- Email: [email protected]
Folic acid intestinal absorption in newborn rats at 21 day postpartum: Effects of maternal ethanol consumption
Life Science,
ELSEVIER
PI1 SOO24-3205(99)00147-2
Vol. 64, No. 22, pp. 2001~2010,19!B CIopyTight0 1999 Eckievierscience Inc. Printed in the USA. All rights rcsclvcd 0024-32OS/99/$-see front matter
FOLIC ACID INTESTINAL ABSORPTION IN NEWBORN RATS AT 2 1 DAY POSTPARTUMz EFFECTS OF MATERNAL ETHANOL CONSUMPTION ‘Eva Tavares , Ana Gomez-Tubio,
Maria Luisa Murillo and Olimpia Carreras.
Departamento de Fisiologia y Biologiu Animal. Facultad de Farmacia. Universidad de Sevilla. ESpaliCZ (Received in final form February 12, 1999)
Summary This study was designed to examine the effects of prenatal and postnatal exposure of ethanol in the in vivo absorption of free folic acid in the small intestine in pups rats at the 21’ day after birth. The rats were accustomed to increasing amounts of ethanol (5 to 20 %, voVvo1) in tap water for 1 month. During pregnancy and suckling period, ethanol-fed dams were assigned again to ethanol 20 % in drink& water. Two sets of experiments were performed. In the fist set, jejunal tree folic acid absorption in control group and litters nursed by dams receiving ethanol showed a gradual increase In general, along with the increase of perfusion time at all the assayed concentrations. in litters of ethanol-fed dams, jejunal free folic acid absorption expressed as nmol/intestinal surface, nmol/ g tissue wet weight and nmoll g tissue dry weight were higher than in control animals. In the second set of experiments, in distal ileum loops, free folic acid absorption did not occur in control pups, but appeared in litters exposed to ethanol. MiIk folk acid levels ate sig&cautly decreased in ethanol-treated dams However, only a slight decrease in the serum folic acid levels occurs in litters of ethanol-fed dams. In conchrsion, the results obtained in the present work suggested a diierent pattern of free folic acid absorption in distal ileum for the two groups. The exposure of rats to ethanol during the pregnancy and suckling period, can affect postnatal development of intestinal functions and could play a role in the genesis of malnutrition observed in the infant. Key Words: folic acid, alcohol, lactation, offspring, intestinal absorption
Pregnant women who drink alcohol heavily have a high risk of giving biih to cl&hen with growth retardation, congenital malformations and mental deficiency, known collectively as the Fetal Alcohol Syndrome (FAS) (l-5). Animal experiments also indicate that alcohol is a teratogen (611). However, the mechanism by which ethanol exerts its effect on fetal development is still unclear. Folic acid is an essential cofactor in the synthesis of purines and pyrimidmes, components of DNA and RNA (7,9). The jejunum is the site of maxim um absorption of free folate where the absorption occurs by a pH-dependent, carrier-mediated system (12). In the adult rat a saturable transport
’Corresponding author: Dra. Eva Tavares, Departamento de Fisiologta y Biologta Animal, Facultad de Farmacia, Universidad de &villa. Tramontana s/n, 4 1012, Sevilla. Espafk
2002
Maternal Alcohol on Pup Folic Acid Absorption
mechanism is responsible for nearly all the absorption physiologic concentrations of the nutrient ( 12 j.
Vol. 64, No. 22,1999
of f?ee folic acid in the small intestine at
Mason and Selhub (13) demonstrated that in neonatal animals the characteristics of folate-binding protein (FBP)-bound folic acid absorption are very different from those of free folic acid. Absorption of free folic acid in the suckling rat was observed to occur more avidly in the jejunum than in the ileum, a reversal of the pattern seen with bound folic acid (13). Folate deficiency during pregnancy has produce fetal malformations (8,9). Folate congenital defect in folate absorption in chronic diarrhea and incomplete maturation
been shown to interfere with fetal development and is an essential micronutrient for neonatal mammals. A human infants is associated with growth retardation, of the central nervous system (13).
Ethanol is known to interfere with the absorption deficiency is common among alcoholics (18). It is wide range of gastrointestinal effects in adult rats arising through ethanol administration have been (19,21,22,23) but not in suckling exposed to ethanol
and metabolism of folate (14-17) and folate well known that ethanol ingestion produces a (19-22). Alterations in folic acid absorption documented by several studies in adult rats during gestation and lactation period.
The purpose of this work was study the effects of maternal ethanol consumption during gestation and suckling period in the small intestinal absorption of free folic acid in o&spring rats at the 21’ day after the birth. Methods
Animals and alcohol administration. Male and female rats weighing between 200 and 250 g were randomized into two groups. In the first group, ethanol was administered with tap water by a previously described method (24). Ethanol-treated rats received ad libitum 5,10, and 15 % of ethanol in the d&king livid during 3 weeks successively. A consumption of 20 % was maintained in this group for 5 additional weeks. Ethanol-treated rats were mated. The presence of sperm in the vaginal smear the next morning denoted day 1 of pregnancy. Pregnant drinking period. lactation
females were replaced individually in their cages and water as the sole source of liquid with food ad libitum, The day of parturition was designated as day 1 of period. During the suckling period, the pups had free
assigned again to 20 % ethanol in during the pregnancy and suckling lactation and day 21 the final of access to the nipples.
Grouptwo~~ascontrolandreceivedontywaterandastaradardratchowdiet(Panlab04)ad lii cOntrolratswerebandledinthesamewayastheother~groups. Theaverage intake of ethanol (ml), food (g), water (ml) and body weight (g) were controlled on day 1 after birth andagainonday7andthereaftereveryweekuntiltheendoftheexperimentaiperiod. Each measurement was recorded at 9.00 AM to avoid changes due to circadian rhythms. The rats were maintained under automatically light-dark cycles with lights on at 07.00 h.
controlled
temperature
(22-23 “C) and 12 hour
Multiple-Pass Perfusion. Twenty-one days after the birth, the pups were used in this study. The o&pring from ethanol-treated and control dams were anaesthetized with subcutaneous uretane 10 %. Two sets of experiments were performed. In the first set, the abdominal cavity was opened by
Vol. 64, No. 22, 1999
Maternal Alcohol on Pup Folic Acid Absorption
2003
a longitudinal incision and the jejunum was cam&ted with polyethylene tubing. Inflow and out flow cannulas were tied into jejunum. Thus, a jejunal loop was isolated from continuity with the lumen. After cannulation, the loop was rinsed v&h 0.9 % NaCl solution and replaced inside the body wall and perfused as previously described by Ponz et al. (25). A flow rate of 3 ml/min during 5 min period was used, for each concentration in all animals (26). In the second set of experiments, in order to achieve the same experimental parameters on the distal ileum, we following the same procedures described for the first set, in different offspring l?om the first set of animals. Folic acid absorption. ‘H-folic acid (0.01 pCi / ml) was prepared in a solution containing: 154 mM NaCl and cold folic acid at 0.25 pM, 0 5 pM, 1.O uM, 1.5 pM and 2.5 pM concentrations. This solution was pre-warmed (37°C) before perfusion. Folk acid uptake was measured in different experimental animak. PerfUsions were performed with increasing concentrations and immediate washes with sterile saline solution were made between successive perfusions. The perfusion time for all the o@qing studied was divided into an equilibrium period of 15 min and one period of 5 min at each substrate concentration. Folk acid absorption was determined as the difference between the initial and final amount of substrate obtained in the perfusates. Offspring were maintained under controlled temperature (37’C) with a heating pad. The absorption was calculated and expressed as nmol I intestinal weight and nmol / g tissue dry wt.
surface, nmol I g tissue wet
Morphometric Tissue Evaluation. At the end of perhkions, pups were sacr&zd and the jejunum and ileumt&ueswereremovedtodeterm&theirwetanddryweights. Thetissueiblctionswerewe~ driedinanovenat 100°C toconstantweight(24h)andreweighed,toobtaintksuedryweights. To determ&thetotalserosalareqtheoutercircun&ence was measured according to winne (27). Milk and bloodpreparation. In order to obtaiu the maximumamountofmilkwithoutmod@ingthe physiologicalconditionsofthesubjectswithanesthetics,onday21oflactation3hafterremovingthe litters~mthemothers,damswere~bydecapitationandmilk~~swere~ cOllected.Mill
Pups body weight. Fii 1 show the body weight of pups rats during the exper&ntal bir&therewasnosigmfkam~betweenthebodyweightofthelittersnursedbydams
period. At
Maternal Alcohol on Pup Folic Acid Absorption
2004
Vol. 64, No. 22,199!9
receiving ethanol
during the gestation period compared to control group. However, during the lactationalperiod, s~differencewereo~~betweenbothcontrolaradethanolgroups.
40
?
T
35
& --c
c I 2
3o
5
25
> 0
20
0 m
CONTROL ETHANOL
75
10
5
1
7
14
21
DAYS
Fig. 1
Body weight (g) in of&p&g of ethanol-fed dams and control group. The results are expmssesasmean~SEM.N=10indkatethenumberofanimalsineachgmup. p
Effects of ethanol on several intestinal parameters. Pups from ethanol-treated mothers showed at 21 day postpartum (Table I) decreased values in jejunum of total area F( 1, 19) = 8.41 ,p < 0.02, tissue wet wt. g F(1, 19) = 1.27, p < O.Ol), tissue dry wt. g F(1, 19) = 5.83, p < 0.01 compared with control group. In Table I showed similar results in the ileum those described for the jejtmum. Total areafll, 19) = 14.9, p < 0.001, tissue wet wt. g F(1, 19) = 17.3, p < 0.001, tissue dry wt. g F(1, 19) = 15.5, p < 0.001. .
TABLE I
Jejunal and distal ileum parameters measured in 2 1-d-old rats born from ethanoltreated mothers and controls. The results are expresses as mean f SE&I. N=lO indkate the number ofanimals in each group. * p < 0.05; ** p < 0.02; *** p < O.Ol;#p < 0.001. Ethanol group vs Control group.
Tissue wet wt. (g)
2005
Maternal Alcohol on Pup Folic Acid Absorption
Vol. 64, No. 22,1999
Jejunal free folic acid absorption. The results obtained for jejunal free folk acid absorption in ofEspring from the ethanol-fed dams and control, expressed as nmolkm2, nmol/tissue wet wt. g and nmolltissue dry wt. g at diI&rent substrate concentrations: 0.25 uM, 0.5 uM, 1 PM, 1.5 uM and 2.5 uM are shown in Fig. 2 and Fig. 3. Jejunal free folk acid absorption in control group and litters nursed by dams receiving ethanol during pregnancy and suckling period showed a gradual increase along with the increase of perfusion time at all the assayed concentrations. In general, in offspring from ethanol-fed dams, jejunal free folk acid absorption was higher than in control animals (Fig. 2 and 3). In pups of ethanol-fed dams the jejunal free folk acid absorption was significantly increased at 0.25 uM (F(1, 19) = 12.7, p < 0.001); 0.5 l.tM (F(1, 19) = 26.7, p < O.OOl), 1.5 uM (fll, 19) = 15.6, p < 0.001) and 2.5 uM (F(1,19) = 9.37, p < 0.01) with respect to control group, when the absorption was expressed as nmoYintestinal surface (Fig. 2).
z 0 c
0 cn
** 0
e
0.10
I PZZZZJ
CONTROL ETHANOL
m ,’ * 0 0 4:
0, -I
:
*
0.06
* N
-
0
0 .06
: -
0.04
*
0 Y
0.02 0 .oo
0.2 5 FOLIC
ACID
0.5
1 .o
CONCENTRATIONS
1 .5
2.5 (PM)
Fig. 2 Jejunal free folk acid absorption (nmoVcm2) in oftspring of ethanol-fed dams and control group, at the 2 1’ day atter birth. The results are expresses as mean k SEM. N=1Oindicatethenumberofanimalsineachgroup. p < O.OOl*;p < O.Ol**. Ethanol group vs Control group.
Similar results were obtained when the absorption per gram of both wet and dry tissue were measured (Fig. 3). In Figure 3A, jejunal free folk acid absorption expressed as the absorption per gram of wet tissue was significantly increased in litters of ethanol ted-dams at 0.25 uM (F(1,19) = 7.13, p < 0.01); 0.5 @I (F(1, 19) = 18.9; p < 0.001) and 2.5 uM (fll, 19) = 7.76, p < 0.02) with respect to the control group. A significant increase in absorption was also shown in ofhqking of ethanol treated dams at 0.25 @I (01, 19) = 8.15, p < 0.01); 0.5 uM (F(1, 19) =
2006
Maternal Alcohol on Pup Folic Acid Absorption
Vol. 64, No. 22, 1999
26.7, p < O.OOlj; 1.5 PM (F(1, 19) = 34, p < 0.001) and 2.5 pM (F(1, 19) = 9.14, p < 0.01) expressed as the absorption for tissue dry wt. g (Fig. 3B).
A U
CONTROL
m
ETHANOL
0.25 FOLIC
1 .o 0.5 1.5 ACID CONCENTRATIONS
2.5 (PM)
B 0 W
0.25 FOLIC
CONTROL ETHANOL
0.5 ACID
1 .o
1 .5
CONCENTRATIONS
2.5 (VU)
Fig. 3 Jejunal Gee folk acid absorption in o&pring of ethanol-fed dams and control group, at the 21’ day after birth A) expressed as nmoY g tissue wet weight and B) aSUXZUlztSEM. elrpressedasnmoYgtissuedryweight.Theresuttsare~ N=10indkatethenuruberofanimalsineachgroup. p
Maternal Alcohol on
Vol. 64, No. 22,1999
Pup Folic Acid Absorption
2007
Distal ileumfiee folic acid absorption. The results obtained for free folk acid absorption in distal ileum in pups at day 21 were nil in the controls at all of the assayed concentrations. However, free fohc acid absorption in distal ileum in ofkpriug of ethanol-fed dams (Table II), did take place. TABLE II
Distal ileum fke folk acid absorption in offspring of ethanol-fed dams, at the 2 1’ day after birth. N=lO indicate the number of animals. FOLIC ACID CONCENTRATIONS l&M 0.25p.M O&M l.OpM
Folic acid absorption
nmoVtissue wet wt. g nmol/tissue drywt.g nmowtotal area
2.sp.M
0.2M.01
0.44z!IO.04
0.56kO.03
0.63ztO.05
1.62kO.2
1.29kO.04
2.79fo.04
3.5fo.16
4.02&O.14
10.3kO.42
4.3*1U3 *lo”
8.g*10‘3 ti*lO”’
12*10-3 ks.4*10q
13*10” ti.2*10A
30*10-3 k2*1U4
Serum and milk foiic acid levels. Figure 4 shows that the levels of folk acid in milk (F (1, 13) = 8.23, p < 0.05) were sigo&an@ decreased in the ethanol-fed dam on day 21 of lactation. However, serum folk acid levels on postpartum day 2 1 were only slightly decreased in litters nursed by dams receiving ethanol during gestation and suckling period.
0 0
CONTROL
1 Q2ZZ
ETHANOL
ul 100
ii
ii >
16
lZ?SZ3
CONTROL
I
ETHANOL
>
W -I
80
n 0, a-
E
y2J .=
00
40
IA
Y
d
ZO
I
0
I
P 3
a ii
4 2 0
Fig. 4
Milk folk acid levels aud serum folk acid levels in o&pring. The results are expmssesasmeau*SEMN=14indicatethenumberofauimalsineachgroup.p< 0.05*. Ethanol group vs Control group.
2008
Maternal Alcohol on Pup Folic Acid Absorption
Vol. 64, No. 22,1999
Discussion
In this study, we measured the “in vivo” free folk acid absorption across the jejunum and ileum distal in litters nursed by dams receiving ethanol during gestation and suckling period. Alterations in folk acid absorption arising through ethanol administration have been documented by several studies in adult rats (14,16,19,21,22) but not in suckling exposed to ethanol during gestation and lactation period. In adult rats, previous studies observed no inhibitory effect of alcohol (29) on the jejunal uptake of folk acid. However, under partial starvation conditions (4,19,2 1) an increase in folate transport has been described. Recently, Buts et al. (6) using a similar experimental model of ethanol administration during pregnancy but not in the suckling period have shown that postnatal maturation of the small intestine in the ethanol offspring was depressed during the early nursing period, even though ethanol had been withdrawn at birth. Pups born from ethanol-treated mothers showed lower intestinal mass, depressed jejunal and ileal DNA contents per unit of length, decreased mucosal rat DNA synthesis. All these parameters returned to control levels by day 15 postpartum. An immature aspect of the enterocytes that persisted until weaning was also found in this study (6). The data obtained in the present study in jejunum and distal ileum showed a decrease in intestinal area, wet weight and tissue dry weight in litters exposed to ethanol as well as an increase in values of free folk acid absorption at difkrent folk acid concentrations in the jejunum with respect to the control group. Morphometric studies have shown that villus height and mucosalsurtace area are reduced in alcoholic as compared to nonalcoholic patients (30,31). Previous studies in rats (19,26) have shown a decrease in intestinal wet and dry weights after ethanol consumption. This hypoplasia probably contributed to the increase in jejunum absorption values detected in litters of ethanol-fed dams when the absorption values are related to tissue wet weight, tissue dry weight and intestinal surthce. Little is known about folate absorption in the suckling animal (32,33) but it is clear that a variety of pancreatic and intestinal absorptive functions of the suckling are immature. Jejunal free folk acid absorption in the control group and litters nursed by dams receiving ethanol showed a gradual increase along with the increase of per&ion time at all the assayed concentrations. In general, in offspring from ethanol-fed dams, jejunal t&e folk acid absorption was higher than in control animals. In distal ileum loops free folk acid absorption did not occur in control pups, but appeared in litters exposed to ethanol during the lactation and suckling period. Our results in litters nursed by ethanol-fed dams shown that dii ileum t?ee folk acid absorption was 5-fold less than jejunum. Mason and Selhub (13) demonstrated that in neonatal animals the absorption of tke folk acid in the suckling rat was observed to occur more avidly in the jejunum than in the ileum a reversal of the pattern seen with bound folk acid (13). One might, therefore, speculate that the suckhng of ethanol-fed dams has an alternative mechanism by which it could absorb t&e folk acid from milk than the control animals. In this study, the ethanol has a negative elect during the suckling period with a retardation on the growth in the
Vol. 64, No. 22, 1999
Maternal Alcohol on Pup Folic Acid Absorption
2m9
ethanol-treated dams. This effect may be due to the toxic direct effect of ethanol and/or its metabolites and/or the decrease of milk uptake in the litters nurtured by ethanol-treated dams. Alcohol-exposed rat pups have been shown to take a longer time to attach to the nipple (34,35), are incapable of exerting adequate suckling pressure and have a reduced number of rapid rhythmic sucks per minute of suckling (29). Our results showed that the amount of milk secreted is decreased in ethanol-treated dams (data not shown). The milk folic acid levels are lower as well, which may contribute to retardation in litters growth. However, serum folic acid levels were only slight decreased in litters exposed to ethanol. This alteration in the small intestinal absorption, as well as, the milk folic acid levels may have contributed to the rnahrutrition status observed in the litters exposed to ethanol treatment at the end of lactation period.
offspring
In conclusion, this alteration formed part of a vicious circle, both the exposure to ethanol and the decrease in folk acid intake an effect of the ethanol exposure itself deficit caused alterations in the firnction of the intestinal mucous, which alters the of time absorption, the intake of folate and development. Free folic acid absorption in jejuntmr in litters exposed to ethanol is higher than control group at all assayed concentrations. It is possible then that the increased bee folate absorption in the jejunum is a compensatory mechanism of jejunal cells to attempt to increase free folate levels to compensate for the imposed decreases, possibly an up regulation of the folate carrier system However, ileal loops did not show free folic acid absorption in control group, but absorption appears in litters exposed to ethanol during pregnancy and the entire period of lactation at the end of lactational period. These results indicate that exposure of rats to ethanol during the pregnancy and suckling period, can tiect postnatal development of intestinal functions and could play a role in the genesis of malnutrition observed in the infant. Acknowledgments This investigation was supported by Grant PB93-1192 From the Direction General de Investigation Cientiflca y Tecnica (DGICYT) (Spain) References 1. K. CARUSO, R. TEN-BENSEL, Minn. Med. 76 25-29 (1993). 2. C. E. COLES, Cli. Obstet. Gynecol. 36 255-266 (1993). 3. J. L. JACOBSON, S. W. JACOBSON, R J. SOKOL, S. S. MARTIER, J. W. AGER, M. G. KAPLAN, Alcohol Clin. Exp. Res. 17 174-183 (1993). 4. K. L. JONES, D. W. SMITH, Lancet 2 999-lOOl(l973). 5. S. P. KUMAR, Ann Clin. Lab. Sci. 12 254-257 (1982). 6. J-P. BUTS, E. M. SOKAL, F. VAN HOOF, Pediatric Res. 32 5 (1992). 7. G. W. J. LIN, Alcohol 8 169- 172 (1991). 8. G. W. J. LIN, Nutr. Res. 11223-230 (1991). 9. G. W. J. LIN,Prog. B&hem. PharmacoL 18 115-121(1981). IO. G.W. J. LIN, Nutrition and alcohol temtogenicity. In Watson, R R, ed. Bio&er&fry and physiology of substance abuse. VOL2. Boca Raton, FL:CRC Press, 199022 I-243. 11. R. M. SANCHIS, M. SANCHO-TELLO, C. GUERRI, Alcohol 21295-305 (1986). 12. I. H. ROSENBERG, Intestinal absorption of folate. In: Johnson, L ed. Physiology of the gastrointestinal tract. New York: Raven Press; 1981: 1221-1230. 13. J. B. MASON, J. SELHUB, Am. J. Clin. Nut. 48 620-625 (1988). 14. T. D. COLLINS, B. H. EISENGA, S. D. BHANDARI, K. E. MCMARTIN, Alcohol Clin. Exp. Res. 16 757-763 (1992). 15. R. S. HILMANN, S. E. STEINBERG, Annu. Rev. Med. 33 345-354 (1982).
ml0
Maternal Alcohol on Pup Folk Acid Absorption
Vol. 64, No. 22,1!#9
16. L. C. RACUSEN, E. L. KRAWITT, Am. J. Dig. Dis. 22 915-920 (1977). 17. A. M. REISENAUER, T. BUFFINGTON, J. A. VILLANUEVA, C. H. HALSTED, Am, J. Cliu. Nutr. 50 1429-1435 (1989). 18. A. WU, I. CHANARIN, G. SLAVIN, A. J. LEVI, Brit. J. Hematol. 29 469-478 (1975). 19. 0. CARRERAS, A. L. VAZQUEZ, J. M. RUBIO, M. J. DELGADO, M. L. MURILLO, Am. Nutr. Metab. 38(4) 22 I-225 (1994). 20.0. CARRERAS, A. L. VAZQUEZ, J. .M. RUBIO, M. J. DELGADO, M. L. MURILLO, Archives Internationales de Physiologie, de Biochirnie et de Biophysique, 101 13-16 (1993). 21.0. FERNANDEZ-BORRACHERO, J:M. RUBIO, M. J. DELGADO, 0. CARRERAS, M. L. MURILLO, Ann. Nutr. Metab. 40 (5) 277-282 (1996). 22. 0. FERNANDEZ-BORRACHERO, J. M. RUBIO, M. J. DELGADO, M. L. MURILLO, 0. CARRERAS, Ann Nutr. Metab. 40 (5) 283-286 (1996). 23. H. M. SAID, D. W. HORNE, C. WAGNER, Arch Biochem. Biophys. 251 114-120 (1986). 24. J. J. HAJJAR, T. TOMICIC, R. L. SCHEIG, Digestion 22 170- 176 (1981). 25. F. PONZ, A. ILUNDAIN, M. LLUCH, Rev. Esp. Fisiol. 35 97-104 (1979). 26. E. TAVARES, 0. CARRERAS, A. GOMEZ-TUBIO, C. HERCE-PAGLIALI, M. L. MURILLO, Life Sciences 62 787-797 (1998). 27, D. WINNE, Experiential 32 1278-1279 (1976). 28. A. ENGUIX, L. GARCIA, M. HERNANDEZ, Rev. Diagn. BioL 41403406 (1992). 29. G. A. ROCKWOOD, E. P. RILEY, Teratology33 145-151(1985). 30. J.C.H. BODE, H. KNUPPEL, W. SCHWERK, H. LORENZ-MEYER, H.K. DURR, Digestion. 23 265-270 (1982). 3 1. J. PERSSON, N.O. BBERG, K. SJOLUND, R. STENLING, P.H. MAGNUSSON, Stand. J. Gastroenterol. 25 172-l 84 (1990). 32. D. N. SALTER, P. BLAKEBOROUGH, Br. J. Nutr. 59 497-507 (1988). 33. J. SELHUB, R AMOLD, A. M. SMITH, M. F. PICCIANO, Nutr. Res. 4 181-187 (1987). 34. J. CHEN, C.D. DRISCOLL, E. P. RILEY, Teratology26 145-153 (1982). 3 5. L. S. HANSEN-TRENCH T. M. SEGAR, S. BARRON, Neurotoxicol TeratoL 18 (6) 651657 (1996).
ELSEVIER
PI1 SOO24-3205(99)00147-2
Vol. 64, No. 22, pp. 2001~2010,19!B CIopyTight0 1999 Eckievierscience Inc. Printed in the USA. All rights rcsclvcd 0024-32OS/99/$-see front matter
FOLIC ACID INTESTINAL ABSORPTION IN NEWBORN RATS AT 2 1 DAY POSTPARTUMz EFFECTS OF MATERNAL ETHANOL CONSUMPTION ‘Eva Tavares , Ana Gomez-Tubio,
Maria Luisa Murillo and Olimpia Carreras.
Departamento de Fisiologia y Biologiu Animal. Facultad de Farmacia. Universidad de Sevilla. ESpaliCZ (Received in final form February 12, 1999)
Summary This study was designed to examine the effects of prenatal and postnatal exposure of ethanol in the in vivo absorption of free folic acid in the small intestine in pups rats at the 21’ day after birth. The rats were accustomed to increasing amounts of ethanol (5 to 20 %, voVvo1) in tap water for 1 month. During pregnancy and suckling period, ethanol-fed dams were assigned again to ethanol 20 % in drink& water. Two sets of experiments were performed. In the fist set, jejunal tree folic acid absorption in control group and litters nursed by dams receiving ethanol showed a gradual increase In general, along with the increase of perfusion time at all the assayed concentrations. in litters of ethanol-fed dams, jejunal free folic acid absorption expressed as nmol/intestinal surface, nmol/ g tissue wet weight and nmoll g tissue dry weight were higher than in control animals. In the second set of experiments, in distal ileum loops, free folic acid absorption did not occur in control pups, but appeared in litters exposed to ethanol. MiIk folk acid levels ate sig&cautly decreased in ethanol-treated dams However, only a slight decrease in the serum folic acid levels occurs in litters of ethanol-fed dams. In conchrsion, the results obtained in the present work suggested a diierent pattern of free folic acid absorption in distal ileum for the two groups. The exposure of rats to ethanol during the pregnancy and suckling period, can affect postnatal development of intestinal functions and could play a role in the genesis of malnutrition observed in the infant. Key Words: folic acid, alcohol, lactation, offspring, intestinal absorption
Pregnant women who drink alcohol heavily have a high risk of giving biih to cl&hen with growth retardation, congenital malformations and mental deficiency, known collectively as the Fetal Alcohol Syndrome (FAS) (l-5). Animal experiments also indicate that alcohol is a teratogen (611). However, the mechanism by which ethanol exerts its effect on fetal development is still unclear. Folic acid is an essential cofactor in the synthesis of purines and pyrimidmes, components of DNA and RNA (7,9). The jejunum is the site of maxim um absorption of free folate where the absorption occurs by a pH-dependent, carrier-mediated system (12). In the adult rat a saturable transport
’Corresponding author: Dra. Eva Tavares, Departamento de Fisiologta y Biologta Animal, Facultad de Farmacia, Universidad de &villa. Tramontana s/n, 4 1012, Sevilla. Espafk
2002
Maternal Alcohol on Pup Folic Acid Absorption
mechanism is responsible for nearly all the absorption physiologic concentrations of the nutrient ( 12 j.
Vol. 64, No. 22,1999
of f?ee folic acid in the small intestine at
Mason and Selhub (13) demonstrated that in neonatal animals the characteristics of folate-binding protein (FBP)-bound folic acid absorption are very different from those of free folic acid. Absorption of free folic acid in the suckling rat was observed to occur more avidly in the jejunum than in the ileum, a reversal of the pattern seen with bound folic acid (13). Folate deficiency during pregnancy has produce fetal malformations (8,9). Folate congenital defect in folate absorption in chronic diarrhea and incomplete maturation
been shown to interfere with fetal development and is an essential micronutrient for neonatal mammals. A human infants is associated with growth retardation, of the central nervous system (13).
Ethanol is known to interfere with the absorption deficiency is common among alcoholics (18). It is wide range of gastrointestinal effects in adult rats arising through ethanol administration have been (19,21,22,23) but not in suckling exposed to ethanol
and metabolism of folate (14-17) and folate well known that ethanol ingestion produces a (19-22). Alterations in folic acid absorption documented by several studies in adult rats during gestation and lactation period.
The purpose of this work was study the effects of maternal ethanol consumption during gestation and suckling period in the small intestinal absorption of free folic acid in o&spring rats at the 21’ day after the birth. Methods
Animals and alcohol administration. Male and female rats weighing between 200 and 250 g were randomized into two groups. In the first group, ethanol was administered with tap water by a previously described method (24). Ethanol-treated rats received ad libitum 5,10, and 15 % of ethanol in the d&king livid during 3 weeks successively. A consumption of 20 % was maintained in this group for 5 additional weeks. Ethanol-treated rats were mated. The presence of sperm in the vaginal smear the next morning denoted day 1 of pregnancy. Pregnant drinking period. lactation
females were replaced individually in their cages and water as the sole source of liquid with food ad libitum, The day of parturition was designated as day 1 of period. During the suckling period, the pups had free
assigned again to 20 % ethanol in during the pregnancy and suckling lactation and day 21 the final of access to the nipples.
Grouptwo~~ascontrolandreceivedontywaterandastaradardratchowdiet(Panlab04)ad lii cOntrolratswerebandledinthesamewayastheother~groups. Theaverage intake of ethanol (ml), food (g), water (ml) and body weight (g) were controlled on day 1 after birth andagainonday7andthereaftereveryweekuntiltheendoftheexperimentaiperiod. Each measurement was recorded at 9.00 AM to avoid changes due to circadian rhythms. The rats were maintained under automatically light-dark cycles with lights on at 07.00 h.
controlled
temperature
(22-23 “C) and 12 hour
Multiple-Pass Perfusion. Twenty-one days after the birth, the pups were used in this study. The o&pring from ethanol-treated and control dams were anaesthetized with subcutaneous uretane 10 %. Two sets of experiments were performed. In the first set, the abdominal cavity was opened by
Vol. 64, No. 22, 1999
Maternal Alcohol on Pup Folic Acid Absorption
2003
a longitudinal incision and the jejunum was cam&ted with polyethylene tubing. Inflow and out flow cannulas were tied into jejunum. Thus, a jejunal loop was isolated from continuity with the lumen. After cannulation, the loop was rinsed v&h 0.9 % NaCl solution and replaced inside the body wall and perfused as previously described by Ponz et al. (25). A flow rate of 3 ml/min during 5 min period was used, for each concentration in all animals (26). In the second set of experiments, in order to achieve the same experimental parameters on the distal ileum, we following the same procedures described for the first set, in different offspring l?om the first set of animals. Folic acid absorption. ‘H-folic acid (0.01 pCi / ml) was prepared in a solution containing: 154 mM NaCl and cold folic acid at 0.25 pM, 0 5 pM, 1.O uM, 1.5 pM and 2.5 pM concentrations. This solution was pre-warmed (37°C) before perfusion. Folk acid uptake was measured in different experimental animak. PerfUsions were performed with increasing concentrations and immediate washes with sterile saline solution were made between successive perfusions. The perfusion time for all the o@qing studied was divided into an equilibrium period of 15 min and one period of 5 min at each substrate concentration. Folk acid absorption was determined as the difference between the initial and final amount of substrate obtained in the perfusates. Offspring were maintained under controlled temperature (37’C) with a heating pad. The absorption was calculated and expressed as nmol I intestinal weight and nmol / g tissue dry wt.
surface, nmol I g tissue wet
Morphometric Tissue Evaluation. At the end of perhkions, pups were sacr&zd and the jejunum and ileumt&ueswereremovedtodeterm&theirwetanddryweights. Thetissueiblctionswerewe~ driedinanovenat 100°C toconstantweight(24h)andreweighed,toobtaintksuedryweights. To determ&thetotalserosalareqtheoutercircun&ence was measured according to winne (27). Milk and bloodpreparation. In order to obtaiu the maximumamountofmilkwithoutmod@ingthe physiologicalconditionsofthesubjectswithanesthetics,onday21oflactation3hafterremovingthe litters~mthemothers,damswere~bydecapitationandmilk~~swere~ cOllected.Mill
Pups body weight. Fii 1 show the body weight of pups rats during the exper&ntal bir&therewasnosigmfkam~betweenthebodyweightofthelittersnursedbydams
period. At
Maternal Alcohol on Pup Folic Acid Absorption
2004
Vol. 64, No. 22,199!9
receiving ethanol
during the gestation period compared to control group. However, during the lactationalperiod, s~differencewereo~~betweenbothcontrolaradethanolgroups.
40
?
T
35
& --c
c I 2
3o
5
25
> 0
20
0 m
CONTROL ETHANOL
75
10
5
1
7
14
21
DAYS
Fig. 1
Body weight (g) in of&p&g of ethanol-fed dams and control group. The results are expmssesasmean~SEM.N=10indkatethenumberofanimalsineachgmup. p
Effects of ethanol on several intestinal parameters. Pups from ethanol-treated mothers showed at 21 day postpartum (Table I) decreased values in jejunum of total area F( 1, 19) = 8.41 ,p < 0.02, tissue wet wt. g F(1, 19) = 1.27, p < O.Ol), tissue dry wt. g F(1, 19) = 5.83, p < 0.01 compared with control group. In Table I showed similar results in the ileum those described for the jejtmum. Total areafll, 19) = 14.9, p < 0.001, tissue wet wt. g F(1, 19) = 17.3, p < 0.001, tissue dry wt. g F(1, 19) = 15.5, p < 0.001. .
TABLE I
Jejunal and distal ileum parameters measured in 2 1-d-old rats born from ethanoltreated mothers and controls. The results are expresses as mean f SE&I. N=lO indkate the number ofanimals in each group. * p < 0.05; ** p < 0.02; *** p < O.Ol;#p < 0.001. Ethanol group vs Control group.
Tissue wet wt. (g)
2005
Maternal Alcohol on Pup Folic Acid Absorption
Vol. 64, No. 22,1999
Jejunal free folic acid absorption. The results obtained for jejunal free folk acid absorption in ofEspring from the ethanol-fed dams and control, expressed as nmolkm2, nmol/tissue wet wt. g and nmolltissue dry wt. g at diI&rent substrate concentrations: 0.25 uM, 0.5 uM, 1 PM, 1.5 uM and 2.5 uM are shown in Fig. 2 and Fig. 3. Jejunal free folk acid absorption in control group and litters nursed by dams receiving ethanol during pregnancy and suckling period showed a gradual increase along with the increase of perfusion time at all the assayed concentrations. In general, in offspring from ethanol-fed dams, jejunal free folk acid absorption was higher than in control animals (Fig. 2 and 3). In pups of ethanol-fed dams the jejunal free folk acid absorption was significantly increased at 0.25 uM (F(1, 19) = 12.7, p < 0.001); 0.5 l.tM (F(1, 19) = 26.7, p < O.OOl), 1.5 uM (fll, 19) = 15.6, p < 0.001) and 2.5 uM (F(1,19) = 9.37, p < 0.01) with respect to control group, when the absorption was expressed as nmoYintestinal surface (Fig. 2).
z 0 c
0 cn
** 0
e
0.10
I PZZZZJ
CONTROL ETHANOL
m ,’ * 0 0 4:
0, -I
:
*
0.06
* N
-
0
0 .06
: -
0.04
*
0 Y
0.02 0 .oo
0.2 5 FOLIC
ACID
0.5
1 .o
CONCENTRATIONS
1 .5
2.5 (PM)
Fig. 2 Jejunal free folk acid absorption (nmoVcm2) in oftspring of ethanol-fed dams and control group, at the 2 1’ day atter birth. The results are expresses as mean k SEM. N=1Oindicatethenumberofanimalsineachgroup. p < O.OOl*;p < O.Ol**. Ethanol group vs Control group.
Similar results were obtained when the absorption per gram of both wet and dry tissue were measured (Fig. 3). In Figure 3A, jejunal free folk acid absorption expressed as the absorption per gram of wet tissue was significantly increased in litters of ethanol ted-dams at 0.25 uM (F(1,19) = 7.13, p < 0.01); 0.5 @I (F(1, 19) = 18.9; p < 0.001) and 2.5 uM (fll, 19) = 7.76, p < 0.02) with respect to the control group. A significant increase in absorption was also shown in ofhqking of ethanol treated dams at 0.25 @I (01, 19) = 8.15, p < 0.01); 0.5 uM (F(1, 19) =
2006
Maternal Alcohol on Pup Folic Acid Absorption
Vol. 64, No. 22, 1999
26.7, p < O.OOlj; 1.5 PM (F(1, 19) = 34, p < 0.001) and 2.5 pM (F(1, 19) = 9.14, p < 0.01) expressed as the absorption for tissue dry wt. g (Fig. 3B).
A U
CONTROL
m
ETHANOL
0.25 FOLIC
1 .o 0.5 1.5 ACID CONCENTRATIONS
2.5 (PM)
B 0 W
0.25 FOLIC
CONTROL ETHANOL
0.5 ACID
1 .o
1 .5
CONCENTRATIONS
2.5 (VU)
Fig. 3 Jejunal Gee folk acid absorption in o&pring of ethanol-fed dams and control group, at the 21’ day after birth A) expressed as nmoY g tissue wet weight and B) aSUXZUlztSEM. elrpressedasnmoYgtissuedryweight.Theresuttsare~ N=10indkatethenuruberofanimalsineachgroup. p
Maternal Alcohol on
Vol. 64, No. 22,1999
Pup Folic Acid Absorption
2007
Distal ileumfiee folic acid absorption. The results obtained for free folk acid absorption in distal ileum in pups at day 21 were nil in the controls at all of the assayed concentrations. However, free fohc acid absorption in distal ileum in ofkpriug of ethanol-fed dams (Table II), did take place. TABLE II
Distal ileum fke folk acid absorption in offspring of ethanol-fed dams, at the 2 1’ day after birth. N=lO indicate the number of animals. FOLIC ACID CONCENTRATIONS l&M 0.25p.M O&M l.OpM
Folic acid absorption
nmoVtissue wet wt. g nmol/tissue drywt.g nmowtotal area
2.sp.M
0.2M.01
0.44z!IO.04
0.56kO.03
0.63ztO.05
1.62kO.2
1.29kO.04
2.79fo.04
3.5fo.16
4.02&O.14
10.3kO.42
4.3*1U3 *lo”
8.g*10‘3 ti*lO”’
12*10-3 ks.4*10q
13*10” ti.2*10A
30*10-3 k2*1U4
Serum and milk foiic acid levels. Figure 4 shows that the levels of folk acid in milk (F (1, 13) = 8.23, p < 0.05) were sigo&an@ decreased in the ethanol-fed dam on day 21 of lactation. However, serum folk acid levels on postpartum day 2 1 were only slightly decreased in litters nursed by dams receiving ethanol during gestation and suckling period.
0 0
CONTROL
1 Q2ZZ
ETHANOL
ul 100
ii
ii >
16
lZ?SZ3
CONTROL
I
ETHANOL
>
W -I
80
n 0, a-
E
y2J .=
00
40
IA
Y
d
ZO
I
0
I
P 3
a ii
4 2 0
Fig. 4
Milk folk acid levels aud serum folk acid levels in o&pring. The results are expmssesasmeau*SEMN=14indicatethenumberofauimalsineachgroup.p< 0.05*. Ethanol group vs Control group.
2008
Maternal Alcohol on Pup Folic Acid Absorption
Vol. 64, No. 22,1999
Discussion
In this study, we measured the “in vivo” free folk acid absorption across the jejunum and ileum distal in litters nursed by dams receiving ethanol during gestation and suckling period. Alterations in folk acid absorption arising through ethanol administration have been documented by several studies in adult rats (14,16,19,21,22) but not in suckling exposed to ethanol during gestation and lactation period. In adult rats, previous studies observed no inhibitory effect of alcohol (29) on the jejunal uptake of folk acid. However, under partial starvation conditions (4,19,2 1) an increase in folate transport has been described. Recently, Buts et al. (6) using a similar experimental model of ethanol administration during pregnancy but not in the suckling period have shown that postnatal maturation of the small intestine in the ethanol offspring was depressed during the early nursing period, even though ethanol had been withdrawn at birth. Pups born from ethanol-treated mothers showed lower intestinal mass, depressed jejunal and ileal DNA contents per unit of length, decreased mucosal rat DNA synthesis. All these parameters returned to control levels by day 15 postpartum. An immature aspect of the enterocytes that persisted until weaning was also found in this study (6). The data obtained in the present study in jejunum and distal ileum showed a decrease in intestinal area, wet weight and tissue dry weight in litters exposed to ethanol as well as an increase in values of free folk acid absorption at difkrent folk acid concentrations in the jejunum with respect to the control group. Morphometric studies have shown that villus height and mucosalsurtace area are reduced in alcoholic as compared to nonalcoholic patients (30,31). Previous studies in rats (19,26) have shown a decrease in intestinal wet and dry weights after ethanol consumption. This hypoplasia probably contributed to the increase in jejunum absorption values detected in litters of ethanol-fed dams when the absorption values are related to tissue wet weight, tissue dry weight and intestinal surthce. Little is known about folate absorption in the suckling animal (32,33) but it is clear that a variety of pancreatic and intestinal absorptive functions of the suckling are immature. Jejunal free folk acid absorption in the control group and litters nursed by dams receiving ethanol showed a gradual increase along with the increase of per&ion time at all the assayed concentrations. In general, in offspring from ethanol-fed dams, jejunal t&e folk acid absorption was higher than in control animals. In distal ileum loops free folk acid absorption did not occur in control pups, but appeared in litters exposed to ethanol during the lactation and suckling period. Our results in litters nursed by ethanol-fed dams shown that dii ileum t?ee folk acid absorption was 5-fold less than jejunum. Mason and Selhub (13) demonstrated that in neonatal animals the absorption of tke folk acid in the suckling rat was observed to occur more avidly in the jejunum than in the ileum a reversal of the pattern seen with bound folk acid (13). One might, therefore, speculate that the suckhng of ethanol-fed dams has an alternative mechanism by which it could absorb t&e folk acid from milk than the control animals. In this study, the ethanol has a negative elect during the suckling period with a retardation on the growth in the
Vol. 64, No. 22, 1999
Maternal Alcohol on Pup Folic Acid Absorption
2m9
ethanol-treated dams. This effect may be due to the toxic direct effect of ethanol and/or its metabolites and/or the decrease of milk uptake in the litters nurtured by ethanol-treated dams. Alcohol-exposed rat pups have been shown to take a longer time to attach to the nipple (34,35), are incapable of exerting adequate suckling pressure and have a reduced number of rapid rhythmic sucks per minute of suckling (29). Our results showed that the amount of milk secreted is decreased in ethanol-treated dams (data not shown). The milk folic acid levels are lower as well, which may contribute to retardation in litters growth. However, serum folic acid levels were only slight decreased in litters exposed to ethanol. This alteration in the small intestinal absorption, as well as, the milk folic acid levels may have contributed to the rnahrutrition status observed in the litters exposed to ethanol treatment at the end of lactation period.
offspring
In conclusion, this alteration formed part of a vicious circle, both the exposure to ethanol and the decrease in folk acid intake an effect of the ethanol exposure itself deficit caused alterations in the firnction of the intestinal mucous, which alters the of time absorption, the intake of folate and development. Free folic acid absorption in jejuntmr in litters exposed to ethanol is higher than control group at all assayed concentrations. It is possible then that the increased bee folate absorption in the jejunum is a compensatory mechanism of jejunal cells to attempt to increase free folate levels to compensate for the imposed decreases, possibly an up regulation of the folate carrier system However, ileal loops did not show free folic acid absorption in control group, but absorption appears in litters exposed to ethanol during pregnancy and the entire period of lactation at the end of lactational period. These results indicate that exposure of rats to ethanol during the pregnancy and suckling period, can tiect postnatal development of intestinal functions and could play a role in the genesis of malnutrition observed in the infant. Acknowledgments This investigation was supported by Grant PB93-1192 From the Direction General de Investigation Cientiflca y Tecnica (DGICYT) (Spain) References 1. K. CARUSO, R. TEN-BENSEL, Minn. Med. 76 25-29 (1993). 2. C. E. COLES, Cli. Obstet. Gynecol. 36 255-266 (1993). 3. J. L. JACOBSON, S. W. JACOBSON, R J. SOKOL, S. S. MARTIER, J. W. AGER, M. G. KAPLAN, Alcohol Clin. Exp. Res. 17 174-183 (1993). 4. K. L. JONES, D. W. SMITH, Lancet 2 999-lOOl(l973). 5. S. P. KUMAR, Ann Clin. Lab. Sci. 12 254-257 (1982). 6. J-P. BUTS, E. M. SOKAL, F. VAN HOOF, Pediatric Res. 32 5 (1992). 7. G. W. J. LIN, Alcohol 8 169- 172 (1991). 8. G. W. J. LIN, Nutr. Res. 11223-230 (1991). 9. G. W. J. LIN,Prog. B&hem. PharmacoL 18 115-121(1981). IO. G.W. J. LIN, Nutrition and alcohol temtogenicity. In Watson, R R, ed. Bio&er&fry and physiology of substance abuse. VOL2. Boca Raton, FL:CRC Press, 199022 I-243. 11. R. M. SANCHIS, M. SANCHO-TELLO, C. GUERRI, Alcohol 21295-305 (1986). 12. I. H. ROSENBERG, Intestinal absorption of folate. In: Johnson, L ed. Physiology of the gastrointestinal tract. New York: Raven Press; 1981: 1221-1230. 13. J. B. MASON, J. SELHUB, Am. J. Clin. Nut. 48 620-625 (1988). 14. T. D. COLLINS, B. H. EISENGA, S. D. BHANDARI, K. E. MCMARTIN, Alcohol Clin. Exp. Res. 16 757-763 (1992). 15. R. S. HILMANN, S. E. STEINBERG, Annu. Rev. Med. 33 345-354 (1982).
ml0
Maternal Alcohol on Pup Folk Acid Absorption
Vol. 64, No. 22,1!#9
16. L. C. RACUSEN, E. L. KRAWITT, Am. J. Dig. Dis. 22 915-920 (1977). 17. A. M. REISENAUER, T. BUFFINGTON, J. A. VILLANUEVA, C. H. HALSTED, Am, J. Cliu. Nutr. 50 1429-1435 (1989). 18. A. WU, I. CHANARIN, G. SLAVIN, A. J. LEVI, Brit. J. Hematol. 29 469-478 (1975). 19. 0. CARRERAS, A. L. VAZQUEZ, J. M. RUBIO, M. J. DELGADO, M. L. MURILLO, Am. Nutr. Metab. 38(4) 22 I-225 (1994). 20.0. CARRERAS, A. L. VAZQUEZ, J. .M. RUBIO, M. J. DELGADO, M. L. MURILLO, Archives Internationales de Physiologie, de Biochirnie et de Biophysique, 101 13-16 (1993). 21.0. FERNANDEZ-BORRACHERO, J:M. RUBIO, M. J. DELGADO, 0. CARRERAS, M. L. MURILLO, Ann. Nutr. Metab. 40 (5) 277-282 (1996). 22. 0. FERNANDEZ-BORRACHERO, J. M. RUBIO, M. J. DELGADO, M. L. MURILLO, 0. CARRERAS, Ann Nutr. Metab. 40 (5) 283-286 (1996). 23. H. M. SAID, D. W. HORNE, C. WAGNER, Arch Biochem. Biophys. 251 114-120 (1986). 24. J. J. HAJJAR, T. TOMICIC, R. L. SCHEIG, Digestion 22 170- 176 (1981). 25. F. PONZ, A. ILUNDAIN, M. LLUCH, Rev. Esp. Fisiol. 35 97-104 (1979). 26. E. TAVARES, 0. CARRERAS, A. GOMEZ-TUBIO, C. HERCE-PAGLIALI, M. L. MURILLO, Life Sciences 62 787-797 (1998). 27, D. WINNE, Experiential 32 1278-1279 (1976). 28. A. ENGUIX, L. GARCIA, M. HERNANDEZ, Rev. Diagn. BioL 41403406 (1992). 29. G. A. ROCKWOOD, E. P. RILEY, Teratology33 145-151(1985). 30. J.C.H. BODE, H. KNUPPEL, W. SCHWERK, H. LORENZ-MEYER, H.K. DURR, Digestion. 23 265-270 (1982). 3 1. J. PERSSON, N.O. BBERG, K. SJOLUND, R. STENLING, P.H. MAGNUSSON, Stand. J. Gastroenterol. 25 172-l 84 (1990). 32. D. N. SALTER, P. BLAKEBOROUGH, Br. J. Nutr. 59 497-507 (1988). 33. J. SELHUB, R AMOLD, A. M. SMITH, M. F. PICCIANO, Nutr. Res. 4 181-187 (1987). 34. J. CHEN, C.D. DRISCOLL, E. P. RILEY, Teratology26 145-153 (1982). 3 5. L. S. HANSEN-TRENCH T. M. SEGAR, S. BARRON, Neurotoxicol TeratoL 18 (6) 651657 (1996).