The Effect of Maternal Restraint on Developmental Toxicity of Aluminum in Mice

Neurotoxicology and Teratology, Vol. 20, No. 6, pp. 651–656, 1998 © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0892-0362/98 $19.00 ⫹ .00

PII S0892-0362(98)00025-7

The Effect of Maternal Restraint on Developmental Toxicity of Aluminum in Mice M. TERESA COLOMINA,* JOSE L. ESPARZA,† JACINTO CORBELLA† AND JOSE L. DOMINGO† *Department of Psychology, Psychobiology Unit, “Rovira i Virgili” University, Tarragona, Spain †Laboratory of Toxicology and Environmental Health, School of Medicine, “Rovira i Virgili” University, Reus, Spain Received 20 January 1998; Accepted 2 June 1998 COLOMINA, M. T., J. L. ESPARZA, J. CORBELLA AND J. L. DOMINGO. The effect of maternal restraint on developmental toxicity of aluminum in mice. NEUROTOXICOL TERATOL 20(6) 651–656, 1998.—Both aluminum (Al) and maternal restraint have been reported to cause developmental toxicity in mammals. This study assessed in pregnant mice the potential interaction between Al and maternal restraint. Four groups of plug-positive female mice were given IP injections of AlCl3 at 37.5 and 75 mg/kg/day on days 6–15 of gestation. Two of these groups were also subjected to restraint for 2 h/day during the same gestational days. Control groups included restrained and unrestrained pregnant mice nonexposed to Al. Cesarean sections were performed on gestation day 18, and the fetuses were weighed and examined for morphological defects. Maternal toxicity was significantly enhanced by restraint at 75 mg AlCl3/kg/day. No increases in the number of resorptions or dead fetuses per litter were observed following exposure to Al, maternal restraint, or combined Al and restraint. However, a significant decrease in fetal body weight, as well as a significant increase in the number of litters with morphologic defects, was observed in the group exposed to 75 mg AlCl3/kg/day plus maternal restraint. The current results suggest that maternal restraint could enhance the metal-induced developmental toxicity (reduced fetal body weight, increase in the number of litters with morphologic defects) only at high doses of the metal, which are also toxic to the dam. © 1998 Elsevier Science Inc. Aluminum chloride

Restraint

Maternal toxicity

Developmental toxicity

Mice

Despite the relevance of the above findings, their significance for human health still requires a better understanding of the amount and bioavailability of Al in sources of exposure such as food and drinking water, or some medications that are consumed by pregnant women (16). In relation to medications, a small trial was performed to assess whether Al-containing antacids were safe during pregnancy (32). The Al concentration in serum from the mother and her newborn child were analyzed in consecutive cases where the mothers had used antacids during pregnancy and in randomly selected cases where the mothers had not used antacids (control group). The use of low-dose Al-containing antacids did not seem to cause hyperaluminemia in newborns. However, consumption of high-dose Al-containing antacids could increase Al in

ALTHOUGH it is well established that parenteral exposure to aluminum (Al) during pregnancy can cause a developmental syndrome that includes resorptions and deaths, skeletal and soft tissue abnormalities, and growth retardation (1,8,33), until recently there was little concern about embryo/fetal consequences of Al ingestion because bioavailability was considered low (10,16). However, a number of studies have shown that oral Al exposure during gestation can provoke growth restriction and delayed ossification, as well as an increased incidence of gross, internal, and skeletal abnormalities at doses of Al that also lead to reduced maternal weight gain (10,16). Moreover, adverse effects on neuromotor development have been also reported as a result of exposures of some species of mammals to Al (3,14,15,34).

Requests for reprints should be addressed to Dr. Jose L. Domingo, Laboratory of Toxicology and Environmental Health, School of Medicine, “Rovira i Virgili” University, San Lorenzo 21, Reus 43201, Spain. Tel: ⫹34-77-759380; Fax: ⫹34-77-759322; E-mail: [email protected]

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serum of healthy pregnant women. Taking into account that whether this element would increase the fetal Al was unknown, it was suggested that consumption of high-dose antacids should not be recommended during pregnancy (32). On the other hand, work in rodents has shown that, during pregnancy, maternal stress from restraint, noise, light, and heat among others may be also associated with adverse effects on embryo/fetal development (29). Of special concern is the finding that interaction between maternal stress and some chemical teratogens can enhance the teratogenicity of those chemicals. However, although some studies have shown that restraint could significantly exacerbate the maternal and embryo/fetal toxicity of various environmental toxicants such as arsenate (26) or methylmercury (5), other investigations did not find synergistic or antagonistic modification of the developmental toxicity of cadmium by noise (20), or arsenite by maternal restraint (6). Because Al is ubiquitous, exposure to this element is in fact unavoidable. It means that pregnant women may be potentially exposed to Al in food, drinking water, soil ingestion, and some medications, whereas they may be also concurrently exposed to various types of stress, either at home or in the workplace. Because Al and maternal stress during pregnancy have been shown to produce adverse developmental effects in mammals, the purpose of this study was to assess the developmental toxicity in mice of a combined exposure to both Al and maternal restraint. Among the animal models to examine the effects of maternal stress on the embryo/fetal toxicity of a chemical, restraint has been widely used (2,5–7,21,26–28). Aluminum, as Al chloride, was administered IP during the period of organogenesis. METHOD

Subjects and Husbandry Sexually mature male and female Swiss mice (28–32 g) were obtained from Interfauna Iberica (Barcelona, Spain). Animals were housed in plastic cages in a fully air-conditioned facility with a constant day–night cycle (light: 0800– 2000 h) at a temperature of 22 ⫾ 2⬚C, and a relative humidity of 50 ⫾ 10%. After a quarantine period of 7 days female mice were mated with males (2:1) overnight and examined the following morning for copulatory plugs. Day 0 was defined as the day in which the vaginal plug was found. Food (Panlab rodent chow, Barcelona, Spain) and tap water were available ad lib. Aluminum concentrations in diet and water were 0.257 g/ kg and 0.052 ␮g/ml, respectively. Chemical Aluminum chloride (AlCl3) was obtained from Sigma (Alcobendas, Spain). It was administered in aqueous solutions at doses of 37.5 and 75 mg/kg. The choice of these doses was based on the results of a previous study on developmental toxicity of Al in mice (8). Solutions were injected IP at pH 4.0, and adjusted so that a 30-g mouse would receive a volume of 0.20 ml. Treatment Plug-positive female mice were randomly divided into six groups and received the following treatments on gestational days 6–15. The first group of animals (unrestrained control group) was unrestrained and injected with 0.9% saline acidified with HCl to a pH of 4 (25). The second group of mice (restrained control group) was restrained daily for 2 h (1000–

1200 h) by placing the animals in plastic cylindrical holders that were lined with foam padding. The restrained mice were held in a prone position and were monitored periodically. Because it has been reported that injection alone during pregnancy could have long-lasting effects on the development of the offspring (23,24), restrained animals were also injected with 0.9% saline acidified with HCl to a pH of 4. Mice in the third and fifth groups (aluminum-treated groups) were given IP doses of 37.5 and 75 mg/kg/day, respectively, of aqueous AlCl3 solutions. Plug-positive female mice in the fourth and sixth groups (combined AlCl3 and restraint groups) received IP injections of 37.5 or 75 mg/kg/day of AlCl3, followed by restraint stress for 2 h/day. All mice, including those in the restrained groups, were allowed free access to food and water. Maternal and Fetal Observations All animals were observed daily for food consumption, body weight gain, and clinical signs of toxicity. Mice were killed with diethyl ether on day 18 of gestation. Maternal body, liver, kidney, and uterine weights were recorded. All females were evaluated for the status of uterine implantation sites (i.e. number of sites, early, and late resorptions, and dead and live fetuses.) Live fetuses were weighed, sexed, and examined for external abnormalities. Approximately one-half of the fetuses were fixed in Bouin’s solution for internal examination. The remaining fetuses were fixed in 95% ethanol, cleared with 1% KOH, stained with Alizarin red S, and examined for skeletal malformations and variations (31). Statistics The unit of comparison was the dam or the litter. Results of the quantitative continous variables (e.g., maternal body weights, organ weights, fetal weights) were compared by a twoway (Al dose ⫻ restraint) analysis of variance (ANOVA) with significant F-values further analyzed with the LSD method to assess differences between groups. Nonparametric data were statistically evaluated using the Kruskal–Wallis test when appropiate. Data on internal and skeletal anomalies were analyzed using chi-square test. Significance was set at the 0.05 probability level. RESULTS

Maternal Toxicity Table 1 summarizes the distribution and fate of all mated mice in the study. There were no deaths, abortions, early deliveries, or resorbed litters in the Al-untreated groups. However, 8.1% (37.5 mg/kg/day of AlCl3), 14.2% (37.5 mg/kg/day of AlCl3 plus restraint and 75 mg/kg/day of AlCl3), and 11.7% (75 mg/kg/day of AlCl3 plus restraint) of the dams died due to Al exposure or combined Al and restraint during organogenesis. Moreover, concurrent exposure to Al and restraint caused a significant number of abortions (28.5% and 41.1% at 37.5 and 75 mg/kg, respectively) compared with Al-untreated dams or with the group exposed to 37.5 mg/kg/day of AlCl3 only. A two-way (Al dose ⫻ restraint) ANOVA did not reveal significant effects of Al and/or restraint on maternal food consumption, body weight change, body weight at termination, and absolute or relative liver and kidney weights (Table 2). Although corrected body weight (body weight at sacrifice minus gravid uterine weight) was lower in the group given acidified saline plus restraint than in the group concurrently exposed to the highest Al dose and restraint, a two-way (Al dose ⫻ restraint) ANOVA did not show significant effects that might be attrib-

MATERNAL RESTRAINT AND Al IN PREGNANT MICE

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TABLE 1 THE DISTRIBUTION AND FATE OF ALL MATED MICE ON STUDY

No. of plug-positive females (day 0) Deaths Abortions Early deliveries Evaluated at term Resorbed litters No. of litters

Acidified Saline

Acidified Saline ⫹ Restraint

AlCl3 Alone (37.5 mg/kg)

AlCl3 (37.5 mg/kg) ⫹ Restraint

AlCl3 Alone (75 mg/kg)

AlCl3 (75 mg/kg) ⫹ Restraint

12 0 0 0 12 0 12

9 0 0 0 9 0 9

11 1 0 0 10 0 10

14 2 4 0 8 0 8

14 2 2 0 10 0 10

17 2 7 0 8 0 8

utable to Al exposure, F(2, 50) ⫽ 2.10, p ⫽ 0.134, restraint, F(1, 50) ⫽ 1.16, p ⫽ 0.288, or the interaction between Al and restraint, F(2, 50) ⫽ 2.03, p ⫽ 0.143. An overall effect of restraint, F(1, 47) ⫽ 4.48, p ⫽ 0.04, and an interaction between Al exposure and restraint, F(2, 47) ⫽ 3.49, p ⫽ 0.028, were observed on corrected body weight change (corrected body weight minus body weight on day 0 of gestation). However, no effects of Al alone were found. In turn, although a combined effect (addition of single effects) of Al treatment and restraint, F(3, 49) ⫽ 7.53, p ⫽ 0.000, as well as an overall effect of Al dose, F(2, 49) ⫽ 11.22, p ⫽ 0.000, was observed on gravid uterine weight, no effects of restraint alone or interactions between restraint and Al could be observed on this parameter. Embryo/Fetal Toxicity Table 3 shows a series of reproductive data for dams exposed to AlCl3, restraint, or combined AlCl3 plus restraint during organogenesis. There were no significant differences among groups in the number of total implants, early and late resorptions, dead and live fetuses, and sex ratio. However, fetal body weight was significantly reduced in all Al-treated groups. A two-way ANOVA showed a combined effect of Al and restraint, F(3, 56) ⫽ 25.10, p ⫽ 0.000, as well as an overall effect of Al alone, F(2, 56) ⫽ 32.99, p ⫽ 0.000, and restraint

only, F(1, 56) ⫽ 8.18, p ⫽ 0.006. In relation to it, fetuses in the groups whose dams were concurrently exposed to Al and restraint showed a lower body weight than those in the group subjected to restraint only (Table 3). There were no external anomalies due to Al exposure, restraint only, or combined administration of Al and restraint. The type and frequency of internal and skeletal anomalies observed in the current study are given in Table 4. Although no significant differences in the number of litters with fetuses showing internal malformations or variations were noted, there was a significant increase in the number of litters with assymetrical sternebrae in the groups exposed to 75 mg/kg/ day of AlCl3 plus restraint. In addition, the number of litters with skeletal anomalies, as well as the total number of litters with internal and skeletal defects, was significantly higher in this group than in the remaining groups. DISCUSSION

The importance of the route of exposure and the chemical form of the Al compound on the developmental toxicity of this element are now well established (10,16). Although parenteral exposure to high doses of Al chloride caused a developmental toxicity syndrome in rats and mice (1,8,33), no evidence of maternal and embryo/fetal toxicity was observed when high doses of Al hydroxide were given by gavage to

TABLE 2 EFFECTS OF AlCl3, RESTRAINT, AND COMBINED AlCl3 AND RESTRAINT ON MATERNAL TOXICITY IN PREGNANT MICE*

No. of dams Food consumption (g/dam) on gestation days 0–18 Body weight change (g) on gestation days 0–18 Body weight at termination (g) Gravid uterine weight (g) Corrected body weight (g) Corrected body weight change (g) Liver weight (g) Relative liver weight (%) Kidney weight (g) Relative kidney weight (%)

Acidified Saline

Acidified Saline ⫹ Restraint

AlCl3 Alone (37.5 mg/kg)

AlCl3 (37.5 mg/kg) ⫹ Restraint

AlCl3 Alone (75 mg/kg)

AlCl3 (75 mg/kg) ⫹ Restraint

12

9

10

8

10

8

96.60 ⫾ 21.22

100.50 ⫾ 3.50

100.84 ⫾ 16.79

90.43 ⫾ 3.37

97.02 ⫾ 13.44

81.50 ⫾ 21.85

27.21 ⫾ 4.54 54.26 ⫾ 5.69 18.51 ⫾ 3.93a 36.08 ⫾ 5.43ab 9.26 ⫾ 2.24ab 2.39 ⫾ 0.34 4.54 ⫾ 0.49 0.43 ⫾ 0.46 0.81 ⫾ 0.12

26.34 ⫾ 3.61 51.02 ⫾ 3.64 18.77 ⫾ 3.20a 32.24 ⫾ 0.92b 7.57 ⫾ 9.54b 2.21 ⫾ 0.18 4.35 ⫾ 0.35 0.40 ⫾ 0.06 0.76 ⫾ 0.09

23.44 ⫾ 6.18 51.94 ⫾ 1.90 16.44 ⫾ 0.92ab 35.18 ⫾ 1.19ab 9.44 ⫾ 2.14ab 2.53 ⫾ 0.28 4.74 ⫾ 0.43 0.40 ⫾ 0.04 0.76 ⫾ 0.07

22.58 ⫾ 4.71 49.76 ⫾ 4.82 14.59 ⫾ 3.68b 36.23 ⫾ 5.77ab 10.52 ⫾ 4.40a 2.35 ⫾ 0.42 4.76 ⫾ 1.17 0.40 ⫾ 0.04 0.80 ⫾ 0.14

24.51 ⫾ 3.40 51.47 ⫾ 2.52 14.65 ⫾ 2.76b 37.03 ⫾ 2.62a 11.27 ⫾ 1.74a 2.09 ⫾ 0.34 4.09 ⫾ 1.20 0.38 ⫾ 0.08 0.79 ⫾ 0.06

22.47 ⫾ 3.69 51.53 ⫾ 3.71 14.85 ⫾ 2.60b 36.43 ⫾ 2.21a 7.44 ⫾ 2.09b 2.57 ⫾ 0.42 5.18 ⫾ 0.78 0.39 ⫾ 0.04 0.78 ⫾ 0.09

*Data are presented as means ⫾ SD. Values in the same row not showing a common superscript (a,b) are significantly different ( p ⬍ 0.05).

654

COLOMINA ET AL. TABLE 3 EFFECTS OF AlCl3, MATERNAL RESTRAINT, AND COMBINED AlCl3 AND RESTRAINT ON GESTATIONAL PARAMETERS IN PREGNANT MICE*

No. of dams Implants/litter Live fetuses/litter Early resorptions/litter Late resorptions/litter Dead fetuses/litter Postimplantation loss (%) Sex ration (M/M⫹F) Fetal body weight (g)

Acidified Saline

Acidified Saline ⫹ Restraint

AlCl3 Alone (37.5 mg/kg)

AlCl3 (37.5 mg/kg) ⫹ Restraint

AlCl3 Alone (75 mg/kg)

AlCl3 (75 mg/kg) ⫹ Restraint

12 13.32 ⫾ 2.91 11.00 ⫾ 1.76 0.16 ⫾ 0.39 0.08 ⫾ 0.29 0.08 ⫾ 2.29 2.56 ⫾ 5.01 0.51 ⫾ 0.08 1.30 ⫾ 0.11a

9 12.22 ⫾ 2.27 11.78 ⫾ 2.10 0.33 ⫾ 0.71 0.10 ⫾ 0.31 0.00 ⫾ 0.00 3.57 ⫾ 5.54 0.43 ⫾ 0.12 1.26 ⫾ 0.12ab

10 11.10 ⫾ 1.45 10.00 ⫾ 2.23 0.00 ⫾ 0.00 0.30 ⫾ 0.67 0.70 ⫾ 1.34 9.42 ⫾ 17.49 0.44 ⫾ 0.15 1.13 ⫾ 0.08bc

8 11.29 ⫾ 2.43 9.71 ⫾ 3.77 0.00 ⫾ 0.00 1.29 ⫾ 1.70 0.29 ⫾ 0.76 16.15 ⫾ 21.63 0.50 ⫾ 0.19 1.05 ⫾ 0.16cd

10 10.90 ⫾ 2.46 9.60 ⫾ 3.20 0.70 ⫾ 1.34 0.50 ⫾ 0.85 0.10 ⫾ 0.31 13.33 ⫾ 15.64 0.49 ⫾ 0.15 1.06 ⫾ 0.11cd

8 12.88 ⫾ 1.13 11.38 ⫾ 2.26 0.00 ⫾ 0.00 1.12 ⫾ 2.47 0.38 ⫾ 0.52 11.32 ⫾ 16.67 0.59 ⫾ 0.13 0.91 ⫾ 0.15d

*Data are presented as means ⫾ SD. Values in the same row not showing a common superscript (a,b,c,d) are significantly different ( p ⬍ 0.05).

these species during the period of organogenesis (11,17). However, signs of maternal and developmental toxicity were found in mice when Al hydroxide was given concurrently with citric (18) or lactic (4) acids, whereas oral administration of Al nitrate to pregnant rats also resulted in embryo/fetal toxic effects (22). In the current study, when AlCl3 was administered IP on gestation days 6–15, maternal toxicity was evidenced by a number of deaths at 37.5 (8.1%) and 75 mg/kg/day (11.7%), as well as by the presence of abortions at 75 mg/kg/day. The relatively notorious number of deaths in the Al-treated groups was not attributed to the low pH of Al solutions. Both control groups (acidified saline only and acidified saline plus restraint) received solutions given at a pH of 4 and no deaths were observed in these groups. In turn, developmental toxicity was evidenced by a significant decrease in fetal body weight in the groups exposed at 37.5 and 75 mg/kg/day of AlCl3. Although there were no significant differences in the number of deaths among the Al-treated groups, the number of

dams with abortions was significantly increased by maternal restraint. In addition, corrected body weight change was significantly lower in the group given 75 mg/kg/day of AlCl3 plus restraint than in that given 75 mg AlCl3/kg/day alone. Although no signs of embryotoxicity caused by maternal restraint were noted in the Al-treated groups, there was a decrease (p ⬍ 0.05) in fetal body weight in the groups concurrently subjected to restraint. A significant increase in the number of litters with fetuses affected by skeletal defects was also observed in the group exposed to 75 mg AlCl3/kg/day plus maternal restraint. Because stress, by its very definition, is a disturbance in the normal physiology of the maternal organism, it seems likely that maternal restraint can result in developmental toxicity (9). However, taking into account that the term maternal stress can comprise a variety of conditions, each of which may or may not have the capacity to adversely affect the development of offspring, in the present study restraint was used as a potential model of maternal stress. Maternal restraint has been often utilized as an archetype easily controlled stressor

TABLE 4 EFFECTS OF AlCl3, MATERNAL RESTRAINT, AND COMBINED AlCl3 AND RESTRAINT ON MORPHOLOGIC DEFECTS IN MOUSE FETUSES* Acidified Saline

No. of fetuses examined internally (litters) Cleft palate Septonasal assimetry Total fetuses with internal anomalies (litters) No. of fetuses examined skeletally (litters) Assymetrical sternebrae Metartasus, diminished ossification Total fetuses with skeletal anomalies (litters) Total fetuses with internal or skeletal defects (litters)

Acidified Saline ⫹ Restraint

AlCl3 Alone (37.5 mg/kg)

AlCl3 (37.5 mg/kg) ⫹ Restraint

AlCl3 Alone (75 mg/kg)

AlCl3 (75 mg/kg) ⫹ Restraint

73 (12)

40 (9)

41 (10)

38 (8)

40 (10)

40 (8)

1 (1) 0 (0)

0 (0) 4 (2)

1 (1) 0 (0)

1 (1) 0 (0)

0 (0) 1 (1)

2 (2) 1 (1)

1 (1)

4 (2)

1 (1)

1 (1)

0 (0)

3 (3)

60 (10)

50 (9)

50 (10)

40 (8)

45 (10)

44 (8)

(2)a

2 4 (4)ac

(1)a

1 1 (1)b

(3)a

4 0 (0)b

(2)a

2 0 (0)b

(2)a

4 1 (1)b

22 (8)b 16 (5)c

4 (4)a

3 (2)a

4 (3)a

2 (2)a

4 (2)a

28 (8)b

5 (4)a

7 (3)a

5 (3)a

3 (3)a

*Number of litters in the same row showing different superscripts (a,b,c) are significantly different ( p ⬍ 0.05).

5 (3)a

31 (8)b

MATERNAL RESTRAINT AND Al IN PREGNANT MICE that imposes both physical and psychological demands on the subject (9,19,21). In recent years, a number of investigations have assessed the influence of maternal stress on the adverse embryo/fetal effects of some developmental toxicants. Goldman and Yakovac (13) showed that maternal immobilization during pregnancy played a profound additive role in salicylate-induced teratogenesis in rats, whereas Shiota and coworkers (30) demonstrated that ethanol and hypothermia could be synergistically teratogenic in mice when the doses of each agent were below the teratogenic threshold. In turn, Rasco and Hood (27,28) found that restraint combined with a near threshold teratogenic dose of all-trans-retinoic acid produced an increase in the incidence of morphological defects in mice. The influence of maternal stress on the developmental toxicity of some metals has been also examined. Rasco and Hood (26) reported that maternal restraint combined with an apparently nonmaternally toxic dose of sodium arsenate increased the fetotoxicity induced in pregnant mice by exposure to arsenate. By contrast, Murata and coworkers (20) did not find any modification of the teratogenicity due to cadmium (20) following combined exposure of pregnant mice to noise and cadmium. On the other hand, Ferm and Kilham (12) showed synergistic teratogenic effects of arsenic and hyperthermia in hamsters concurrently exposed at minimal teratogenic levels of each. It clearly indicated that the teratogenic potential of the two could be additive (12). Recently, we assessed the influence of maternal restraint on the embryo/fetal effects of prenatal exposure to methylmercury and arsenite. Maternal restraint enhanced the embryo/ fetal toxicity of a single dose of methylmercury in mice only at doses that were highly toxic to the dams, whereas at levels of

655

methylmercury that were less acutely toxic the role of maternal restraint was not significant (5). In turn, restraint did not enhance the arsenite-induced developmental toxicity in mice at single oral doses of arsenite that were not teratogenic by themselves (6). Moreover, a significant interaction between maternal restraint and methylmercury or arsenite could not be established for developmental toxicity in mice when the xenobiotics were given concurrently with restraint on gestation days 15–18 (7). In accordance with these data, in the current study we found that the influence of maternal restraint on the Alinduced embryo/fetal adverse effects was more evident at the highest Al dose. The present results, along with those of previous studies (5,7), support the hypothesis that although maternal restraint may exacerbate the effects of exposure to trace metals with potential developmental toxicity, the interaction only would be clearly noted at doses of the elements that are also toxic to the dam. It could explain the apparent disagreement with results of previous studies in which exposure to the xenobiotics was conducted at doses that were teratogenic by themselves (12,26). Taking into account that pregnant women are not currently exposed to Al doses that could cause adverse effects on health, it seems evident that the potential influence of maternal stress would not mean an additional real risk for those women consuming current quantities of Al during pregnancy.

ACKNOWLEDGEMENTS

This work was supported by the DGICYT, Ministry of Education, Spain, through grant PM96-0030.

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