Effect of Prenatal Lead in the Cross-fostered Mice Offspring

Saudi Journal of Biological Sciences 13 (1) 80-90: 2006 ISSN 1319-562X The Official Journal of the Saudi Biological Society htt:www.saudibiosoc.com Effect of Prenatal Lead in the Cross-fostered Mice Offspring Qasim M. Abu-Taweel, Jamaan S. Ajarem*and Mohammad Ahmad Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451 Saudi Arabia Abstract. Lead (Pb) at the dose of 0.2% (w/v) containing 1100 ppm of Pb was given to female Swiss-Webster strain mice in their drinking water. Treatment started from the first day of pregnancy until the day of delivery. Thereafter, the dams were switched to plain tap water. The control as well as the treated mothers and offspring were cross-fostered on first day of delivery in a way that four groups were created, viz : control mothers v/s control pups (Cm-Cp); control mothers v/s treated pups (Cm-Lp); treated mothers v/s control pups (Lm-Cp) and treated mothers v/s treated pups (Lm-Lp). All cross-fostered offspring were subjected to various tests. The affected parameters were a decline in the body weight gain, delayed opening of the eyes and retarded development of the sensory motor reflexes in the pups throughout their postnatal developing weaning period. Alterations were noticed in the levels of acid and alkaline phosphatases in liver, and acetylcholinesterase in the brain tissues of the developing offspring. Significant increase in various parameters of 'locomotory test', and a significant decrease in attacking behaviour and alteration in the acts and postural behaviour in the ‘Standard Opponent Test’ was observed in the Pb exposed adult male offspring. The 'tube restraint test' showed a significant change in the latency to the first bite and in the number of target biting by the Pb exposed female offspring. Overall observations on behavioural and biochemical parameters showed a significant effect of Pb in the cross-fostered offspring in the order of Lm-Lp > Lm-Cp > Cm-Lp as compared to the control (Cm-Cp). The present Pb effects in cross-fostered offspring are possibly via in utero action and/or via mother’s milk. Key words: Lead; prenatal exposure; mice offspring; cross fostering; behaviour; sensory motor reflexes; esterases; locomotory behaviour, standard opponent test; tube restraint test. Introduction Research has shown that even low levels of lead (Pb) exposure are dangerous (Silergeld, 1990). Thus, Pb exposure/toxicity continues to be a leading environmental health issue for children and women of childbearing age (Mushak, 1992). A variety of toxic effects caused by Pb exposure during gestation and early childhood have been reported in both human and animal studies. Many neurological and behavioural anomalies have been attributed to preand postnatal Pb exposure, such as aggressiveness, decreased IQ, learning disabilities, hyperactivity, and impulsiveness, as well as aberrant neuromotor coordination function (Kishi et al., 1998; Needleman, 1987, 1993). In both humans and experimental animals, Pb readily crosses the placental-fetal barrier (Goyer, 1990; Lataillade et al., 1995; Donald et al., 1986), causing a direct relation between the Pbexposed mother and the possibility of irreversible developmental damage to her offspring. Keller and Doherty (1980) reported that Pb-exposed mouse dams transmitted a significantly greater amount of Pb to their offspring through their milk than by in utero exposure (transplacentally). Earlier studies in humans and experimental animals have shown that not only does Pb accumulate in the fetus from the second tremester onwards (Bhattacharayya, 1983), but during lactation, it is excreted into the milk, which continues the risk to nursing offspring (Namihira et al., 1993; Correspondence author: Jamaan S. Ajarem* Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia 80 Saudi Journal of Biological Sciences Vol. 13, No.1, 2006 Effect of prenatal lead in the cross-fostered mice offspring Halten et al., 1995a,b, 1996). The detrimental effects of Pb occur regardless of the developmental time exposure, although gestational exposure appears more sensitive to the Pb effects (Dearth et al., 2002). To compare the differential hazards of gestational and lactational Pb exposure, pregnant mice were exposed to Pb in their drinking water beginning at day 1 of gestation until the day of delivery, and crossfostering of their litters at parturition was performed. A variety of parameters were assessed during the weaning period and at adolescent age to determine if Pb-induced alteration was, in fact, observed due to gestational exposure, or if suckling offspring were at a higher risk of the adverse effects of Pb (Snyder et al., 2000). Cross-fostering is a widely used laboratory practice serving experimental purpose in developmental teratology, to determine the relative contribution of placental and milk transmission of a compound from the mother to the pups (Howdeshell et al., 1999). Despite its extensive use, there is a relative paucity of studies investigating the effect of cross-fostering per se on the behaviour and the physiology of laboratory animals (Bartolomucci et al., 2004). The aim of the present cross-fostering study is to provide a comprehensive behavioural and biochemical investigation of the effects of prenatal Pb exposure on the postnatal developing offspring. It is also intended to examine as to if such cross-fostering designs produce lasting actions via changes in the maternal behaviour and/or lacatational efficiency. Materials and Methods Experimental animals Male and female Swiss-Webster strain mice (89 weeks old) were housed in opaque plastic cages (three females to one male in each cage) measuring 30 X12 X 11 cm, in the animal facility of the Zoology Department, King Saud University, Riyadh, Saudi Arabia. Animals were kept under reversed lighting conditions with white lights on from 22.30 to 10.30 hours local time. The ambient temperature was regulated between 18 and 22o C. After pregnancy (appearance of vaginal plug was considered as day one of pregnancy), the males were removed from the cages and the females were subjected to experimental treatments. The wood fillings were changed regularly Saudi Journal of Biological Sciences Vol. 13, No.1, 2006 and food (Pilsbury’s Diet) and water were available ad libitum, unless otherwise indicated. Lead administration Lead acetate (analytical grade, Riedel de Haen, Germany) was dissolved in deionized distilled water to give a dose of 0.2% (w/v), containing 1100ppm of Pb. This lead dose formed the sole drinking fluid source for the experimental group of mice during the required period of the experiment. Fresh drinking fluid containing lead dose was replaced every five days. In order to preclude the precipitation of insoluble lead salts, 12- drops of acetic acid (vehicle of lead) were added to all bottles including controls. No vehicle control group was included in this study. The control group received deionized distilled water only. All pregnant mice were housed individually. Treatment was started from the day 1 of pregnancy and was continued until the day of delivery and thereafter the mothers were switched to plain deionized distilled water. Cross-fostering On the day of birth (postnatal day 0, PD0), the control and treated groups of dams and offspring were cross-fostered in such a way that four groups were created, viz: control mothers v/s control pups (Cm-Cp); control mothers v/s treated pups (Cm-Lp); treated mothers v/s control pups (Lm-Cp) and treated mothers v/s treated pups (Lm-Lp). Behavioral observations The pups of each cross-fostered groups were culled to only eight per dam on PD0 and were left with their allocated mothers until PD22. During this weaning period, three pups of each litter were colour marked from the others and were subjected to various behavioural tests (described below) under dim lighting (ca 8 lux). In all, 21 pups belonging to seven litters from each treatment category were considered. All observations were recorded on PD1 and repeated every other day until PD21 in the same three colour marked pups of each litter. These observations were used to measure the early development of sensory motor coordination reflexes together with morphological development in the pups. For statistical analysis, the mean of all three colour marked pups per litter was considered as a single 81 Qasim M. Abu-Taweel, Jamaan S. Ajarem and Mohammad Ahmad score. Thus, seven replicates from each treatment category were considered in these observations. Body weight: Weight is an useful indicator of development. Thus, the pups were weighed every alternate day from PD1 until PD21. Righting reflex: The time taken by a pup placed on its back to turn over and place all four paws on the substrate was recorded. An upper limit of 2 min being set for this test. Cliff avoidance: Pups were placed on the edge of a table top with the forepaws and face over the edge. The time taken by the pup to back away and turn from the “cliff”was recorded. Again an upper limit of 2 min was chosen. A latency of 2 min was attributed when the animal fell from the “cliff”. Rotating reflex : The surface used to measure the rotating reflex was the same as that used for righting reflex, except that it was inclined at an angle of 30o. The pups were placed on this surface with their heads pointed downwards. The time elapsed until the pup rotate its body through 180o geonegatively and face its head upwards, was recorded as the rotating time. The upper limit of this test was also set at 2 min. Eye opening and hair appearance: The day at which the body hair fuzz appeared, and the eyes opened were also recorded. These two parameters are also useful morphological indicators of development. Biochemical Studies: During the weaning period, on PD7, PD14, and PD21, one pup was picked up at random from each litter, apart from those three colour marked pups that were used for the behavioural tests. Thus, seven pups were collected from each experimental group on each postnatal day (PD7, 14 and 21) without any consideration to its sex and were killed by decapitation. Their brain and liver were removed and gently rinsed in physiological saline (0.9% NaCl), and then blotted on Whatman filter paper. Their fresh weights were recorded, and organs were then frozen. Tissue homogenate preparation: A 10% (w/ v) homogenate of each frozen tissue was prepared in teflon-glass homogenizer at 4 ± 1oC, centrifuged at 1000 X g for 10 min. to remove cell debris and the supernatant was used for enzyme assays. The brain homogenate was prepared in an ice-cold phosphate buffer, (0.067M, pH7.2) and the liver was homogenized in chilled 0.25M sucrose solution. 82 Enzyme estimations: The acetylcholinesterase (AChE) activity in the homogenised brain tissue was estimated by the method of Hestrin (1949) using acetylchloline chloride as the substrate. The specific activity of AChE was expressed as µ moles acetylcholine chloride hydrolysed per gram wet tissue weight per hour at 37 ± 1oC. The levels of total acid phosphatase (AcP) and alkaline phosphatase (AlP) were estimated in the liver tissue homogenates using sodium p-nitrophenol phosphate as the substrate (Bergmeyer et al., 1974). The protein content in the homogenates was estimated according to the method of Lowry et al. (1951). The specific activities of these phosphomonoesterases were expressed as n-moles p-nitrophenol liberated per mg protein per minute at 37 ± 1oC. Locomotory Tests of young adult males: The offspring were weaned on PD21 and thereafter, the males and females were isolated and kept in groups of two or three, for 14 days. Subsequently, 10 males from each treated group ( including representatives from each 7 litters) were subjected to locomotor activity tests. The young adult males were placed in an experimental wooden arena measuring 80 X 80 X 30 cm and the floor was divided into 64 equal sized squares. Various behavioral elements were observed as described by Ajarem (1987). Elements of locomotory activity included the number of squares crossed and the number of wall rears as well as the duration of locomotion and immobility. The visual observations in the arena lasted 300 sec for each animal. “Standard Opponent” Test: After weaning at 21days of age, 10 male subjects from each treatment category, were individullay housed in ftresh cages for 14 days. After this isolation period, these male offspring(test animals) from each category were subjected to “standard opponent” tests under dim red lighting (ca. 8 lux) as described elsewhere (Brain et al., 1989). The docile and age-matched male “standard opponents” were rendered anosmic by applying 25µl of 4% zinc sulphate solution to the nasal tract under ether anaesthesia for three days prior to encounters (Brain et al.,1981). The anosmic ‘standard opponent’ intruders were introduced in the home cages of the ‘test animals’ and the “standard opponent” test of each Saudi Journal of Biological Sciences Vol. 13, No.1, 2006 Effect of prenatal lead in the cross-fostered mice offspring ‘test animal’ was observed visually for 500 seconds. The opponents were used only once and the selected “elements” of behaviour were based on the studies of Brain et al. (1987) and Ajarem and Ahmed (1991). Tube restraint test of young adult females:The females isolated after weaning period were subjected to the 'tube restraint test'. Ten females from each treated group (including representatives from each 7 litters) were used for this test. The apparatus was based on the equipment described by Ajarem and Ahmed (1992) and consisted of a cylindrical transparent perspex tube 13 cm in length and with an internal diameter of 3.1 cm. One end of the tube was blocked by a perforated perspex wall through which a 2 cm long metal target was attached to a telegraph key/electronic counter arrangement. This enabled one to record the number of bites directed by the restrained mouse towards the target. The test was conducted visually as outlined by Ajarem and Ahmad (1994) for 500 seconds under normal laboratory white lighting and temperature. Statistical Analysis: The data of body weight, dates of morphological developments, data of sensory motor reflexes and data of biochemical analyses were compared within the experimental groups by the analysis of variance (ANOVA) using minitab computer programme, and were subsequently analysed by Student’s t-test (Yamane, 1973). Data of 'Locomotory', 'Standard opponent' and 'tube restraint' tests were compared within the experimental groups by the analysis of variance (ANOVA) and subsequently were analysed using Mann-Whitney U tests (Sokal and Rohlfe,1981). their own treated mothers. However, the eye-opening was affected in the other groups also in the order of Lm-Lp >Lm-Cp> Cm-Lp (Fig. 2). The body hair appearance was not affected in any of the groups, however, it was delayed slightly but insignificantly only in the pups born and crossed with their own treated mothers (Lm-Lp) (Fig. 2). Prenatal exposure of mice to lead, followed by the postnatal cross-fostering design, showed that the presence of lead either in mother or in the pups during their fetal or postnatal developing stages, had a significant effect on all the sensory motor reflexes of the pups. However, the lead exposed pups fostered with their own treated mothers (Lm-Lp), showed the most significant (p<0.001) decrease in their righting Fig 1. Effect of prenatal lead exposure on the body weight gain of the cross-fostered mouse pups. * indicates significance at p< 0.01 as compared to control (by Student’s t-test). Results The ANOVA on body weights show that the pups born and brought up with their treated mothers remain retarded very significantly (p<0.001) in their body weight gain throughout their weaning period (Fig. 1). Further, it was observed that Pb whether present in mothers or in pups, had a varied retarding effect on the body weight gain of the pups depending on the cross-fostering design. However, Pb exposure affected the body weight gain in the cross-fostering groups, in the order of Lm-Lp> Lm-Cp> Cm-Lp. The eye-opening was delayed significantly (p<0.001) in the treated pups that were crossed with Saudi Journal of Biological Sciences Vol. 13, No.1, 2006 Fig 2. Effect of prenatal lead exposure on the hair appearance and eye opening in the cross-fostered mouse pups. *, ** and *** indicate significance at p<0.05, p<0.01 and p< 0.001, respectively, as compared to control (by Student’s t-test). 83 Qasim M. Abu-Taweel, Jamaan S. Ajarem and Mohammad Ahmad Table 1. Effect of prenatal lead exposure on the locomotor activity of cross-fostered adult male offspring. Median number (with ranges) of acts and postures Treatment group Number of squares crossed Wall Rears Rears Wash cleaning Locomotion duration (sec) Immobility duration (sec) Cm - Cp 182 (134-227) 25 (16-35) 3 (0-6) 10 (0-21) 134.6 (89.3186.6) 165.4 (117.7212.7) Cm - Lp 220 (155-225) 30 (27-35) 6* (2-10) 4 (2-6) 212.4** (190.9226.3) 87.6** (73.7110.2) Lm - Lp 230** (153-240) 33 (29-37) 7** (3-12) 2 (1-6) 216.6** (135.7229.3) 83.4** (70.7110.2) Lm - Cp 215 (168-220) 27 (24-30) 4 (2-9) 5 (3-8) 210.6* (185.2220.1) 89.4** (79.9115.9) *, ** and *** shows statistically significant at P<0.05, P<0.01 and P<0.01 and P<0.001 respectively from the control by Mann-Whitney U-test. C: Control; L: Lead treated; m: mother; p: pups. Table 2. Effect of prenatal lead exposure on the behaviour of cross-fostered adult male offspring in a standard opponent test. Median (with ranges) number of seconds allocated to behaviours like Treatment group Nonsocial investigation Social investigation Defense Threat Attack Displacement Cm - Cp 120.8 (40.5238.1) 216.2 (63.4289.1) 7.1 (1.4-18.5) 7.4 (4.9-21.2) 120.1 (96.2158.8) 24.9 (3.7-53.8) Cm - Lp 107.5 (63.4200.9) 238.1 (200.2290.5) 7.1 (3.8-24.6) 13.1 (3.8-24.6) 93.4 (39.6158.8) 15.4 (14.7-53.3) Lm - Lp 200.3 (63.4241.4) 215.2 (200.3238.1) 5.5 (0.3-23.6) 5.6 (3.2-20.3) 51.9** (25.6125.0) 15.4 (3.7-53.8) Lm - Cp 170.9 (100.2207.2) 206.9 (190.4253.9) 8.1 (5.3-26.2) 6.5 (2.5-21.8) 65.2** (44.4107.4) 18.2 (3.7-23.7) ** shows statistically significant at P< 0.01 from the control by Mann-Whitney U-test. C: Control ; L: Lead treated; m: mother ; p: pups. (Fig. 3) and rotating (Fig. 4) reflexes as well as cliff avoidance activity (Fig. 5) during their weaning period. These effects were observed in the other cross-fostering groups also but in the order of LmLp>Lm-Cp>Cm-Lp. The levels of phosphomonoesterases enzymes in the liver of the treated pups did not remained the same as in the control groups(Cm-Cp). The Alk (Fig. 6) and AcP (Fig. 7) were stimulated significantly in the treated pups fostered with their own treated mothers (Lm-Lp). The other groups were also affected but at a comparatively lower level. The AChE activity in the brain tissue of the treated pups was found to be disturbed throughout the postnatal developing stages. A significant (p<0.001) decrease in the level of AChE activity was observed in the pups of all groups whether Pb was present in mothers or pups at PD7 and PD14 stages. However, at PD21 stage, AChE activity was stimulated, but all alterations in 84 the AChE activity were in the order of Lm-Lp> LmCp> Cm-Lp, (Fig. 8). The results of locomotor activity test in the male offspring (Table 1) shows that postnatal lead exposure had a significant stimulatory effect on the numbers of squares crossed, rears, as well as the duration of locomotion. On the other hand, the duration of immobility had significantly (p<0.001) decreased. Maximum effect was observed in the Lm-Lp group as compared to other groups (Table 1). The behavioural data (median with ranges) in the ‘standard opponent’ test of the four cross-fostered groups of male offspring are given in Tables 2 and 3. All statistically significant results, particularly an increase in the attacking behaviour and decline in the other acts and postural behaviour was found only in the groups where the treated mothers were either crossed with their own treated offspring (Lm-Lp), or with the male offspring that were born to the control mothers (Lm-Cp). Saudi Journal of Biological Sciences Vol. 13, No.1, 2006 Effect of prenatal lead in the cross-fostered mice offspring Table 3. Effect of prenatal lead exposure on the adult behaviour of cross-fostered male offspring in a standard opponent test. Median number (with ranges) of acts and postures Treatment group Latency to threat (sec) Latency to attack (sec) Number of fights Number of Naso-Nasal contact Number of Naso-Genital contact Wall rears Rears Cm - Cp 70 (3-130) 64 (12-215) 28 (15-37) 37 (23-48) 35 (27-43) 22 (16-35) 17 (14-20) Cm - Lp 160* (3-230) 210 (53-295) 29 (14-35) 30 (23-39) Lm - Lp 190*** (65250) 252** (56315) 8 (3-30) 14*** (6-26) Lm - Cp 140 (3-200) 200 (50-290) 16 (50-290) 26 (24-28) 29 (17-38) 22 (12-28) 16 (8-19) 15*** (7-37) 13 (8-38) 8 (4-230 26 (24-39) 19 (15-27) 13 (8-29) *,** and *** shows statistically significant at P< 0.05, P < 0.01 and P < 0.001 respectively from the control by Mann-Whitney U-test. C: Control; L: Lead treated; m: mohter; p: pups. Table 4. Effect of prenatal lead exposure on the adult female behaviour of cross-fostered offspring in a Tube-restraint test. Treatment group Measures (Median values with ranges) Latency to first bite (sec) Number of bites Cm - Cp 5 (2 - 12) 74 (60 - 90) Cm - Lp 25 (3 - 135) 70 (43 - 80) Lm - Lp 45** (5 - 295) 60 (45 - 71) Lm - Cp 60 (3 - 220) 51** (20 - 65) **shows statistically significant at P< 0.01 from the control by Mann-Whitney U-test. C: Control; L: Lead treated; m: mother; p:pups. The present results suggest that the prenatal exposure (from the day 1 of pregnancy until the day of birth) of female mice to Pb, influences the rate of physical maturation, sensory motor reflexes and level of enzyme activities in liver and brain tissues of the pups at different developmental stages during the weaning period. Furthermore, after the weaning period, in the male and female offspring, various behavioural indices are also affected in the standard opponent test, locomotory test and tube restraint test. Cross fostering the treated offspring to their original treated mothers (Lm-Lp group) incurred the most significant effects on all the parameters studied Fig 3: Effect of prenatal lead exposure on the mean righting reflex of the cross-fostered mouse pups. *, ** and *** indicate significance at p<0.05, p<0.01 and p< 0.001, respectively, as compared to control (by Student’s t-test). Fig 4: Effect of prenatal lead exposure on the mean rotating reflex of the cross-fostered mouse pups. * and ** indicate significance at p<0.05 and p< 0.01 , respectively, as compared to control (by Student’s t-test). The data of ‘tube-restraint’ test (Table 4) showed that the latency to first bite increased and the number of bites on the target, were decreased significantly (p<0.001), only in the female offspring that were exposed to lead (Lm-Lp and Lm-Cp groups). Discussion Saudi Journal of Biological Sciences Vol. 13, No.1, 2006 85 Qasim M. Abu-Taweel, Jamaan S. Ajarem and Mohammad Ahmad Fig 5. Effect of prenatal lead exposure on the mean cliff avoidance activity of the cross-fostered mouse pups. * and ** indicate significance at p<0.05 and p< 0.01, respectively, as compared to control (by Student’s t-test). Fig 6. Effect of prenatal lead exposure on the activity of AcP in liver of the cross-fostered mouse pups at various developing weaning ages. * and ** indicate significance at p<0.05 and p< 0.01 , respectively, as compared to control (by Student’s t-test). Fig 7. Effect of prenatal lead exposure on the activity of AlP in liver of the cross-fostered mouse pups at various developing weaning ages, * and ** indicate significance at p<0.05 and p< 0.01 , respectively, as compared to control (by Student’s t-test). 86 Fig 8. Effect of prenatal lead exposure on the activity of AChE in brain of the cross-fostered mouse pups at various developing weaning ages, * and ** indicate significance at p<0.05 and p< 0.01 , respectively, as compared to control (by Student’s t-test). herein. It may be possibly due to the fact that Pb was available to the developing fetus in utero as well as in the milk of the lactating mothers during the weaning period. The untreated offspring when cross fostered to treated mothers(Lm-Cp group), still confounded effects in the offspring on all parameters studied herein, but comparatively at a lower level. This may be possibly due to exposure of the offspring to Pb postnatally, only through the milk of the lactating treated mothers. It is known that pregnant females have greater capacity to transmit Pb to their offspring through their milk than by in utero exposure (Keller and Dougherty, 1980). On the contrary, Dearth et al.(2002) reported that gestational exposure appears more sensitive to the Pb effects in the offspring. However, it is observed from the present study that the detrimental effects of Pb occur regardless of the developmental time exposure. Further, in new born, absorption of metals from the gut is much higher than in adults, partly because of immaturity of the absorption process and partly because of the nature of the diet, that is, milk (Kostial et al., 1984). Also, it is well established that significant quantities of compounds that are given to mothers in late pregnancy may be transmitted to the offspring in utero and/or during lactation (Ordy et al., 1966; Fabro and Sieber, 1969; Mereu et al., 1987; Ajarem and Ahmed, 1991,1998; Ajarem and Brain, 1993; Ajarem, 1999). During the susceptible developmental period even a low Pb exposure may be of concern (Silergeld, 1990). Saudi Journal of Biological Sciences Vol. 13, No.1, 2006 Effect of prenatal lead in the cross-fostered mice offspring The offspring of rodents exposed to Pb during pregnancy are reported to display many neurological and behavioural anomalies such as aggressiveness, decreased IQ, learning disabilities, hyperactivity, and impulsiveness, as well as aberrant neuromotor coordination function (Kishi et al., 1998; Needleman, 1987, 1993). Such significant effect of lead has previously been reported in rodents (Grant et al., 1980; Draski et al., 1989; Ferguson et al.,1998). Abnormalities in response to teratogens may be due to several factors that causes alterations in normal cell metabolism, especially in enzymes and their substrates (Wilson, 1973), or due to combination of several factors (Coyle et al., 1976). The hepatic enzymes AcP and AlP are known good indicators of liver status (Corpas et al., 2001) and are frequently associated with transport mechanisms across the biological membranes (Starling, 1975, Ajarem and Ahmad, 1991). In rodents, lead level in maternal milk correlates with lead level in the blood of developing pups (Palminger and Oskarsson, 1993). Thus, alterations in the level of these phosphomonoesterases in liver of the developing pups, due to prenatal lead exposure, might have led to variations in their phosphate pool. This probably lead to disturbed energy source available to the animal with the consequent disturbance in its metabolism (Wilson, 1973), which is reflected in the form of altered physical maturation and sensory motor reflexes. An important neurotransmitter that has been implicated in behaviour process is AChE (Allikmets, 1974). Changes in the behaviour due to toxicants are presumably due to alterations in the availability of neurotransmitters (Kruck and Pycok, 1979; Kellog et al., 1980; Lee, 1980). It is known that a major portion of brain cells (70%) of the closely related rats are formed after birth (Patel, 1983) and an alteration in the level of AChE was detected in all lead exposed pups irrespective of their crossing over with any kind of dams. This could possibly explain as to why the sensory motor reflexes of such Pb exposed pups were disturbed during the first fortnight of their postnatal development. Also, it has been established that the cerebellum in the brain may be most vulnerable to the neurotoxicity of lead in the the very rapid growth period during the first 20 postnatal days (Gietzen and Wooley, 1984). Thus, lead could have Saudi Journal of Biological Sciences Vol. 13, No.1, 2006 produced developmental abnormalities in the brain as well as an alteration in the level of AChE, that might have brought about the observed effects on the motor reflexes of the developing pups in the present study. It has been reported that alterations in brain enzymes are among the factors responsible for disturbances in behavioural activities of affected animals (Branchey and Friedhoff, 1976; Kellog et al.,1980; Johnson et al.,1981; Ajarem and Ahmad,1991). The locomotory test on the male offspring and the tube restraint test on the female offspring after the weaning period, at the adulthood stage, suggests for a lasting and significant effect in their behavioural activities brought about by prenatal lead exposure. The present results strongly support the earlier conclusion of Ajarem and Ahmad (1991, 1998) that the enzymes AcP , AlP and AChE could be used as convenient markers in teratological studies of adults as well as the developing pups during the weaning period. Further, the present results also support the earlier findings (Peters et al., 1994; Flora and Seth, 2000) that lead exerts neurotoxic effects by altering certain membrane bound enzymes and may cause oxidative stress which ultimately alters the cellular processes. Such effects might be affecting the overall morphological developments and sensory motor reflexes of the developing pups, and behaviour of the young adult offspring, irrespective to the fact whether their exposure is transplacental (in utero) and/or via their mother's milk during lactation. Acknowledgement The authors are grateful to the Chairman of the Department for providing necessary laboratory facilities. References Ajarem, J.S. 1987. 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Ajarem and Mohammad Ahmad‬‬ ‫ﺗﺄﺛﲑ ﺍﻟﺘﻌﺮﺽ ﻟﻠﺮﺻﺎﺹ ﻗﺒﻞ ﺍﻟﻮﻻﺩﺓ ﻋﲆ ﺻﻐﺎﺭ ﺍﻟﻔﺌﺮﺍﻥ ﺍﳌﺘﺒﺎﺩﻟﺔ ﺍﻟﺮﺿﺎﻋﺔ‬ ‫ﻗﺎﺳﻢ ﳏﻤﺪ ﺃﺑﻮ ﻃﻮﻳﻞ ‪ ،‬ﲨﻌﺎﻥ ﺳﻌﻴﺪ ﻋﺠﺎﺭﻡ ﻭﳏﻤﺪ ﺃﲪﺪ‬ ‫ﻗﺴﻢ ﻋﻠﻢ ﺍﳊﻴﻮﺍﻥ – ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ – ﺟﺎﻣﻌﺔ ﺍﳌﻠﻚ ﺳﻌﻮﺩ ‪.‬‬ ‫ﺹ ‪ .‬ﺏ ‪ . ٢٤٥٥ .‬ﺍﻟﺮﻳﺎﺽ – ‪. ١١٤٥١‬‬ ‫ﺍﳌﻤﻠﻜﺔ ﺍﻟﻌﺮﺑﻴﺔ ﺍﻟﺴﻌﻮﺩﻳﺔ ‪.‬‬ ‫ﺍﳌﻠﺨﺺ‪ :‬ﻋﺮﺿﺖ ﺇﻧﺎﺙ ﺍﻟﻔﺌﺮﺍﻥ ﺍﻟﺒﻴﻀﺎﺀ ﺍﻟﺴﻮﻳﴪﻳﺔ ﺍﳊﻮﺍﻣﻞ ﻟﻠﺮﺻﺎﺹ ﻋﻦ ﻃﺮﻳﻖ ﺍﻟﴩﺏ ﺑﱰﻛﻴﺰ ‪ ) ( w/v ) % ٠٫٢‬ﲢﺘﻮﻱ‬ ‫‪ ppm ١١٠٠‬ﻣﻦ ﺍﻟﺮﺻﺎﺹ ( ﻣﻦ ﺑﺪﺍﻳﺔ ﺍﻟﻴﻮﻡ ﺍﻷﻭﻝ ﻟﻠﺤﻤﻞ ﻭﺣﺘﻰ ﻳﻮﻡ ﺍﻟﻮﻻﺩﺓ ‪ ،‬ﺛﻢ ﲢﻮﻝ ﺍﻷﻣﻬﺎﺕ ﺇﱃ ﻣﺎﺀ ﺍﻟﴩﺏ ﺍﻟﻌﺎﺩﻱ ‪.‬‬ ‫ﹴ‬ ‫ﺳﻮﺍﺀ‬ ‫ﲤﺖ ﻋﻤﻠﻴﺔ ﺗﺒﺎﺩﻝ ﺍﻟﺮﺿﺎﻋﺔ ﺑﻌﺪ ﺍﻟﻮﻻﺩﺓ ﻣﺒﺎﴍﺓ ) ﺃﻭ ﺧﻼﻝ ‪ ٢٤‬ﺳﺎﻋﺔ ( ‪ ،‬ﻭﺗﻀﻤﻨﺖ ﻫﺬﻩ ﺍﻟﻌﻤﻠﻴﺔ ﺑﻘﺎﺀ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﳌﻮﺍﻟﻴﺪ‬ ‫ﺍﳌﺠﻤﻮﻋﺔ ﺍﻟﻀﺎﺑﻄﺔ ) ‪ (Cm-Cp‬ﺃﻭ ﺍﳌﻌﺎﳉﺔ ﺑﺎﻟﺮﺻﺎﺹ ) ‪ ( Lm-Lp‬ﻣﻊ ﺃﻣﻬﺎﲥﺎ ﺍﻷﺻﻠﻴﺔ ‪ ،‬ﻭﳎﻤﻮﻋﺔ ﺃﺧﺮ￯ ﻣﻦ ﺍﳌﻮﺍﻟﻴﺪ ﺍﳌﻌﺎﳉﺔ ﺃﻣﻬﺎﲥﺎ‬ ‫ﺑﺎﻟﺮﺻﺎﺹ ﻧﻘﻠﺖ ﺇﱃ ﺍﻷﻣﻬﺎﺕ ﻏﲑ ﺍﳌﻌﺎﳉﺔ ) ﺍﻟﻀﺎﺑﻄﺔ ( ) ‪ ، ( Cm-Lp‬ﻭﳎﻤﻮﻋﺔ ﻣﻦ ﺍﳌﻮﺍﻟﻴﺪ ﻏﲑ ﺍﳌﻌﺎﳉﺔ ﻧﻘﻠﺖ ﺇﱃ ﺍﻷﻣﻬﺎﺕ ﺍﳌﻌﺎﳉﺔ‬ ‫ﺑﺎﻟﺮﺻﺎﺹ ) ‪ . ( Lm-Cp‬ﺃﺟﺮﻳﺖ ﺍﻟﻌﺪﻳﺪ ﻣﻦ ﺍﻻﺧﺘﺒﺎﺭﺍﺕ ﺍﻟﺴﻠﻮﻛﻴﺔ ﻭﺍﻟﻜﻴﻤﻮﺣﻴﻮﻳﺔ ﻋﲆ ﺻﻐﺎﺭ ﺍﳌﺠﻤﻮﻋﺎﺕ ﺍﳌﺨﺘﻠﻔﺔ ﺧﻼﻝ ﻓﱰﺓ‬ ‫ﺍﻟﻨﻤﻮ ﻭﺣﺘﻰ ﻭﻗﺖ ﺍﻟﻔﻄﺎﻡ ‪.‬‬ ‫ﺃﺷﺎﺭﺕ ﺍﻟﻨﺘﺎﺋﺞ ﺍﳌﻮﺭﻓﻮﻟﻮﺟﻴﺔ ﺇﱃ ﺍﻧﺨﻔﺎﺽ ﰲ ﻣﻌﺪﻝ ﻭﺯﻥ ﺍﳉﺴﻢ ﻭﺇﱃ ﺗﺄﺧﺮ ﰲ ﻭﻗﺖ ﺗﻔﺘﺢ ﺍﻷﻋﲔ ﻭﺇﱃ ﺗﺜﺒﻴﻂ ﰲ ﻧﻤﻮ ﺍﻻﻧﻌﻜﺎﺳﺎﺕ‬ ‫ﺍﳊﺴﻴﺔ ﺍﳊﺮﻛﻴﺔ ﻟﺼﻐﺎﺭ ﺍﳌﺠﻤﻮﻋﺎﺕ ﺍﳌﻌﺎﳉﺔ ﺑﺎﻟﺮﺻﺎﺹ ﺍﻟﺜﻼﺙ ) ‪ ( Lm-Lp > Lm-Cp > Cm-Lp‬ﺧﻼﻝ ﻓﱰﺓ ﺍﻟﻨﻤﻮ ‪.‬‬ ‫ﺃﻇﻬﺮﺕ ﻧﺘﺎﺋﺞ ﺍﺧﺘﺒﺎﺭ ﺍﻟﺴﻠﻮﻙ ﺍﳊﺮﻛﻲ ﻭﺍﻻﺟﺘﲈﻋﻲ ﻟﻠﺬﻛﻮﺭ ﺍﳌﻌﺎﳉﺔ ﺑﺎﻟﺮﺻﺎﺹ ﰲ ﺍﳌﺠﻤﻮﻋﺎﺕ ﺍﻟﺜﻼﺙ ﺯﻳﺎﺩﺓ ﻣﻌﻨﻮﻳﺔ ﰲ ﻋﻨﺎﴏ‬ ‫ﺍﻟﺴﻠﻮﻙ ﺍﳊﺮﻛﻲ ﻭ ﺍﻧﺨﻔﺎﺽ ﰲ ﺍﻟﺴﻠﻮﻙ ﺍﳍﺠﻮﻣﻲ ﻭﺗﻐﻴــﺮﺍﺕ ﰲ ﺳــﻠﻮﻙ ﺍﻟﺘﻨﺤﻲ ) ‪ ( Lm-Lp > Lm-Cp > Cm-Lp‬ﻣﻘــﺎﺭﻧــﺔ‬ ‫ﺑﺎﻟﻜﻨﺘــﺮﻭﻝ )‪. (Cm-Cp‬‬ ‫ﺃﺷﺎﺭﺕ ﺍﻟﻨﺘﺎﺋﺞ ﺍﻟﻜﻴﻤﻮﺣﻴﻮﻳﺔ ﺇﱃ ﺣﺪﻭﺙ ﺗﻐﲑﺍﺕ ﻣﻠﺤﻮﻇﺔ ﰲ ﻣﺴﺘﻮ￯ ﺇﻧﺰﻳﻢ ﺍﻟﻔﻮﺳﻔﺎﺗﺎﺯ ﺍﳊﺎﻣﴤ ﻭﺍﻟﻘﺎﻋﺪﻱ ﰲ ﺍﻟﻜﺒﺪ ﻭﻛﺬﻟﻚ‬ ‫ﰲ ﻧﺸﺎﻁ ﺇﻧﺰﻳﻢ ﺍﻷﺳﺘﻴﻞ ﻛﻮﻟﲔ ﺇﺳﺘﲑﻳﺰ ﰲ ﺃﻧﺴﺠﺔ ﺍﳌﺦ ﻟﻠﺼﻐﺎﺭ ﺍﳌﻌﺎﳉﺔ ﺃﻣﻬﺎﲥﺎ ﺑﺎﻟﺮﺻﺎﺹ ) ‪(Lm-Lp > Lm-Cp > Cm-Lp‬‬ ‫ﻋﻨﺪ ﻣﻘﺎﺭﻧﺔ ﺑﺎﳌﺠﻤﻮﻋﺔ ﺍﻟﻀﺎﺑﻄﺔ )‪. (Cm-Cp‬‬ ‫ﺃﻇﻬﺮ ﺍﺧﺘﺒﺎﺭ ﺍﻷﻧﺒﻮﺑﺔ ﺍﻟﻜﺎﺑﺤﺔ ﻟﻠﺤﺮﻳﺔ ﺍﺧﺘﻼﻓﺎﺕ ﻣﻌﻨﻮﻳﺔ ﰲ ﺯﻣﻦ ﺑﺪﺍﻳﺔ ﺍﻟﻌﺾ ﰲ ﺍﳌﺠﻤﻮﻋﺔ ) ‪ ( Lm-Lp‬ﻭﻋﺪﺩ ﻣﺮﺍﺕ ﺍﻟﻌﺾ‬ ‫ﻟﻠﻤﺠﻤﻮﻋﺔ ) ‪ ( Lm-Cp‬ﻟﻺﻧﺎﺙ ﻣﻘﺎﺭﻧﺔ ﺑﺈﻧﺎﺙ ﺍﳌﺠﻤﻮﻋﺔ ﺍﻟﻀﺎﺑﻄﺔ ) ‪. ( Cm-Cp‬‬ ‫ﻭﻗﺪ ﺃﻭﺿﺤﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻋﻤﻮﻣ ﹰﺎ ﺃﻥ ﺍﻟﺘﻌﺮﺽ ﻟﻠﺮﺻﺎﺹ ﺧﻼﻝ ﻓﱰﺓ ﺍﻟﻨﻤﻮ ﺍﳌﺒﻜﺮﺓ ﺃﺩﺕ ﺇﱃ ﺗﻐﲑﺍﺕ ﺳﻠﻮﻛﻴﺔ ﻭ ﻛﻴﻤﻮﺣﻴﻮﻳﺔ ﰲ ﺍﻟﻔﺌﺮﺍﻥ‪،‬‬ ‫ﻭﺃﻥ ﻫﺬﻩ ﺍﻟﺘﻐﲑﺍﺕ ﺃﺷﺎﺭﺕ ﺇﱃ ﺳﻬﻮﻟﺔ ﻭﺻﻮﻝ ﺍﻟﺮﺻﺎﺹ ﺇﱃ ﺻﻐﺎﺭ ﺍﻟﻔﺌﺮﺍﻥ ﺍﳌﻌﺎﳉﺔ ﺃﻣﻬﺎﲥﺎ ﺑﺎﻟﺮﺻﺎﺹ ﻋﻦ ﻃﺮﻳﻖ ﺍﻟﺮﺣﻢ ﺃﻭ ﻋﻦ ﻃﺮﻳﻖ‬ ‫ﺣﻠﻴﺐ ﺍﻷﻡ ﺃﻭ ﺑﻜﻠﻴﻬﲈ ‪ ،‬ﳑﺎ ﻳﻮﺣﻲ ﺑﺨﻄﻮﺭﺓ ﺍﻟﺘﻌﺮﺽ ﻟﻠﺮﺻﺎﺹ ﺧﻼﻝ ﻓﱰﺓ ﺍﻟﻨﻤﻮ ﺍﳌﺒﻜﺮﺓ ﻣﻦ ﺣﻴﺎﺓ ﺍﳊﻴﻮﺍﻥ ‪.‬‬ ‫‪Saudi Journal of Biological Sciences Vol. 13, No.1, 2006‬‬ ‫‪90‬‬