg (1977a, b). This study was undertaken io determine whether chronic maternal ethanol ingestion by pregnant rats’ affects the serum GH level in the offspring. The effect of a single dose of ethanol given to developing rats on serum GH and brain and heart ODC activity was also studied. METHODS Chronic
*Supported by NIAAA Grant No. 5 ROI AA0293502. ‘i’Postdoctoral Fellow supported by Research Training Program in Toxicology (NIEHS). Grant No. 5 T32 ESO7002. $ Present address: Mount Sinai Medical School. City University of New York, Institute of Computer Science. New York. U.S.A. aRecipient of an NIMH Research Scientist Development Award, USPHS Grant No. 50K 5 MH 06489.
ethanol
experiments
Timed pregnant Sprague-Dawley rats (Zivic-Miller Laboratories, Allison Park, Pennsylvania) were housed individually in breeding cages without access to water and were fed a nutritionally complete liquid diet (sustacal) from the 1ith day of gestation. On the 13th day of gestation and thereafter, the experimental group received ethanol (6.87; v/v) in sustacal while controls received sustacal, made isocaloric and isonutritional to the ethanol diet by the .addition of sucrose. To ensure that both groups ate the same amount. the control intake was restricted to that consumed by the ethanol group throughout the study; consumption was measured daily and averaged approximately 65 ml/day. In cross-fostering studies, some pups born of ethanol-treated mothers were transferred at birth to control mothers and control pups, to ethanoltreated mothers, giving rise to four groups of pups: (1) control (2) those exposed to ethanol.both prenatally and postnataliy (3) those withdrawn at birth (4) those exposed to ethanol postnatally starting at birth. Maternal care in both control and ethandl groups appeared normal and no rejection of pups by foster
811
822
PUSHPAV. THADANIand S. M. SCHANBERG
mothers was evident. In every case, the litter size was made equivalent in control and experimental groups. After weaning, rats were housed two to three per cage and allowed standard chow and water ad libitum. The pups were killed by decapitation at intervals of several days beginning from the 20th day of gestation to the 37th day of postna~l age. Blood samples were collected in polystyrene tubes, allowed to clot at 4”C, centrifuged and the serum samples stored at -20°C until analyzed for GH. All experiments were carried out between 9:00 and 1O:OOa.m. To minimize the effects of stress, the rats were brought to the laboratory at least 16 hr before the experiment. Acute ethanol experiments
Timed pregnant Sprague-Dawley rats (Zivic-Miller Laboratory) were obtained at least 4 days before delivery and housed individually in breeding cages with access to food and water ad ~~itum. Pups along with their mothers were brought to the laboratory at least 16 hr before the experiment and each litter was divided into three groups. One group was injected subcutaneously with ethanol 2g/kg as 20% saline solution, the second, received the same amount of saline and the third group was used as control and received no treatment. Thirty minutes after the injection, half of the pups from each group were killed by decapitation. The remaining pups were killed 85 min after ethanol injection and the brain and heart was analyzed for ODC activity. Blood was collected in polystyrene tubes, allowed to clot at 4”C, centrifuged and serum was analyzed for GH.
Materials
DL(~-‘~C) ornithine monohydrochloride (sp. activity 43.1 mCi/mmol) was obtained from New England Nuclear Corporation, L-ornithine monohydrochloride and pyridoxal S-phosphate from Sigma Chemical Company and dithiothreitol from Bachem Feinchemikalien AC. The GH kit was supplied by NIAMDD Rat Pituitary Program.
RESULTS Eflect of ethanol, acute ‘and chronic, on serum growth hormone levels
In control pups; the serum GH levels decreased from 522 ng/ml in 20 day old fetus to 230ng/ml a few hours after birth (Fig. 1). A further decline in GH level was observed until postnatal day 3 after which the levels remained Unchanged through 14 days of age: At 20 postnatal day, GH level was si~ificantly less than at 14 days of age. These values were stiil higher than those found. at day 37. In neonates exposed to ethanol from day 13 of gestation, serum GH levels were significantly elevated compared to controls (Fig. 1) in 20 days old fetus and in pups 2 and 3 postnatal days of age. The levels were normal
SERUM GROWTH HORMONE LEVELS
cmmi
Assays
The brain and heart were weighed, homogenized in 20 vol of ice-cold 10mM Tris-HCl (pH 7.2) and the homdgenate centrifuged at 26,OOOg for 20min. An aliquot of the supernatant was assayed for ODC activity by generating 14C0, from DL(1-i4C)-ornithine using a modification of the method of Russell and Snyder (1968). The incubation medium for brain contained final concentrations of 0.5 mM dithiothreipyridoxal S-phosphate, 0.25 &i toi, 0.5 mM DL(l-14C~ornithine and 125 pM unlabelled t-ornithine. In order to increase the sensitivity of the assay for heart, no unlabelled ornithine was added to the incubation medium. The 14C02 was trapped using hyamine hydroxide and counted by liquid scintillation spectrometry. Growth hormone was assayed in each serum sample by radioimmunoassay with reagents supplied by NAIMDD Rat Pituitary Program. Assays were performed according to the recommendations sup plied with NIAMDD Kit. Results are expressed in terms of NIAMDD Rat GH-RP 1. Data are presented as means and standard errors; levels of significance calculated by paired and unpaired student’s t-test.
Birlh
POSTNATAL AGE ( days)
Linq
Fig. 1. Serum growth hormone levels in developing rats. Continuous ethanol ingestion from 13th day of gestation and continued for the duration of the experiment. Pups born or ethanol-treated niothers were transferred to control mothers at birth (withdrawn group) and control pups were reared by ethanol-treated mothers (started from 13th day of gestation) mothers (postnatal group) as indicated. Points and bars represent mean + SE of 5-15 determinations at each age; asterisks denote significant differences vs control (at least P < 0.05 by unpaired r-test).
823
Ethanol and serum growth hormone SERUM
GROWTH
HORMONE
LEVELS
AFTER
ACUTE
ETHANOL
DOSE
0 Control
63 Solinc q 30 minr,ofln
Eloti dose 85 mins. ofler CIoH dose
days
5 days
II days
3 days
30 days
Fig. 2. Etfect of acute ethanol dose (2 g/kg) on serum growth hormone levels in developing rats after 30 or 85 min. Bars represent mean + SE of 12-24 determinations at each age; asterisks denote significant difference vs saline/untreated-control unpaired f-test).
between 5 and 6 days and then declined significantly until 13 postnatal day after which the levels became supranormal again. However, no change was seen in GH levels after weaning. in cross-fostering studies, withdrawal at birth produced similar changes in GH levels to those seen in the pups continuously exposed to ethanol. Exposure of pups to ethanol postnatally also produced a signific~t decrease in serum GH levels (paired t-test P c 0.01 through 13 days). As there was no statistical difference in the serum GH levels in the pups injected with saline 30 or 85 BRAIN
0
(at least P -C 0.05 by
minutes before killing, the data for these two groups were pooled together (Fig. 2). The results for control group were also pooled. A single dose of ethanol given 30min prior to killing produced a significant decrease in GH levels of S-, 7-, ll- or 13-day-old rats compared to their respective control or saline groups (Fig. 2). Similar decreases were also observed in neonates given a single dose of ethanol 85 minutes prior to killing. While the GH levels measured tended to be lower in 3O-day-old rats after ethanol injection, the changes were not significant statistically. ODC
ACTIVITY
Control
hp Soline 30 mins after OoH in&lion q 85 mins oflet EtoHinjection 4.0 .g 5
3.2
9 2
2.4
g c I .6
0.8 -i I
days
I doys
13 doys
Fig. 3. Effect of acute ethanol dose (Zgikg) on brain omithine decarboxylase activity in developing rats after 30 or 85 min. Bars represent mean + SE of 4-12 determinations
at each age; asterisks denote significant difference vs control (untreated)/saIine (at least P c 0.05 N.P.
18/1&-F
by unpaired t-test).
824
PUSHPAV. THADAN~and S. M. SCHANBERG
(1975). fn additions some of these children show a bypers~cr~tio~ of GH after insulin-indeed hypoglycemia and arginine infusion, Tze et al. (1976). In animinsofler Eloti mjeetion mals, chronic maternal ethanol ingestion alters. in the mins after Eto H injtclron offspring, both biochemical and morphological development in the brain and heart, Sandor (1968); Sandor and Amels (1971); Branchey and Friedhoff (1975); Rawat (1975); Thadani et al. (1977a, b. c), including ODC activity and development of synaptic function in central noradrenergic systems. Previously, this laboratory has shown that GH administration induces an increase in ODC activity in brain and heart, Roger et al. (1974); Butler and Sch~~~~ (1975) and its decrease appears to mediate the decline in ODC activity in brain and heart observed after maternal deprivation, Kuhn et al. (1978). As alcohol administration also alters ODC activity, Thadani et al. (1977a b, c) the present study 7 days days 3 days was undertaken to investigate whether acute and/or chronic (from 13th day of gestation) exposure to ethaFig. 4. Effect ‘of acute ethanol dose (2g/kg) on heart ornithine decarboxylase activity in developing rat 30 or nol affects serum GH levels. In control rat pups, 85 min after ethanol injection. Bars represent mean + SE serum GH levels were high at 20th gestation day and of 512 determinations at each age; asterisks denote signi- then a dramatic decline was observed until 20th postficant difference vs saline~u~treated cantrol (at least natal day con~rmi~g previous findings, Blazquez, P < 0.05 by unpaired t-test). Simon, Blazquez and Foa (1974); Rieutort (1974); Walker, Dussault, Alvarado-~rbina and Dupont (1977). However, the age at which serum GH concentrations reach the basal adult level appear to be different in various laboratories probably due to difference As there was no statistical difference in brain ODC in species and nutritional status, Blazquez et al. activity of the pups injected with saline 30 or 85 min (1974); Rieutort (1974); Walker ef al. (1977). The prior to killing, the data for both groups were pooled maternal ethanol ingestion from 13th day af gestation together (Fig. 3). The results for control groups were caused an initial increase and then a decrease in also pooled. In neonates given a single dose of ethaserum GH concentration in the offspring as compared nol 30 min prior to killing brain ODC activity was to control pups indicating that there may be an altermarkedly decreased at 5, 7 or 11 postnatal days compared to respective control or saline groups [Fig. 3). ation in the development of hypothalamic regulation on GH secretion during the early neonatal period. Similar decreases were also observed in pups given These abnormal levels of serum GH seen in neonates ethanol 85 minutes before killing with an exception exposed to ethanol throughout deveIopme~t were not that, itt this time period, a .marked decrease in ODC prevented by ter~natio~ of ethanol at birth (withactivity was seen also in 13-day-old neonatal rats drawal group). whereas no change was observed after 30 min. Heart ODC activity for pooled control and saline To determine whether a significant effect of ethanol could be achieved by solely postnatal exposure, anigroups is shown in Figure 4. Thirty minutes after a single dose of ethanol, heart ODC activity was demals born to control mothers were transferred to creased only in 7-day-old rats compared to control ethanol-treated mothers (from 13th day of gestation) or saline rats (Fig. 4). However, 85 min after ethanol, at birth. A decrease in serum GH concentration was a marked decline in the activity was observed in all observed starting from day 5. Similarly, a single dose ethanol-treated groups (7-, 1l- and 13-day-old}. of ethanol given to the developing rats produced a marked decrease in serum GH level within 3~min (except in 3~ay-olds) indicating that ethanol can interfere .~~utely with GH secretion. However, it is not clear from these data whether this action of ethEthanol crosses the ~la~nta~ barrier, Ho, Fritchie, anol is due to a direct effect on the bypotbalami~Idaupan-He~kkila and Moissac (I972); Waltman and pituitary axis regulation of GH secretion or to the Iniquez (1972) and, therefore, could effect the developrelease of biogenicbamines or whether it is secondary ment of the fetus directly. Studies in humans suggest that infants born to alcoholic mothers experience disto a ‘“nonspecific” stress effect produced by ethanol. Earlier investigations have shown that in adult and turbances in development and function of the central developing rats, GH secretion is regulated by the bionervous system as well as retardation in general genie amines, Muller, Dal Pra and Pecile (1968); Ng, growth, Jones and Smith (1973); Jones, Smith, StreissChase, Colburn and Kopin (1970); Collu, Fraschini, guth and Myrianthopolitus (1974); Jones and Smith
Ethanol and setun 1 growth hormone Visconti and Martini (1972); Smythe and Lazarus (1973); Smythe, Br~dstate~ and Lazarus (1975); Stuart, Lazarus, Smythe, Moore and Sara (1977). In adult rats, acute and chronic ethanol administration has been shown to alter biogenic amines in brain, Corrodi, Fuxe and Hokfelt (1966); Duritz and Truitt (1964); Tyce, Flock, Taylor and Gwen (1970); Hunt and Majchrowicz (1974); Pohorecky (1974); Thadani, Kulig, Brown and Beard (1976); Thadani and Truitt (1977d). However, it is of interest that adult rats differed from neonatal pups and that acute ethanol admi~is~ation to the adult was relatively ineffective in lowering serum GH ~u~ntration as compared to its effect on pups, Tba~~~ and ~hau~rg (unp~blished observations), It has been reported previously (Thadani et al., 1976, 1977a-d) that maternal ethanol exposure causes a perturbation in the developmental pattern of ODC activity in heart and brain of the pups, with the most persistant effect being a decrease in activity. In the present experiments, decreases in brain and heart ODC activity also occurred after acute ethanol administration to the pups. This decrease correlates both in time and effect with the s~ppressioo of GH levels in the serum. It has been reported that GH functions in the regulation of ODC activity in various tissues (Shaw, 1979). Similarly, the present data is consistent with the hypothesis that decreased secretion of GH might mediate the alcohol-induced alterations in organ tissue ODC activity reported previously, Thadani et al. (1976); Thadani et al. (1977a-d). Such a mechanism would appear to be similar to that proposed for the mediation of the decrease in ODC activity occurring in various organs after maternal deprivation, Kuhn et al. (1978). In conclusion, these results indicate that maternal ethanol exposure causes an alteration in serum GH secretion in the offspring which persists until we~ing. This al~ratjo~ is not reversed by terrn~n~t~on of ethanol at birth. Postnatal exposure to ethanol alone also causes a decline in serum GH levels. These data also show that a single dose of ethanol administered to developing rats causes a decline in serum GH levels which correlates with a decrease in brain and heart ODC activity. It is possible that a suppression of GH release by ethanol may contribute to the “failure to thrive” growth pattern evident in the foetal alcohol
syndrome.
Acknowledgements-The
authors wish to thank Mrs Edith Harris for technical assistance. We also wish to thank Dr Parlow ~~~t~i~ry hormone ~bor~tories, ~IA~D~) for supplying us with the Growth Hormone Kit. REFERENCES
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