Following the development of modern transportation and industry, the environmental lead pollution is getting ever more severe and the situation with regard to the lead poisoning of children is serious. The nervous system of children is in a stage of rapid development and maturation and is thus particularly susceptible to the toxic effects of lead exposure. Once the blood lead concentration exceeds 0.483 μmol/L (100 μg/L) in children, learning and memory abilities can be impaired even though noticeable clinical symptoms may not be present. In recent years, much research has been done on the neurotoxic mechanism of lead. It was found that lead inhibits NOS and NMDA receptor activity in the central nervous system and thus influences learning and memory functions (Zhang et al., 2002
; Chetty et al., 2001
). Yet those results have not yet been finally verified. No studies on the selective influence of low-level lead exposure on the NOS activity in different brain regions or on the dosage-dependency of that inhibitory effect have been reported so far. Furthermore, it is still unclear whether lead has an influence on the expression of NMDA-R mRNA in different subunits and whether there is a connection between the effects and different stages of development.
In this study, we successfully established a rat animal model for low-level lead exposure over different stages of development. Apart from the 0.075% group, in which the blood lead concentration reached 500–600 μg/L on the 21st and 28th day, all other measurements ranged from 200 to 400 μg/L, a level that is comparable to the present diagnostic standard of lead poisoning in Chinese children. In the process of lead exposure, no gastroenteral, neurological or other symptoms of lead poisoning were observed in the maternal rats nor in the pups. The body weight between the different groups did not show significant differences, either, so that our animal model represents a model of subclinical lead poisoning.
The blood lead concentration in all experimental groups followed a characteristic pattern. It was relatively high on the 7th day, slightly lower on the 14th day, markedly elevated on the 21st day and showed a slight decrease again on the 28th day. This result is comparable to that reported by Guilarte and McGlothan (1998
). As the renal capacity of lead excretion is relatively low within the first week after birth, blood lead levels may be high on the 7th day and then decrease as the renal excretion functions mature until the 14th day. Starting from the 19th day, the rat pups learn to take in food by themselves, while the milk feeding has not yet been discontinued. Thus, on the 21st day after birth the pups are exposed to lead from the mother’s milk as well as from water, so that the blood lead concentration was found to be markedly elevated at that point of measurement. And as the 28th day approaches, the small bowl of the pups matures and gradually absorbs less lead until a level similar to adult rats is reached, thus resulting in decreasing blood lead concentrations.
In this study, we found that the NOS activity in hippocampus and cerebellum was not affected by lead exposure on the 7th and 14th day. But on the 21st and 28th day, the NOS activity in the hippocampus and cerebellum of lead-exposed groups was significantly lower than that of the control one. These data suggested that the inhibition of low level lead exposure on the NOS activity in hippocampus and cerebellum depended on the exposure time. This was consistent with a previous study (Chetty et al., 2001
). On the other hand, the inhibition of lead exposure on the NOS activity in hippocampus and cerebellum of the 0.05% and 0.075% groups was not so great and evident as that of the 0.025% group. It needs to be further investigated whether there is a compensation mechanism that will be activated when the lead level of hippocampus and cerebellum is too high to seriously inhibit NOS activity.
NOS activity in the cerebral cortex of the 0.075% group significantly decreased as compared with the control group at all four day spans (P<0.05). But the other lower level lead-exposed groups were not affected. This indicated that the lead inhibition on NOS activity in the cerebral cortex related to the exposure level of lead. The decrease of NOS activity in cerebral cortex was consistent with the report that lead exposure decreased the number of NOS positive neurons in the cerebral cortex.
There was no significantly difference of NOS activity in the brain stem between any lead-exposed group and the control group on the four day spans (P>0.05). This indicated that NOS activity in the brain stem might not be significantly affected by lead exposure during developmental period. The influence of low level lead exposure on the NOS activity in the brain stem needs further study.
The hippocampus is a central neuronal structure for learning and memory. The human cerebral cortex receives information processed by the hippocampus and other regions of the brain. The cerebral cortex plays an important role in the storage of information. Like the hippocampus and the cerebellum, the cerebral cortex may also be affected by lead exposure. Not only does experimental data provide evidence for the damage done to cerebral neurons, clinical observations also show that lead exposure can lead to impaired cognitive functions with loss of attention, impaired coordination abilities and other symptoms. Furthermore, structural abnormalities in the cerebellum can result from lead exposure and influence long-term depression (LTD). The results of this study show that low levels of lead have certain inhibitory effect on the NOS activity in different brain regions of rats in the developing period. This inhibitory effect was found to be selective for different regions and was shown to depend on the time of exposure and/or the concentration of lead the subjects were exposed to. The decrease of NOS activity explained the lead-mediated cognitive deficits because NO regulates long-term potentiation (LTP) and other neurophysiological events in the developing nervous system (O’Dell et al., 1991
In this work, we investigated the mRNA expression of the NMDA receptor subunits NR2A and NR2B and the influence that lead exposure has on this expression. Compared with the normal control group, the level of NR2A expression was significantly higher in the 0.05% and 0.075% groups on the 7th and the 14th day of age. In the 0.025% group, a significant difference was found on the 7th day only. No significant results for NR2A mRNA expression were found at all other points of measurements. The level of NR2B mRNA expression did not differ between the control and the experimental groups. This shows that low-level lead exposure during the developmental period can increase the mRNA expression for the NR2A subunit of the NMDA receptor in the hippocampus of rat pups. This effect was particularly noticeable at an early stage of development (7th day, 14th day). The same level of lead exposure did not affect the mRNA expression for the NR2B subunit of the NMDA receptor.
The normal physiological and pharmacological function of the NMDA receptor depends on its proportional composition of all subunits (Monyer et al., 1992
). The increased expression of NR2A mRNA in the hippocampus may lead to a change in the number or in the structure of NMDA receptors and eventually could influence the LTP conductance for which the NMDA receptor is the physiological basis (Zhang et al., 2002
). The hippocampus LTP, in turn, is known to be one of the important neurological factors in learning and memory functions. The main results derived from this study direct attention on the first two weeks after the birth of the pups. These two weeks after birth have been characterized as the key period for the development of the function of the hippocampus, of learning and memory in mammals (Altmann et al., 1993
). Therefore, we conclude that the influence on NR2A mRNA expression may be one toxicological mechanism through which lead affects learning and memory. This mechanism needs to be further investigated.