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Efectos del acido acetilsalicilico sobre la restriccion de crecimiento intrauterino inducida por etanol en ratas.

RESUMEN: Fueron estudiados los efectos del acido acetilsalicilico (AAS) sobre la restriccion de crecimiento intrauterino inducida por el consumo materno de etanol. Una unica dosis intraperitoneal de etanol (2,96 g/kg de peso corporal), inyectada en el 10[grados] dia de prenez (PD10), disminuyo significativamente la ganancia de peso materno, peso fetal, volumen del liquido amniotico y longitud del cordon umbilical. En cambio, el etanol no afecto el peso de la placenta ni el tamano de la nidada. Una unica dosis intraperitoneal de AAS (200 mg/g de peso corporal), inyectada en el PDI0, no modifico la ganancia de peso materno, tamano de la nidada, los pesos letal y placentario, ni el volumen del liquido amniotico, pero disminuyo significativamente la longitud del cordon umbilical. La inyeccion intraperitoneal de AAS (200 mg/g de peso corporal) y de etanol (2.96 g/kg de peso corporal) en el PD 10, demostro que el AAS antagoniza, por lo menos en parte, los efectos del etanol sobre la ganancia de peso materno, peso fetal y volumen del liquido amniotico. Al contrario, el AAS potencializa el efecto del etanol sobre la longitud del cordon umbilical.

PALABRAS CLAVE: 1. Rata; 2. Feto; 3. AAS; 4. Etanol: 5. Crecimiento intrauterino.

EFFECTS OF ACETYLSALICYLIC ACID ON ETHANOL-INDUCED INTRAUTERINE GROWTH RESTRICTION IN RATS

SUMMARY: The effects of acetylsalicylic acid (ASA) on intrauterine growth restriction induced by maternal ethanol consumption were studied. A single intraperitoneal dose of ethanol (2.96 g/kg body weight), injected on the 10th day of pregnancy (PD10), significantly reduced maternal weight gain, fetal weight, amniotic fluid volume and umbilical cord length. Meanwhile, ethanol did not affect placental weight of litter size. A single intraperitoneal dose of ASA (200 mg/g body weight), injected on the PD10, did not modify maternal body weight gain, litter size, fetal and placental weights and amniotic fluid volume, but significantly diminished umbilical cord length. Intraperitoneal injections of either ASA (200 mg/g body weight) or ethanol (2.96 g/kg body weight) on the PD10, demonstrated that ASA antagonized, in part at least, the effects of ethanol on maternal weight gain, fetal weight, and amniotic fluid volume. Contrarily, ASA potentiated the effect of ethanol on umbilical cord length.

KEY WORDS: 1. Rat; 2. Fetus: 3. ASA; 4. Ethanol; 5. Intrauterine development.

INTRODUCTION

It is well known that high doses of ethanol administered during pregnancy result in adverse pregnancy outcome. The manifestations of prenatal ethanol exposure range flora obvious marked growth deficiency and organ malformations to more subtle effects on central nervous system (CNS) development and abnormal function. These developmental alterations, apparent at birth, characterized by growth retardation and severely compromised mental capabilities, collectively have been labeled fetal alcohol syndrome (FAS) (Jones & Smith, 1973).

Jollie (1990) sustained that prostaglandins (PG) may be involved in the etiology of FAS, due to the stimulating action of ethanol on PG levels. Accordingly, drugs which inhibit PG synthesis, as acetylsalicylic acid (ASA), could have a protecting action on ethanol effects (Randall et al., 1991a).

The purpose of this study is to assess the possible protective effect of ASA on the harmful action of ethanol on intrauterine growth.

MATERIAL AND METHOD

Animals, chemicals and treatment: Virgin female Wistar rats (Rattus norvegicus), weighing between 200 and 250 g, were used. After a period of two weeks of acclimatation, the animals were mated overnight with fertile males, and the day on which sperm was detected in the vaginal smear was designated pregnancy day 1 (PD1). Pregnant rats were kept individually in polypropylene cages throughout pregnancy under controlled conditions of light (12 h-light, 12 h-dark cycle), humidity (50 [+ or -] 10%) and temperature (22 [+ or -] 2[degrees]C), and red with a commercial pelleted rodent diet and water ad libitum.

After 10 days, twenty pregnant rats were randomly distributed in the following four experimental groups:

Group I. Five rats injected with a single intraperitoneal dose of 2.96 g/kg body weight of ethanol, between the 9:00 and 10:00 h.

Group II--Five rats injected with a single intraperitoneal dose of 200 mg/kg body weight of Aspisin[R] (ASA solution of 200 mg/ml), between the 9:00 and 10:00 h.

Group III--Five rats receiving an intraperitoneal injection of 200 mg/kg body weight of Aspisin[R], between the 9:00 and 10:00 h., and, after one hour, ah intraperitoneal injection of 2.96 g/kg body weight of ethanol.

Group IV--Control group, constituted by five animals injected on the GD10 with an equivalent volume of sterile saline, between the 9:00 and 10:00 h.

Ethanol was obtained from Merck (Darmsiadt, Germany), and Aspisin[R] was obtained from Farmasa--Laboratorio Americano de Farmacoterapia S.A. (Sao Paulo, Brazil).

Morphometrical determinations: On the morning of the PD20, rats were killed by anesthetic ether inhalation. The uterine horns were examined for resorption sites, live and dead fetuses. Each fetus was scrutinized for external anomalies, and then fixed in a solution of 85 ml 80% alcohol, 10 ml formalin and 5 ml glacial acetic acid. After 24 h of fixation, fetuses and placentas were blotted dry and weighed in a precision scale, and the umbilical cords measured.

Statistical analysis: Statistical analysis was performed using one-way analysis of variance (ANOVA). Where supported by significant treatment effects, comparisons among groups were performed using the Tukey's test (Sahai & Ageel, 2000).

RESULTS

The maternal body weight gain from the PD10 to the PD20 showed significant differences among groups (Table I). The intraperitoneal injection of ethanol (groups I and III) significantly reduced the maternal weight gain in pregnant rats in comparison either to the control group or to those injected only with ASA (group II). Animals injected only with ethanol, on the other hand, showed smaller body weight gain than those animals treated with ASA plus ethanol.

The litter size showed no differences among groups (Table I).

The fetal body weight showed significant differences among groups (Table I). Fetuses belonging to the two groups injected with ethanol weighed less than those of the control group. Fetuses from pregnant rats injected only with ethanol were lighter than those from animals previously injected with ASA. On the other hand, fetal weight showed no differences between group II (ASA) and either group III (ASA plus ethanol) or IV (control).

The placental weight showed no significant differences among the four groups (Table I).

The amniotic fluid volume showed significant differences among groups (Table I). Fetuses from both ethanol-injected groups (groups I and III) had significantly smaller amniotic fluid volume than either the control fetuses of the ASA injected group fetuses. On the other hand, the amniotic fluid volume was significantly higher in the ASA plus ethanol-injected group than in that only injected with ethanol.

The length of the umbilical cord showed significant differences among groups (Table I). Fetuses from all the treated groups had significantly shorter umbilical cords than the control fetuses (Table 1). Similarly, umbilical cord was shorter in the ASA plus ethanol-injected group than in the ethanol-injected group. On the other hand, no significant differences were found between umbilical cord length of fetuses flom the group II (ASA) and those from both groups I (ethanol) and III (ASA plus ethanol).

DISCUSSION

Pregnant rats injected with ethanol showed a significantly smaller weight gain during the last 10 days of pregnancy than either the control animals or those injected only with ASA. However, no significant difference of maternal weight gain was observed between animals injected with ASA and the control ones. Besides, body weight gain was higher in the animals injected with ASA plus ethanol than in those injected only with ethanol. In this way, ASA demonstrated a partial protecting effect on the action of ethanol in the weight gain during pregnancy.

It is well known that malnutrition and undernourishment are associated to the chronic consumption of ethanol. In these cases, intrauterine growth retardation was attributed to a secondary effect of the nutritional deficit due to ethanol consumption. Abel (1978) claimed that the effects of chronic treatment with ethanol (1 or 2 g/kg body weight) throughout pregnancy, appeared to be related to the reduction of the maternal intake of calories rather to a direct effect of ethanol on the fetus. In the present study, however, ethanol was administered in a single dose, on the PD10, being harmed the interpretation that decreased weight gain would be owed to a reduced ingestion of nutrients.

Warkany & Takacs (1959) demonstrated a loss between 15 and 25 g in the body weight of pregnant rats injected with methyl salicylate, but the animals recovered their weights after 3 or 4 days. Esperidiao et al. (1998) demonstrated that maternal weight gain was not affected in rats receiving up to 100 mg/kg ASA, via gavage, during the whole pregnancy.

In the present study, the litter size did not show difference among groups, as previously reported in mice (Pilstrom & Kiessling, 1967; Randall et al., 1991a,b). Tze & Lee (1975), on the other hand, observed litter size reduction after in utero exposure to ethanol.

Lubawy & Garrett (1977) demonstrated increased number of resorptions after oral administration of ASA (125 of 250 mg/kg/day), between the PD8 and the PD20. Espiridiao et al., after daily administration of ASA (100 to 400 mg/kg), during all the pregnancy, via gavage, found smaller litter size.

Analysis of the fetal weight data indicated that both experimental groups of pregnant rats injected with ethanol showed lighter fetuses. Meanwhile, fetuses from rats injected with ASA plus ethanol were heavier than those from rats injected only with ethanol. It is well known that prenatal exposure to ethanol directly acts on the embryogenesis, causing growth alterations, characterized mainly by retarded intrauterine growth (Randall et al., 1991a,b). After Wunderlich et al. (1979), ethanol inhibits the synthesis of the chorionic somatomammotropin. ASA (150 or 300 mg/ kg), administered on the PD10, 1 h before ethanol (5.8 g/ kg), showed a protective action on the reduction of the fetal weight caused by ethanol in mice (Randall et al., 1991a,b). Our results demonstrated only a partial protective effect of ASA on fetal weight reduction caused by ethanol in rats.

According to Pennington et al. (1985), intrauterine growth retardation caused by ethanol is mediated by the increase of prostaglandin levels. Thus, the simultaneous administration of inhibitors of PG synthesis would protect against the ethanol-induced hypoplasia. Anton el al. (1990) and Cook & Randall (1997) demonstrated that ethanol (6 g/ kg body weight, on the PD17) induces premature labor in mice, and that this action is mediated by PGE and PGF2. Pretreatment with ASA on the PD 17, inhibited PG synthesis and blocked premature labor. The authors concluded that premature labor, shortening the gestational age, could have important implications for the concept, as well as for the normal letal growth and development.

After oral administration of 125 of 250 mg/kg/day of ASA to pregnant rats between the PD8 and the PD20, Lubawy & Garrett demonstrated decrease of both fetal weight and length. Similarly, daily doses of 100 or 400 mg/ kg/day of ASA to pregnant rats significantly decreased the fetal weight (Espiridiao et al.).

In the present work no significant effects on the placental weight was demonstrated, after ethanol, ASA or ASA plus ethanol administration. Wunderlich et al. did not observe difference in the placental weight of fetuses treated with ethanol. Even without affect the placental weight, it is possible that ethanol acts on the fetal weight decreasing the placental transfer capacity, inhibiting enzymes or hormones, as the chorionic somatomammotropin.

There are no references about any toxic action of ASA on the placenta. Lubawy and Garrett, after oral ASA administration (125 or 250 mg/kg/day), between the PD8 and the PD20, and Espiridiao et al., with daily doses of 400 mg/kg ASA, during all the pregnancy, demonstrated decreased placental weight. On the other hand, Hamed et al. (1994) claimed that ASA did not affect the placental weight, both in well fed fetuses and in not well nurtured fetuses, whereas Wallenburg & Rotmans (1987), administering low doses of ASA, prevented the idiopathic uteroplacental insufficiency.

In the present study, it was verified that ethanol, administered only or together with ASA, provoked a significant reduction of the amniotic fluid volume, in comparison to both the control group and the ASA injected group. Administration of ASA during pregnancy provokes no significant alteration of the amniotic fluid volume. There were significant difference between the group injected only with ethanol and that injected with ASA plus ethanol, which shows that ASA possess a partial protecting action against the effects of ethanol on the volume of amniotic fluid.

Randall et al. (1991b) demonstrated renal malformations in 45% of the mouse fetuses treated with ethanol on the PD10, while in animals receiving ASA 1 h before ethanol, the incidence of renal malformations dropped to 29%. Gage & Sulik (1991) demonstrated that IP administration of two ethanol doses (2.9 g/kg) on the PD9 provoked anomalies on the mouse urinary tract, mainly hydronephrosis and hydroureter, in 40,7% of the fetuses. Once the fetal urine is the most important constituent of the amniotic fluid, it is of waiting a decreased urinary excretion and amniotic fluid volume in fetuses from mothers receiving ethanol during the organogenic period. Randall et al. (1991a) showed a high incidence of urinary malformations in fetuses of mice injected with ethanol on the PD 10, and demonstrated a protective action of 150 or 300 mg/kg ASA, administered 1 h before ethanol. The authors claimed that ASA pretreatment dramatically reduces the risks of the prenatal exposition to ethanol, even if does not prevent the ethanol-induced congenital defects.

On the other hand, treatment with ASA or with ASA plus ethanol during pregnancy significantly reduced umbilical cord length when confronted with the control group. Similarly, fetuses from the group injected with ASA plus ethanol have shorter umbilical cords than fetuses from the group injected only with ethanol. Thus ASA acts synergically with ethanol in shortening the umbilical cord length. The length of the umbilical cord reflects, mainly, the fetal movement (Miller et al., 1981). According to the results, ASA would have larger power of limitation of the fetal movements that ethanol, besides to potentiate the effects of ethanol on the intrauterine movement.

In conclusion, ASA seems to reduce, in the pregnant rat, the deleterious effects of ethanol on some of the morphometric fetal parameters, confirming the results obtained by Randall et al. (1991 a,b), in mice.

This work was supported by "Fundacao de Apoio a Pesquisa do Estado de Sao Paulo" (FAPESP--Proc. 00/00360-7), Brasil.

Recibido: 2801-2003

Aceptado: 04-03-2003

REFERENCES

Abel, E. L. Effects of ethanol on pregnant rats and their offspring. Psychopharmacology, 57:5-11, 1978.

Anton, R. F.; Becker, H.C. & Randall, C.L. Ethanol increases PGE and thromboxane production in mouse pregnant uterine tissue. Life Sci., 46:1145-53, 1990.

Cook, J. L. & Randall, C. L. Early onset of parturition induced by acute alcohol exposure in C57BL/OJ mice: role of uterine PGE and PGF2alpha. Repr. Fertil. Dev., 9:815-23, 1997.

Espiridiao, S.; Ajeje, R.; Kopelman, B. I., Simoes. M. J.; Evencio-Neto, J. & Kulay, Jr. L. Acao cronica do acido acetilsalicilico na prenhez da rata. Rey. Bras. Ginec. Obstet., 20: 245-9, 1998.

Gage, J.C. & Sulik, K.K. Pathogenesis of ethanol-induced hydronephrosis and hydroureter as demonstrated following in vivo exposure of mouse embryos. Teratology, 44:299-312, 1991.

Hamed, M. R.; Al-Assy, Y.S. & Ezzeldin, E., Influence of protein malnutrition on teratogenicity of acetylsalicylic acid in rats. Hum. Exper. Toxicol., 13:83-8, 1994.

Jollie, W. P. Effect of sustained dietary ethanol on the ultrastructure of the visceral yolk-sac placenta of the rat. Teratology, 42:541-2, 1990.

Jones, K. L. & Smith, D.W. Recognition of the letal alcohol syndrome in early infancy. Lancet, 2:999-1001, 1973.

Lubawy, W.C. & Garrett, R. J. Effects of aspirin and acetaminophen on fetal and placental growth in rats. J. Pharm. Sci., 66:111-3, 1977.

Miller, M.E.; Higginbottom, M.C. & Smith, D.W. Short umbilical cord: Its origin and relevance. Pediatrics, 67: 618-21, 1981.

Pennington, S.; Allen, Z.; Tunion, J.; Farmer, P.; Rowland, L. & Kalmus, G. Prostaglandin synthesis inhibitors blocks alcohol-induced fetal hypoplasia. Alc. Clin. Exper. Res., 9:433-7, 1985.

Pilstrom, L. & Kiessling, K. H. Effects of ethanol on the growth and on the liver and brain mitochondrial functions of the offspring of rats. Acta Pharmacol. Toxicol., 25: 225-32, 1967.

Randall, C. L.; Anton, R. F.; Becker, H.C.; Hale, R. L. & Ekblad, U. Aspirin dose-dependently reduces alcohol-induced birth defects and prostaglandin E levels in mice. Teratology, 44: 521-9, 1991a.

Randall, C. L.; Becker, H.C. & Anton, R.F. Effect of ibuprofen on alcohol-induced teratogenesis in mice. Ale. Clin. Exper. Res., 15:673-7, 1991b.

Sahai, H. & Ageel, M. I. The analysis of variance. Fixed, random and mixed models. Boston, Birkhauser, 2000.

Tze, W. J. & Lee, M. Adverse effects of maternal alcohol consumption on pregnancy and foetal growth in rats. Nature, 257: 479-80, 1975.

Wallenburg, H.C. & Rotmans, N. Prevention of recurrent fetal growth retardation by low-dose aspirin and dipyridamole. Amer. J. Obstet. Gynecol., 157: 1230-5, 1987.

Warkany, J. & Takacs, E. Experimental production of congenital malformations in rats by salicylate poisoning. Amer. J. Pathol., 35:315-31, 1959.

Wunderlich, S. M.; Surendrabaligga, B. & Munro, H. N. Rat placental protein synthesis and peptide hormone secretion in relation to malnutrition from protein deficiency on alcohol administration. J. Nutr., 109:1534-42, 1979.

Direccion para correspondencia:

Prof. Dr. Miguel A. Sala

Facultad de Odontologia de Ribeirao Preto

Universidad de Sao Paulo

CEP 14040-904

Ribeirao Preto--SP

BRASIL

* Sandra Aparecida Marinho; ** Miguel Angel Sala; ** Ruberval Armando Lopes; ** Marcio Fernando de Moraes Grisi; ** Arthur Belem Novaes Jr.; ** Sergio Luis Scombatti de Souza & ** Mario Taba Jr.

* Faculdade de Odontologia da Universidade de Alfenas (UNIFENAS), Alfenas, MG, Brasil.

** Faculdade de Odontologia de Ribeirao Preto da Universidad de Sao Paulo (FORP-USP), Ribeirao Preto, SP, Brasil.
Table I. Mean ([+ or -] S.D.) of the morphometric parameters of fetuses
and adnexae of the ethanol, ASA, ASA plus ethanol and control groups.
ANOVA and Tukey's post-hoc test.

Parameter                  Group I                Group II
                           (ethanol)              (ASA)

Maternal weight gain (g)   41.2 [+ or -] 4.8 **   64.3 [+ or -] 5.4
                                                   ([double dagger])
Litter (n)                 11 [+ or -] 1.3        14 [+ or -] 1.3
Fetal weight (g)           2.15 [+ or -] 0.20 **  2.40 [+ or -] 0.15
                                                   ([double dagger])
Placenta weight (mg)       460 [+ or -] 50        500 [+ or -] 45
Amniotic fluid (ml)        0.8 [+ or -] 0.1 **    1.0 [+ or -] 0.1
                                                   ([double dagger])
Umbilical cord length
 (mm)                      30 [+ or -] 3 **       29 [+ or -] 3 **

Parameter                  Group III              Group IV
                           (ASA plus ethanol)     (control)

Maternal weight gain (g)   45.2 [+ or -] 3.3 **   63.0 [+ or -] 2.6
                            ([dagger])
                            ([subsection])
Litter (n)                 13 [+ or -] 1.5        13 [+ or -] 1.0
Fetal weight (g)           2.30 [+ or -] 0.15     2.42 [+ or -] 0.28
                            ** ([dagger])
Placenta weight (mg)       470 [+ or -] 46        540 [+ or -] 43
Amniotic fluid (ml)        0.9 [+ or -] 0.1 *     1.0 [+ or -] 0.1
                            ([dagger])
                            ([section])
Umbilical cord length      27 [+ or -] 3 **
 (mm)                      ([dagger])             37 [+ or -] 4

Parameter                  F                      p

Maternal weight gain (g)   4.52                   < 0.01
Litter (n)                 2.30                   ns
Fetal weight (g)           4.45                   < 0.01
Placenta weight (mg)       2.08                   ns
Amniotic fluid (ml)        3.95                   < 0.05
Umbilical cord length
 (mm)                      3.58                   < 0.05

* p < 0.05p (comparison with group IV - control)

** p < 0.01 (comparison with group IV - control)

([dagger]) p < 0.05 (comparison with group I - ethanol)

([double dagger]) p < 0.01 (comparison with group I - ethanol)

([section]) p < 0.05 (comparison with group II - ASA)

([subsection]) p < 0.01 (comparison with group II - ASA)
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Author:Aparecida Marinho, Sandra; Sala, Miguel Angel; Lopes, Ruberval Armando; de Moraes Grisi, Marcio Fern
Publication:International Journal of Morphology
Date:Jun 1, 2003
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