developmental toxicity evaluation of oral aluminum … · 2008-08-03 · developmental toxicity...

DEVELOPMENTAL TOXICITY EVALUATION OF ORAL ALUMINUM IN RATS

Abd El- Azeim A. Khalaf; Ashraf M. Morgan; Mohey M. Mekawy and Maged F. Ali

J. Egypt. Soc. Toxicol. (Vol. 37: 11-26 July 2007)WWW.estoxicology.org

Toxicology and Forensic Medicine Department, Faculty of Veterinary Medicine, Cairo University, Egypt.

ABSTRACT The present study was designed to elucidate the adverse effects of the orally

administered aluminum (Al) on the growing fetus and consequently on the animal wealth in our country. This aim has been achieved by studying the teratogenic, perinatal and postnatal effects of aluminum chloride when administered orally at 345 mg/kg body weight to female rats during organogenesis, fetal and/ or lactation periods. The results showed that Al chloride exposure on days 6-15 of gestation produced a significantly higher percentage of postimplantation death, resorptions, morphological, visceral and skeletal anomalies in the obtained fetuses compared to the control group. In addition, the live fetuses' percentage, mean fetal body weight and placental weights were significantly decreased. The obtained data revealed also that Al chloride exposure on 6th day of gestation till weaning induced significant increase in the percentage of dams showed delayed birth date and signs of dystocia. In addition, it induced a significant increase in the percentage of postimplantation loss, dead fetuses; fetuses showing neurobehavioral and respiratory symptoms and those born with morphological abnormalities. Moreover, it decreased the live/ birth, survival and viability indices and weight gain of these fetuses compared with control. The Al- induced effects on the obtained fetuses from Al chloride treated dams through lactation period included significant increase in the percentage of postnatal deaths, fetal stunted growth with a significantly increased percentage of nervous and respiratory symptoms prior to death. Consequently, the survival and viability indices were reduced. Moreover, the weight gain during lactation was significantly reduced. Brain examination of the obtained fetuses from all exposed dams throughout this study showed different histopathological changes. It can be concluded that Al chloride exposure of female rats during gestation and/ or lactation periods caused teratogenic, perinatal and postnatal adverse effects on their progeny. Keywords: Aluminum chloride, Teratogenicity, Perinatal, Postnatal, Rats, Lactation,

Survival, Viability.

INTRODUCTION The greatly increased use of metals has

introduced a new noxious hazard to animals and humans. Therefore, the persistence of metals in the environment has led to growing concern about the direct and indirect exposures of pregnant females and their unborn babies. Placental transfer of most metals places the health of unborn at the highest risk. Also, the newborn can be affected through breast feeding or direct exposure (Sastray, 1995 and WHO, 1997). Therefore, prolonged exposure to some of these contaminants may induces teratogenic effects, abortion, reproductive failure and/ or immunotoxicity (Nordal et al., 1989; Piramanayagam et al., 1996;

Carson, 2000 and Sharma and Mishra, 2006) both by their direct cellular toxic action and by interfering indirectly with the hypothalamo- hypophyseal function (Krause, 1977; Levine et al., 1992 and Flora et al., 2003).

Nowadays, aluminum (Al) is widely used in treatment of drinking water, drugs (e.g., Antacids), deodorants and antiperspirants preparations, preservation of wood; the disinfection of stables and slaughter houses and in manufacture of alloys (Jackson, 1986; WHO, 1997 and ATSDR, 2006). Al is present also in many food products, vegetables, cereals and beverages (Filipek et al., 1987; U. S. Public Health Service, 1992; Beliles, 1994 and

Khalaf, et al. (2007) Developmental Toxicity Evaluation Of Oral Aluminum In Rats 12

ATSDR, 2006). It has been reported that parenteral exposure to Al chloride caused a developmental toxicity syndrome in rats and mice (Benett, 1975; Wide, 1984 and Cranmer et al., 1986). Moreover, oral Al exposure during pregnancy can produce a syndrome including growth retardation, delayed ossification and perhaps malformations in the offspring at Al doses that also led to maternal toxicity (Paternain et al., 1988; Gomez et al., 1991; Colomina et al., 1992; Muller et al., 1992; Domingo, 1995; Golub and Domingo, 1996 and Colomina et al., 2002). Few studies have shown also that Al exposure during gestation at lower doses than that which produces maternal toxicity cause developmental syndromes including resorptions, deaths, growth retardation, skeletal and soft tissue abnormalities and retardation of neurological development in the offspring (Elbina et al., 1984 Yokel, 1985 and Sharma and Mishra, 2006). It has been found also that interactions between maternal stress and some metals including Al can enhance the potential developmental toxicity of these elements (Domingo et al., 2004 and Colomina et al., 2005).

From the literature, Al is a developmental toxicant when administered parenterally (Yokel, 1985; Cranmer et al., 1986; Golub et al., 1987 and Domingo, 1995). However, until recently, there was little concern about the embryo- and feto- toxic consequences of aluminum ingestion because its bioavailability was considered low. Therefore, the present study was designed to highlight on the adverse effects of the orally administered Al on the growing fetuses and consequently on the animal wealth in our country. This aim has been achieved by studying the developmental toxic effects of aluminum chloride when administered orally to female rats during organogenesis, fetal and/ or lactation periods.

MATERIAL AND METHODS Chemicals:

The tested aluminum salt is aluminum chloride (Alcl3.6 H2O). It was purchased from Sigma Chemicals Co. (St. Louis, USA) with a M.wt. of 241.43. It was given orally to the treated rats at a dose of 345mg/ Kg b.wt (Sharma and Mishra, 2006).

Animals:

Adult male and female albino rats, having body weight 180-200g, were used as Lab. animals in this study. They were obtained from Faculty of Veterinary Medicine, Cairo University. The animals were kept under hygienic conditions and provided with balanced ration and water ad libitum. All animals were kept under observation along two weeks before the start of the experiment for acclimatization.

Experimental design:

Fifty pregnant female rats were used. They were selected after pairing with mature males of proven fertility on a one-to-two basis usually in the early afternoon and left overnight. Pregnancy was confirmed in the following morning by the presence of sperms in the vaginal wash of each female and considered as the zero-day of pregnancy (Manson and Kang, 1994).The pregnant females were divided into 5 equal groups; 10 animals each as following:

Group I: Kept without any treatment, sacrificed at the 20th day of pregnancy and served as control for Group III.

Group II: Kept without any treatment, sacrificed (dams and their offsprings) at 21st day postpartum and served as control for Groups IV and V.

Group III: Received Al chloride orally on days 6-15 of gestation, then sacrificed at the 20th day of pregnancy.

Group IV: Received Al chloride orally on the 6th day of gestation till weaning (21st day postpartum), then sacrificed (dams and their offsprings) at 21st day postpartum.

Group V: Received Al chloride orally on 1st day after birth till weaning, then sacrificed (dams and their ofsprings) in the 21st day postpartum.

The pregnancies were interrupted by sacrifice just prior to the calculated date of delivery (at day 20 of gestation) for animals of Groups I and III. The uterine horns were exposed and the numbers of implants, resorptions and live fetuses were counted. Live as well as dead fetuses were removed from the uterus by means of cesarean sections and blotted dry. The fetuses and placentae were weighed and examined for gross external abnormalities. The brain of each dam; brain and placenta of a fetus / litter were used for histopathological examination. One third of the obtained fetuses were kept in Bouin's fixative for at least one week, after which, fetuses were sectioned using Wilson's free-hand razor blade sectioning technique searching for internal visceral malformations. The other two thirds were kept in ethanol for subsequent preparation for skeletal examination (Manson and Kang, 1994).

Animals of groups II, IV and V were left for normal birth and observed for any delayed birth date, signs of dystocia (groups II and IV) or deaths. The number of live and dead offsprings was counted. Also, the morphological abnormalities, fetal body weights at birth, 4th, 7th, 14th, and 21st days after birth and the live/birth, survival and viability indices were recorded.

Khalaf, et al. (2007) Developmental Toxicity Evaluation Of Oral Aluminum In Rats

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In addition, the neurobehavioral changes on the offsprings during the postnatal (lactation) period were observed. Moreover, brain of both dams and their fetuses were examined for any histopathological changes.

100 X live born pups of No.

PND1 at pups born Live of No.index birth Live =

100 XPND1 at pups born Live of No.PND3 at pups born Live of No. index Survival =

100 X PND1 at pups born Live of No.

7 PND at pups born Live of No. indexViability =

PND means postnatal day

Placentae and brain were fixed in 10% neutral formalin and subjected to histopathological examination according to Bancroft et al. (1996).

Statistical Analysis: The significant differences between treated and

control groups were assessed by Chi2 (Snedecor and Cochran, 1989) and One-Way-Analysis Of Variance (ANOVA) using computer microstat program, copyright (c) 1978-85 by Ecosft,Inc.

RESULTS Developmental and teratogenic effects:

Results of morphological examination of the obtained fetuses from dams treated orally with 345 mg Al chloride /kg b.wt. on days 6-15 of gestation (G III) were recorded in table (1). The postimplantation death (Fig.1) and resorbed fetuses percentages were significantly higher in the treated group than those recorded in control group (16.86 and 6% versus 1.13 and 0 %, respectively). The live fetuses percentage was significantly decreased in the treated group (77%) compared with the control group (98.80%). The mean fetal body weight and placental weights were significantly lower in the treated group than that of the control one (G I) (4.19± 0.63 and 0.54± 0.02 g, versus 5.65± 0.41 and 0.73± 0.01 g, respectively). Aluminum chloride produced a significantly higher percentage of external morphological abnormalities in the obtained fetuses, compared to control group. It included dwarfism (10.93 % versus 1.14%); generalized edema (4.68% versus 0 %), deformed heads (4.68 % versus 0%), micrognathia (Fig.2) (3.12 % versus 0 %) and S/C hemorrhage (3.12 % versus 0 %).

The results of visceral examination of the obtained fetuses from dams treated orally with 345 mg Al chloride/kg b.wt. on 6th to 15th days of gestation (G III) were recorded in table (2). In the treated group, the most prominent abnormalities seen in the examined fetuses were dilated nares (Fig. 3) (38.88

%); hypoplasia of the olfactory bulbs (16.66 %); anophthalmia and/ or microphthalmia (Fig. 4) (11.11 %); hydrocephaly (dilated brain lateral ventricles) with shrunken brain (Fig. 5) (27.77 %); heart, lung hypoplasia (33.33 %); intrathorathic hemorrhages (Fig. 6) (16.66 %); thymus and spleen congestion (11.11 %); dilated renal pelvis (22.20 %) and hydroureter (11.11 %). The percentages of these malformations were significantly higher in the treated group than those recorded in the control group. Slight percentages of visceral malformations were observed in the control group (G I) in the form of dilated nares and lung hypoplasia (3.84 %).

Fig. (1): Uterus of a pregnant rat treated orally with 345 mg Al chloride /Kg b. wt. during organogenesis period (6-15th days of gestation) showing postimplantation death.

Fig. (2): A rat fetus obtained from a mother treated orally with 345 mg Al chloride /Kg b. wt. during organogenesis period (6-15th days of gestation) showing dwarfism, generalized edema, deformed head and micrognathia (right) and a control fetus (left ).

Fig. (3): A transverse section in the head of a rat fetus obtained from a mother treated orally with 345 mg Al chloride /Kg b. wt. during organogenesis period (6-15th days of gestation) showing severe dilated nares (right) and a control one (left).


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Fig. (4): A transverse section in the head of a rat fetus obtained from a mother treated orally with 345 mg Al chloride /Kg b. wt. during organogenesis period (6-15th days of gestation) showing unilateral microphthalmia (right) and a control one (left).

Fig. (5): A longitudinal section in the head of a rat fetus obtained from a mother treated orally with 345 mg Al chloride /Kg b. wt. during organogenesis period (6-15th days of gestation) showing hydrocephaly with shrunken brain(right) and a control one (left).

Fig. (6): A transverse section in the chest of a rat fetus obtained from a mother treated orally with 345 mg Al chloride /Kg b. wt. during organogenesis period (6-15th days of gestation) showing hypoplasia of heart and lung with extensive intrathoracic hemorrhages (right) and a control one (left).

It has been noted that there was a significantly higher percentage of skeletal anomalies recorded in the obtained fetuses from the treated dams during 6th – 15th days of gestation (G III) compared to the control one (G I) (Table 3). They were in the form of large open fontanel (25.0 %); incomplete ossifications of parietal and / or interparietal bones (Fig. 7) (30.55 %); deformed lower jaw (5.56 %); incomplete ossifications and missing sternebrae (41.66 %) deformed ribs (Fig. 8) (8.33%); absence of phalanges of the fore and /or hind limbs (13.88 %) and absence of sacral (5.56 %) and caudal vertebrae (11.11 %). Low percentages of skeletal anomalies were recorded in the control group in the form of incomplete ossifications of parietal and / or interparietal bones (1.96 %); deformed ribs (1.96 %); absence of phalanges of the fore and /or hind limbs (1.96 %) and absence of caudal vertebrae (3.92%).

The histopathological examination of the placentae of the pregnant females orally administered 345 mg Al chloride/Kg b. wt. on days 6- 15 of gestation (G III) showed different degrees of histopathological changes compared to control. The placenta of control group (G I) showed no histopathological alteration while the placenta of Al chloride- exposed group showed Focal haemorrhages and necrosis in the placental surface (Fig. 9).

Fig. (7): A rat fetus obtained from a mother treated orally with 345 mg Al chloride /Kg b. wt. during organogenesis period (6-15th days of gestation) showing large open fontanel and incomplete ossification of parietal and interparietal bones (right) and a control one (left).


Fig. (8): A rat fetus obtained from a mother treated orally with 345 mg Al chloride /Kg b. wt. during organogenesis period (6-15th days of gestation) showing hypoplasia and absence of some sternebrae (right) and a control one (left).

Fig. (9): Cross section in the placenta of a rat fetus obtained from mother treated orally with 345 mg Al chloride /Kg b. wt. during organogenesis period (6-15th days of gestation) showing necrosis and hemorrhages in the placental (PS) surface (arrows) (H & E stain, X 40).

Effects of gestational and lactation (postnatal) exposure:

Results of the reproductive performance, external morphology, weight gain, live/birth, survival and viability indices and neurobehavioral function of the obtained fetuses from Al chloride treated dams on 6th day of gestation – 21st day after birth (weaning) (G IV) were recorded in table (4).

The effects of Al chloride on natural delivery of exposed dams revealed that Al significantly increased

the percentage of dams that delayed from the expected birth date and those showed signs of dystocia, where 30 and 20 % of the pregnant females showed delayed birth date and signs of dystocia, respectively, compared to 0 % for each of them in the control group (G II).

It has been noticed that Al chloride severely affected the pregnancy outcomes of the females treated from 6th day of pregnancy to 21st day of lactation. There was a significant decrease in the percentage of live fetuses in the treated group, compared to the control group (96.96 % versus 100 %). On the contrary, the dead fetuses' percentage was significantly increased in the treated group IV (3.03 %) compared to 0 % in control group. The fetal body weight at 4th , 7th, 14th and 21 st day after birth were significantly lower in the treated group than those in control one. The mean values were 5.10 ± 0.58, 5.72 ± 0.59, 9.46 ± 0.85 and 15.12± 2.30 g versus 6.57 ± 0.50, 7.35 ± 0.54, 12.90 ± 1.22 and 21.80 ± 1.56 g, respectively).

The percentages of the observed external morphological deformities were significantly increased in exposed fetuses of this treated group in comparison with those recorded in control one. It was in the form of dwarfism (26.56 % versus 0 %), generalized edema (32.81 % versus 0 %) and deformed heads (31.25 % versus 0 %). Neurobehavioral, respiratory symptoms (Muscular tremors, ataxia and respiratory distress) and fetal deaths were also observed during lactation period in the fetuses of the treated group in a significantly increased percentage as compared to control (45.31, 23.43 and 10.94% versus 0 % for each of them, respectively). The live/ birth, survival and viability indices were also decreased compared to those recorded in control fetuses (98.44, 96.83 and 90.48 % versus 100 % for each of them, respectively).

Statistically, the obtained data revealed that Al chloride exposure during gestation and lactation periods till weaning induced significant increase in the percentage of dams showed delayed birth date and signs of dystocia. In addition, it induced a significant increase in the percentage of postimplantation loss, dead fetuses; fetuses showing neurobehavioral and respiratory symptoms and those born with morphological abnormalities. Moreover, it decreased the live/ birth, survival and viability indices and weight gain of these fetuses compared with the control ones.

Effects of lactation (postnatal) exposure: Results of the external morphology, weight gain,

survival and viability indices and neurobehavioral function of the obtained fetuses from Al chloride treated dams at 345 mg/ Kg b. wt. on 1st – 21st days after birth (weaning) (GV) were recorded in table (5).


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Aluminum significantly increased the percentage of fetal deaths during lactation period with a significantly increased percentage of nervous and respiratory symptoms prior to death compared with the control (3.50, 25.3 and 10.80 % versus 0 % for each of them, respectively). Consequently, the survival and viability indices were reduced compared with the control (98.80 and 96.50 % versus 100 % for each of them, respectively). Moreover, the weight gain during lactation was significantly reduced as there was a significant reduction in the mean fetal body wt. at 14th and 21st days after birth. The mean values were 10.15 ± 0.90 g and 16.20 ± 2.35 g versus 12.90 ± 1.22 g and 21.80 ± 1.56 g, respectively). The observed fetal stunted growth percentages were significantly increased in the exposed fetuses compared to the non exposed control ones (26.50 versus 0 %). The obtained data revealed that Al chloride exposure during lactation period till weaning induced significant increase in the percentages of stunted growth, neurobehavioral and respiratory symptoms and fetal deaths. Therefore, it decreased the survival and viability indices of these fetuses compared with the control.

The histopathological findings of the brain of exposed dams and their fetuses:

The brain of exposed rat fetuses during organogenesis showed focal astrocytosis and encephalomalacia in the cerebral tissue (Fig. 10) compared with the control one that showed normal histological structure of the cerebral tissue.

Fig. (10): Brain of a rat fetus obtained from mother treated orally with 345 mg Al chloride /Kg b. wt. during organogenesis period (6-15th days of gestation) showing focal encephalomalacia in the cerebral tissue (E) (H & E stain, X 25.2).

However, the brain of the exposed dams showed only congestion in the cerebral blood vessels and capillaries, compared with control. In the brain of rat fetuses obtained from dams exposed to Al chloride from 6th day of gestation – 21st day after birth (GIV), gliosis was noticed in a focal manner at the cerebral tissue (Fig. 11), associated with encephalomalacia of the white matter in the cerebellar tissue (Fig. 12). However, their dam's brain showed mild

degeneration in the purkinje cells at the cerebellar tissue (Fig. 13). Also, the brain of rat fetuses obtained from dams exposed to Al chloride from 1st – 21st day after birth (GV) showed focal gliosis in the cerebral tissue. At the time, the brain of their dams showed no histopathological changes and appeared like control.

Fig. (11): Brain of a rat fetus obtained from mother treated orally with 345 mg Al chloride /Kg b. wt. on the 6th day of gestation till weaning (21st day postpartum), showing focal gliosis (arrow) in cerebral tissue (H & E stain, X 160).

Fig. (12): Brain of a rat fetus obtained from mother treated orally with 345 mg Al chloride /Kg b. wt. on the 6th day of gestation till weaning (21st day postpartum), showing encephalomalacia in the white matter of the cerebellar tissue (E) (H & E stain, X 25.2).

Fig. (13): Brain of a rat mother treated orally with 345 mg Al chloride /Kg b. wt. on the 6th day of gestation till weaning (21st day postpartum), showing mild degeneration of the purkinje cells (arrow) in cerebellum (arrow) (H & E stain, X 40).


DISCUSSION Owing to the extensive use of aluminum (Al) in

treatment of drinking water and industrial purposes (Jackson, 1986; WHO, 1997 and ATSDR, 2006), accidental and/ or prolonged exposure of both animals and human can occurs leading to great economic losses in the animal wealth and toxicological hazards for human health. Moreover, it is known that Al is a developmental and immune- toxicant when administered parenterally (Wide, 1984; Cranmer et al., 1986; Yokel, 1987; Golub et al., 1993 and Domingo, 1995). However, until recently, there was little concern about the embryo- and feto- toxic consequences of aluminum ingestion because bioavailability was considered low. Therefore, the present study was designed to highlight on the adverse effects of the orally administered Al on the growing fetuses and consequently on the animal wealth in our country.

Developmental and teratogenic effects: Our results of morphological examination of the

obtained fetuses from dams treated orally with 345 mg Al chloride /kg b.wt. on days 6-15 of gestation (G III) showed significant variations from the control group (G I). The postimplantation death and resorbed fetuses percentages were significantly higher in the treated group than those recorded in control group. However, the live fetuses' percentage, mean fetal body weight and placental weights were significantly decreased. Moreover, Aluminum chloride produced a significantly higher percentage of morphological, visceral and skeletal abnormalities in the obtained fetuses compared to control group.

It has been recorded that oral Al exposure during pregnancy produce a syndrome including growth retardation, delayed ossification and malformations in the offspring at Al doses that also led to maternal toxicity (Gomez, 1991; Colomina et al., 1992; Muller et al., 1992; Domingo, 1995; Golub and Domingo, 1996 and Colomina et al., 2002). Moreover, in consistent with our results, a number of studies have shown that Al exposure during gestation at lower doses than that which produces maternal toxicity causes resorptions, deaths, decreased litter size, growth retardation, skeletal; soft tissue abnormalities and impairment of the neuromotor maturation of the surviving pups (Elbina et al., 1984; Paternain et al., 1988 and Bernuzzi et al., 1989a).

The recorded teratogenic effects in the obtained fetuses from the treated dams during organogenesis in our results could be attributed to the transplacental passage of Al (Yumoto et al., 2000; 2001 and Sharma and Mishra, 2006). These abnormalities were sustained by the recorded placental histopathological changes. Although

detailed mechanisms of the toxic actions of aluminum are unknown, a number of possible mechanisms have been suggested. Aluminum competes with cations such as magnesium, calcium, and iron and binds to anions such as phosphate and fluoride. Such interactions can affect the uptake, distribution, and excretion of biologically important ions (Spencer and Lender, 1979 and Spencer et al., 1980). Aluminum’s effect on bone formation (Manifested as skeletal anomalies in our study) may be due to Al competition with essential cations and anions responsible for bone formation or due to inhibition of parathyroid hormone secretion and possibly direct inhibition of osteoblast formation (Spencer et al., 1980).

Effects of gestational and lactation (postnatal) exposure:

The obtained data revealed that Al chloride exposure during gestation and lactation periods till weaning induced significant increase in the percentage of dams showed delayed birth date and signs of dystocia. In addition, it induced a significant increase in the percentage of postimplantation loss, dead fetuses; fetuses showing neurobehavioral and respiratory symptoms and those born with morphological abnormalities. Moreover, it decreased the live/ birth, survival and viability indices and weight gain of these fetuses compared with control. Sharma and Mishra (2006) recorded similar developmental anomalies in the progeny of rat dams treated with aluminum chloride at 345 mg/kg /day on days 0 to 16 of gestation and 0 to 16 of post-partum.

In agreement with our results, studies on rats and rabbits have indicated that maternal exposure of Al during gestation/gestation and lactation/ lactation resulted in resorptions, fetal death, growth retardation and alteration in neuromotor maturation with considerably higher level of Al in milk and other organs (Bernuzzi et al 1986; Muller et al., 1992, and Yumoto et al., 2004). Bernuzzi et al. (1986) found that pre-weaning mortality in rats was significantly increased in the Al treated dams' young. It has been recorded also that aluminum lactate administration during gestation and lactation or directly to pups caused adverse effects on neuromotor development (Golub et al., 1987; Bernuzzi et al., 1989a, b and Muller et al., 1993). Toxic effects were seen in mother and infant (Bernuzzi et al., 1989 a, b and Muller et al., 1990). Colomina et al. (1998) found that maternal restraint in pregnant mice could enhance the Al induced embryo/fetal toxicity (reduced fetal body weight, increase in the number of litters with morphological defects). In a subsequent investigation (Colomina et al., 1999) in which all of the pregnant mice were allowed to deliver and wean their offspring, a decrease in the indices of viability and lactation were observed in the pups whose dams were concurrently exposed to Al and restraint stress.


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Our results (Specially those of AL- induced brain and neurobehavioral changes, signs of dystocia and postnatal mortality) confirms also the previous reports that prenatal and postnatal exposure of experimental animals to Al induced symptoms such as memory loss, orientation problems, aggressive behavior, cannibalism, and impairment of neuromotor maturation (Yokel, 1985; Bernuzzi et al., 1989a, b; Muller et al., 1990 and Golub et al., 1995). Higher exposure of fetuses and sucklings of experimental animals to Al increased stillbirths, aborted litters and postnatal mortality, and lowered both body and organ weights, including that of the brain during subsequent development (Cranmer et al., 1986; Paternain et al. 1988 and Muller et al., 1992).

At weaning the diminished body weight of the prenatally treated pups might be caused by the decreased milk production of the dams and the decreased milk consumption of the offspring (Yokel, 1985 and Muller et al., 1990).

Sharma and Mishra (2006) showed that Al crosses the placental barrier and accumulates in the fetal brain. A study using radio isotopic 26Al showed that considerable amount of Al administered to pregnant and/or lactating rats; Al crossed the blood brain barrier and was deposited in the brain of fetuses and sucklings through the transplacental passage and/or maternal milk and remained persistent throughout their lifetime (Yumoto et al., 2000 and 2001). Also, it has been observed that the placenta incorporated a large amount of 26Al, which was approximately 20% the amount distributed into the liver of the pregnant rat and the amount of Al transferred to the fetus was about 100% of the amount of Al measured in the placenta (Yumoto et al., 2000). The placenta plays a very important role for the developing fetuses as it provides nutrition, hormonal regulation and transfers metabolic waste products. Hence, accumulation of Al may alter placental function and thus affect the developing embryo and fetuses. In the present investigation reduction in placental (On exposure during organogenesis period) and fetal body weight (On exposure during all gestation and lactation periods) were recorded. These effects may also be due to the accumulation of the metal in the placenta as well as in the fetuses or to hormonal disorders (Sharma et al., 2002).

Levine et al. (1992) explained the mechanisms of Al cytotoxicity including potential effects on cell membranes, interactions with phosphate or phosphate containing molecules (ATP, DNA, RNA), or with proteins (calmodulin, transferrin, enzymes, microtubules, intermediate filaments). Increased reactive oxygen species (ROS) were reported in previous studies during Al exposure, which was

attributed to electron leakage, enhanced mitochondrial activity and increased electron chain activity (Goloub et al., 2002). Aluminum has long been known to promote Fe-initiated oxidative damage (Gutteridge et al., 1985). Meglio and Oteiza (1999) suggested that Al can promote lipid peroxidation through another ROS generating system, the xanthine/xantine oxidase system. ROS subsequently attack almost all cell components including membrane lipids and produce lipid peroxidation (Flora et al., 2003). Therefore, it can be hypothesized that oxidative stress may be one of the contributing factors for Al-induced central nervous system disorders and neurotoxicity (Christen, 2000 and Goloub et al., 2002). Moreover, Bondy et al. (1998) attributed damage to CNS tissue of rats administered Al gluconate for 21 days to excess NO production from Al- induced increase of nitric oxide synthase (NOS).

The present study suggests participation of free radicals-induced oxidative cell injury in mediating the toxicity of Al in brain of pregnant rats, fetuses and sucklings. There is a high susceptibility of the brain to oxidative insult because it contains a large amount of polyunsaturated fatty acids and consumes 20% of the body’s oxygen. Moreover, in spite of the high rate of oxidative metabolism, brain has a relatively low antioxidant defense system (Masten, 2000).

Brain cell nuclei may be an important target for Al accumulation because of the high density of phosphorus in DNA, RNA and phosphorylated proteins (Lukiw et al., 1989). Al (Al3+) is a trivalent cation, and has a high affinity for negatively charged groups. It has been proposed that Al preferentially interacts with phosphate groups, such as in nucleic acids and phosphorylated proteins (Martin, 1992 and Zatta, 1995). In addition, Al was found to bind to DNA through chelation (Carson, 2000). Therefore, Al incorporated into brain cell nuclei appears to bind firmly to phosphate groups in phosphorylated proteins and DNA, which are comprised of chromatin (Yumoto et al., 1997). In addition, Al has been reported to condense brain chromatin configurations (Walker et al., 1989). Al reduces DNA transcription in nerve cells in vitro (Sarkander et al., 1983), and also inhibits protein synthesis in the brain (Nicholls et al., 1995). In addition, it induces cell death (apoptosis) because Al remarkably decreases DNA synthesis (Berlyne et al., 1970 and Tsubouchi et al., 2001). Therefore, Al may inhibit not only the generation of nerve cells from progenitor cells, but also the cell division of glial cells and endothelial cells, which act to maintain nerve cell functions. Therefore, increased accumulation of Al in the brain cell nuclei of rat fetuses and sucklings may alter the normal growth and development of the brain, and cause neurologic


alterations and neurobehavioral deficits. Therefore, the recorded histopathological changes in brain of the obtained fetuses and sucklings form Al- exposed dams (During gestation and lactation periods) may be due to Al effects on cell membranes, interactions with phosphate, phosphate containing molecules (Specially DNA) or with proteins and/ or due to increased reactive oxygen species (ROS) production.

It has been reported that Al may interfere with neuronal signalling through interactions with glutamate receptor or calcium channels (Plat and Büsselberg, 1994 and Platt et al., 1994) and/or intracellular calcium homeostasis (Gandolfi1 et al., 1998). Alternatively, considerable evidence has been provided for an interaction of Al with the cholinergic system (Meiri et al., 1993). Al was described to interfere with cholinergic transmission and signalling, for example, acetylcholine metabolism may be affected by Al (Bielarczyk et al., 1998), and reduced choline acetyl transferase (ChAT) and acetylcholinesterase (AChE), but not glutamic acid decarboxylase (GAD) activity was observed after Al injections in rabbits (Yates et al., 1980). Moreover, carbachol-induced phosphoinositol signaling was found to be inhibited by Al (Shafer et al., 1993). The cholinergic system (Specially the basal forebrain projections to hippocampus and cortex) is also known to be particularly affected in Alzheimer’s disease (AD) (Whitehouse et al., 1981), and cholinergic signaling is crucially involved in learning and memory mechanisms (Cain, 1998).

Many other mechanisms have been proposed for Al induced developmental neurotoxicity including Al competition for essential elements uptake during development of the nervous system (Spencer and Lender, 1979 and Spencer et al., 1980), deposition of neurofilament aggregates, alteration of cyclic nucleotide levels, effects on glucose metabolism via inhibition of hexokinase and glucose-6-phosphate dehydrogenase and inhibition of brain dihyropteridine reductase (DHPR (Strong et al., 1996 and Kaur et al., 2006). The recorded neurobehavioral changes in the fetuses and sucklings of Al- exposed dams during gestation and lactation may be resulted from Al- induced brain histopathological changes and/ or interference with the signal transduction pathways inside the brain.

Effects of lactation (postnatal) exposure: Results of the Al- induced effects on the obtained

fetuses from Al chloride treated dams through the 1st – 21st days after birth (lactation period) showed that Al significantly increased the percentage of postnatal deaths, fetal stunted growth with a significantly increased percentage of nervous and respiratory symptoms prior to death. Consequently, the survival and viability indices were reduced. Moreover, the weight gain during lactation was significantly

reduced as there was a significant reduction in the mean fetal body weight at 14th and 21st days after birth.

Similar to the over all of our data, Yokel (1985) reported that gestational and lactation exposure of experimental animals to doses of Al, that do not produce maternal toxicity, can result in retardation of neurological development and deficits in neurobehavioral performance in their offspring. In addition, Golub et al. (1987) have shown developmental retardation in offspring of mice following oral exposure to aluminum during gestation, parturition and lactation. Offsprings showed dose-dependent decreases in body weights and crown-rump length at birth and pre weaning. Golub et al. (1989 and 1999) found that high dietary Al during development was associated with impairment of the nursing mouse pups ability to retain absorbed Fe and Mn with a consequent neurobehavioral alteration. Muller et al. (1992) ; Yumoto et al. (2004) and Sharma and Mishra (2006) concluded that studies on rats and rabbits had indicated that maternal exposure to Al during gestation/gestation and lactation/ lactation could result in resorptions, fetal death, growth retardation and alteration in neuromotor maturation and considerably higher level of Al in milk and other organs.

It is worth noting that milk is markedly rich in lactoferrin (Baker et al., 2002), which comprises 20–35% of the protein in breast milk. Most of the Al in the blood binds with the iron binding sites of transferrin. Since lactoferrin has essentially identical iron binding sites to those of transferrin, it is probable that Al in the milk binds with the iron binding sites of lactoferrin. In addition breast-feeding infants are more vulnerable to Al toxicity than adults because of their underdeveloped gastrointestinal barrier to Al absorption and immature renal function to excrete Al from the blood (Bishop et al., 1997 and Baker et al., 2002). The Al – induced effects on postnatal exposure in the present study may be resulted from the toxic effects of the transported Al via milk.

Yumoto et al. (2003) concluded that 26Al subcutaneously injected into lactating rats was incorporated into the cerebrum, cerebellum, hippocampus, brain stem, and spinal cord of suckling rats through maternal milk. Aluminum incorporated into these central nervous tissues of suckling rats via milk may not only inhibit the postnatal development of these tissues, but also impair physiological functions of the tissues throughout their lifetimes as it remained in these tissues till 710 days after weaning. Higher level of Al was also detected in brain of pups when the mother was exposed during lactation. The recorded neurobehavioral changes in our study could be attributed to motor impairment (Bernuzzi et al.,


23

(1989b) which was sustained by the recorded pathological changes in the brain of these suckling fetuses.

It can be concluded that Al chloride exposure of female rats during gestation and/ or lactation periods caused teratogenic, perinatal and postnatal adverse effects on their progeny.

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